RU2829327C2 - Il-2 conjugates and methods of use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to an IL-2 conjugate containing an amino acid sequence having at least approximately 80% identity to SEQ ID NO: 3, where at least one amino acid residue in an amino acid position selected from K34, F41, K42, E61, P64, R37, T40, E67, Y44 and V68, relating to positions of amino acids in SEQ ID NO: 3, is replaced by the structure of formula (I), where the values of X, Y, Z are defined in the claims, and the method for production thereof. Also disclosed are pharmaceutical compositions containing one or more conjugates of IL-2.

EFFECT: conjugates of IL-2 are intended for treating cancer in a patient.

21 cl, 36 dwg, 19 tbl, 15 ex

Description

CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/802,191, filed February 6, 2019, U.S. Provisional Patent Application No. 62/847,844, filed May 14, 2019, U.S. Provisional Patent Application No. 62/870,581, filed July 3, 2019, U.S. Provisional Patent Application No. 62/899,035, filed September 11, 2019, and U.S. Provisional Patent Application No. 62/940,173, filed November 25, 2019, all of which are incorporated by reference in their entireties.

SEQUENCE LIST

[0001.1] This application contains a sequence listing that was filed electronically in ASCII-encoded format and is hereby incorporated by reference in its entirety. A copy of said ASCII-encoded file created on February 4, 2020 is named 46085-729_601_SL.txt and is 124,806 bytes in size.

BACKGROUND OF THE INVENTION

[0002] Distinct populations of T cells modulate the immune system to maintain immune homeostasis and tolerance. For example, regulatory T (Treg) cells prevent inappropriate immune responses by preventing pathological autoreactivity, while cytotoxic T cells target and destroy infected cells and/or cancer cells. In some cases, modulation of different T cell populations provides a treatment option for a disease or trait.

BRIEF DESCRIPTION OF THE INVENTION

[0003] Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (I),

formula (I),

Where

Z is CH ₂ and Y is ;

Y is CH ₂ , and Z is ;

Z is CH ₂ and Y is ; or

Y is CH ₂ , and Z is ;

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has a structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein Z is CH ₂ and Y is , or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein Y is CH ₂ , and Z is , or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein Z is CH ₂ and Y is , or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein Z is CH ₂ and Y is , or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein Y is CH ₂ , and Z is , or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the PEG moiety has an average molecular weight selected from 5 kDa, 10 kDa, 20 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the PEG moiety has an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the PEG moiety has an average molecular weight of 10 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the PEG moiety has an average molecular weight of 15 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the PEG moiety has an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the PEG moiety has an average molecular weight of 25 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the PEG moiety has an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the PEG moiety has an average molecular weight of 35 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the PEG moiety has an average molecular weight of 40 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate in which the PEG moiety has an average molecular weight of 45 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate in which the PEG moiety has an average molecular weight of 50 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate in which the PEG moiety has an average molecular weight of 60 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is selected from K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69 and L72, wherein the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is selected from F42, E62 and P65, wherein the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is K35, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is F42, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is F44, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is K43, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is E62, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is P65, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is R38, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is T41, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is E68, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is Y45, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is V69, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is L72, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0004] Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 4, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (I),

formula (I),

Where

Z is CH ₂ and Y is ;

Y is CH ₂ , and Z is ;

Z is CH ₂ and Y is ; or

Y is CH ₂ , and Z is ;

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has the structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein Z is CH ₂ and Y is , or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein Y is CH ₂ , and Z is , or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein Z is CH ₂ and Y is , or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein Z is CH ₂ and Y is , or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein Y is CH ₂ , and Z is , or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the PEG moiety has an average molecular weight selected from 5 kDa, 10 kDa, 20 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the PEG moiety has an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the PEG moiety has an average molecular weight of 10 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the PEG moiety has an average molecular weight of 15 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the PEG moiety has an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the PEG moiety has an average molecular weight of 25 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the PEG moiety has an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the PEG moiety has an average molecular weight of 35 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the PEG moiety has an average molecular weight of 40 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate in which the PEG moiety has an average molecular weight of 45 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate in which the PEG moiety has an average molecular weight of 50 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate in which the PEG moiety has an average molecular weight of 60 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is selected from K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69 and L72, wherein the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is selected from F42, E62 and P65, wherein the position of the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is K35, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is F42, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is F44, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is K43, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is E62, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is P65, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is R38, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is T41, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is E68, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is Y45, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is V69, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is L72, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is related to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0005] Described herein are IL-2 conjugates comprising the amino acid sequence of any one of SEQ ID NOs: 15-19, wherein [AzK_PEG] has a structure of formula (II), or formula (III), or a mixture of formula (II) and formula (III),

formula (II),

formula (III),

Where

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has the structure

; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein [AzK_PEG] is a mixture of formula (II) and formula (III), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein [AzK_PEG] has the structure of formula (II),

formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 15, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 16, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 17, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 18, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 19, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein [AzK_PEG] has the structure of formula (III),

formula (III); or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 15, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 16, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 17, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 18, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 19, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a linear or branched PEG moiety, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a linear PEG moiety, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a branched PEG moiety, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a methoxy-PEG moiety, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the methoxy-PEG moiety is linear or branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the methoxy-PEG moiety is linear, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate in which the methoxy-PEG moiety is branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. An exemplary structure of a methoxy-PEG moiety is shown in the mPEG-DBCO structure in Scheme 1 of Example 2.

[0006] In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 15, 16, 17, 18, and 19, wherein the [AzK_PEG] comprises a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 60 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 15, 16, 17, 18, and 19, wherein the [AzK_PEG] comprises a PEG moiety having an average molecular weight of 5 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 15, 16, 17, 18, and 19, wherein [AzK_PEG] comprises a PEG moiety having an average molecular weight of 10 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 15, 16, 17, 18, and 19, wherein [AzK_PEG] comprises a PEG moiety having an average molecular weight of 15 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 15, 16, 17, 18, and 19, wherein [AzK_PEG] comprises a PEG moiety having an average molecular weight of 20 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 15, 16, 17, 18, and 19, wherein [AzK_PEG] comprises a PEG moiety having an average molecular weight of 25 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 15, 16, 17, 18, and 19, wherein [AzK_PEG] comprises a PEG moiety having an average molecular weight of 30 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 15, 16, 17, 18, and 19, wherein [AzK_PEG] comprises a PEG moiety having an average molecular weight of 35 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 15, 16, 17, 18, and 19, wherein [AzK_PEG] comprises a PEG moiety having an average molecular weight of 40 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 15, 16, 17, 18, and 19, wherein [AzK_PEG] comprises a PEG moiety having an average molecular weight of 45 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 15, 16, 17, 18, and 19, wherein [AzK_PEG] comprises a PEG moiety having an average molecular weight of 50 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 15, 16, 17, 18, and 19, wherein [AzK_PEG] comprises a PEG moiety having an average molecular weight of 60 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 15, 16, 17, 18, and 19, wherein [AzK_PEG] comprises a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 60 kDa, wherein the PEG moiety is a methoxy-PEG moiety, a linear methoxy-PEG moiety, or a branched methoxy-PEG moiety.

[0007] Described herein are IL-2 conjugates comprising the amino acid sequence of any one of SEQ ID NOs: 20-24, wherein [AzK_PEG5kD] has a structure of formula (II), or formula (III), or a mixture of formula (II) and formula (III),

formula (II),

formula (III),

Where

W is a PEG group having an average molecular weight of 5 kDa; and

X has the structure

; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 20, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 21, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 22, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 23, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 24, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein [AzK_PEG5kD] has the structure of formula (II),

formula (II),

or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 20, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 21, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 22, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 23, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 24, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein [AzK_PEG5kD] has the structure of formula (III),

formula (III); or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 20, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 21, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 22, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 23, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 24, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0008] Described herein are IL-2 conjugates comprising an amino acid sequence of any one of SEQ ID NOs: 25-29, wherein [AzK_PEG30kD] has a structure of formula (II) or formula (III) or is a mixture of structures of formula (II) and formula (III),

formula (II),

formula (III),

Where

W is a PEG group having an average molecular weight of 30 kDa; and

X has the structure

; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 25, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 26, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 27, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 28, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 29, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein [AzK_PEG30kD] has the structure of formula (II),

formula (II), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 25, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 26, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 27, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 28, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 29, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein [AzK_PEG30kD] has the structure of formula (III),

formula (III); or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 25, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 26, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 27, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 28, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 29, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0009] Described herein are IL-2 conjugates comprising the amino acid sequence of any one of SEQ ID NOs: 15-19, wherein [AzK_PEG] is a mixture of structures of formula (II) and formula (III),

formula (II),

formula (III),

Where

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has a structure

; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) contained in the total amount of [AzK_PEG] in the IL-2 conjugate is about 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) contained in the total amount of [AzK_PEG] in the IL-2 conjugate is greater than 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) contained in the total amount of [AzK_PEG] in the IL-2 conjugate is less than 1:1. In some embodiments, described herein is an IL-2 conjugate wherein W is a linear or branched PEG moiety, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a linear PEG moiety, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a branched PEG moiety, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a methoxy-PEG moiety, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the methoxy-PEG moiety is linear or branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate in which the methoxy-PEG group is linear, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate in which the methoxy-PEG group is branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0010] Described herein are IL-2 conjugates comprising the amino acid sequence of any one of SEQ ID NOs: 20-24, wherein [AzK_PEG5kD] is a mixture of structures of formula (II) and formula (III),

formula (II),

formula (III),

Where

W is a PEG group having an average molecular weight of 5 kDa; and

X has a structure

; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (II) to the amount of a structure of formula (III) contained in the total amount of [AzK_PEG5kD] in the IL-2 conjugate is about 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (II) to the amount of a structure of formula (III) contained in the total amount of [AzK_PEG5kD] in the IL-2 conjugate is greater than 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (II) to the amount of a structure of formula (III) contained in the total amount of [AzK_PEG5kD] in the IL-2 conjugate is less than 1:1.

[0011] Described herein are IL-2 conjugates comprising the amino acid sequence of any one of SEQ ID NOs: 25-29, wherein [AzK_PEG30kD] is a mixture of structures of formula (II) and formula (III),

formula (II),

formula (III),

Where

W is a PEG group having an average molecular weight of 30 kDa; and

X has a structure

; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (II) to the amount of a structure of formula (III) contained in the total amount of [AzK_PEG30kD] in the IL-2 conjugate is about 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (II) to the amount of a structure of formula (III) contained in the total amount of [AzK_PEG30kD] in the IL-2 conjugate is greater than 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (II) to the amount of a structure of formula (III) contained in the total amount of [AzK_PEG30kD] in the IL-2 conjugate is less than 1:1.

[0012] Described herein are IL-2 conjugates comprising the amino acid sequence of any one of SEQ ID NOs: 40-44, wherein [AzK_L1_PEG] has a structure of formula (IV), or formula (V), or a mixture of formula (IV) and formula (V),

formula (IV),

formula (V),

Where

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has a structure

; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein [AzK_L1_PEG] is a mixture of formula (IV) and formula (V), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein [AzK_L1_PEG] has the structure of formula (IV),

formula (IV), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 40, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 41, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 42, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 43, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 44, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein [AzK_L1_PEG] has the structure of formula (V),

[0013] formula (V), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 40, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 41, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 42, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 43, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 44, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a PEG moiety having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a linear or branched PEG moiety, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a linear PEG moiety, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a branched PEG moiety, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a methoxy-PEG moiety, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the methoxy-PEG moiety is linear or branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the methoxy-PEG moiety is linear, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate in which the methoxy-PEG moiety is branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] comprises a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 60 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] comprises a PEG moiety having an average molecular weight of 5 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] comprises a PEG moiety having an average molecular weight of 10 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] comprises a PEG moiety having an average molecular weight of 15 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] comprises a PEG moiety having an average molecular weight of 20 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] comprises a PEG moiety having an average molecular weight of 25 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] comprises a PEG moiety having an average molecular weight of 30 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] comprises a PEG moiety having an average molecular weight of 35 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] comprises a PEG moiety having an average molecular weight of 40 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] comprises a PEG moiety having an average molecular weight of 45 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] comprises a PEG moiety having an average molecular weight of 50 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] comprises a PEG moiety having an average molecular weight of 60 kDa. In some embodiments, described herein is an IL-2 conjugate having an amino acid sequence selected from any of SEQ ID NOs: 40, 41, 42, 43, and 44, wherein [AzK_L1_PEG] comprises a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 60 kDa, wherein the PEG moiety is a methoxy-PEG group, a linear methoxy-PEG group, or a branched methoxy-PEG group.

[0014] Described herein are IL-2 conjugates comprising the amino acid sequence of any one of SEQ ID NOs: 45-49, wherein [AzK_L1_PEG5kD] has a structure of formula (IV), or formula (V), or a mixture of formula (IV) and formula (V),

formula (IV),

formula (V),

Where

W is a PEG group having an average molecular weight of 5 kDa; and

X has the structure

; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 45, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 46, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 47, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 48, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 49, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein [AzK_L1_PEG5kD] has the structure of formula (IV),

formula (IV), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 45, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 46, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 47, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 48, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 49, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein [AzK_L1_PEG5kD] has the structure of formula (V),

formula (V), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 45, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 46, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 47, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 48, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 49, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0015] Described herein are IL-2 conjugates comprising the amino acid sequence of any one of SEQ ID NOs: 50-54, wherein [AzK_L1_PEG30kD] has a structure of formula (IV) or formula (V) or is a mixture of structures of formula (IV) and formula (V),

formula (IV),

formula (V),

Where

W is a PEG group having an average molecular weight of 30 kDa; and

X has a structure

; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 50, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 51, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 52, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 53, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 54, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein [AzK_L1_PEG30kD] has the structure of formula (IV),

formula (IV), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 50, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 51, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 52, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 53, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 54, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein [AzK_L1_PEG30kD] has the structure of formula (V),

formula (V), or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 50, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 51, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 52, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 53, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 54, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0016] Described herein are IL-2 conjugates comprising the amino acid sequence of any one of SEQ ID NOs: 40-44, wherein [Azk_L1_PEG] is a mixture of structures of formula (IV) and formula (V),

formula (IV),

formula (V),

Where

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has a structure

; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) contained in the total amount of [AzK_L1_PEG] in the IL-2 conjugate is about 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) contained in the total amount of [AzK_L1_PEG] in the IL-2 conjugate is greater than 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) contained in the total amount of [AzK_L1_PEG] in the IL-2 conjugate is less than 1:1. In some embodiments, described herein is an IL-2 conjugate wherein W is a linear or branched PEG moiety, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a linear PEG moiety, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a branched PEG moiety, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein W is a methoxy-PEG moiety, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the methoxy-PEG moiety is linear or branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate in which the methoxy-PEG group is linear, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate in which the methoxy-PEG group is branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[0017] Described herein are IL-2 conjugates comprising the amino acid sequence of any one of SEQ ID NOs: 45-49, wherein [AzK_L1_PEG5kD] is a mixture of structures of formula (IV) and formula (V),

formula (IV),

formula (V),

Where

W is a PEG group having an average molecular weight of 5 kDa; and

X has a structure

; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (IV) to the amount of a structure of formula (V) contained in the total amount of [AzK_L1_PEG5kD] in the IL-2 conjugate is about 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (IV) to the amount of a structure of formula (V) contained in the total amount of [AzK_L1_PEG5kD] in the IL-2 conjugate is greater than 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (IV) to the amount of a structure of formula (V) contained in the total amount of [AzK_L1_PEG5kD] in the IL-2 conjugate is less than 1:1.

[0018] Described herein are IL-2 conjugates comprising the amino acid sequence of any one of SEQ ID NOs: 50-54, wherein [AzK_L1_PEG30kD] is a mixture of structures of formula (IV) and formula (V),

formula (IV),

formula (V),

Where

W is a PEG group having an average molecular weight of 30 kDa; and

X has a structure

; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (IV) to the amount of a structure of formula (V) contained in the total amount of [AzK_L1_PEG30kD] in the IL-2 conjugate is about 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (IV) to the amount of a structure of formula (V) contained in the total amount of [AzK_L1_PEG30kD] in the IL-2 conjugate is greater than 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (IV) to the amount of a structure of formula (V) contained in the total amount of [AzK_L1_PEG30kD] in the IL-2 conjugate is less than 1:1.

[0019] Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (VI) or (VII) or a mixture of (VI) and (VII),

formula (VI),

formula (VII),

Where

n is an integer in the range from approximately 2 to approximately 5000; and

X has the structure , or a pharmaceutically acceptable salt, solvate or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulae (VI) and (VII) is in the range of from about 5 to about 4600, or from about 10 to about 4000, or from about 20 to about 3000, or from about 100 to about 3000, or from about 100 to about 2900, or from about 150 to about 2900, or from about 125 to about 2900, or from about 100 to about 2500, or from about 100 to about 2000, or from about 100 to about 1900, or from about 100 to about 1850, or from about 100 to about 1750, or from about 100 to about 1650, or from about 100 to about 1500, or from about 100 to about 1400, or from about 100 to about 1300, or from about 100 to about 1250, or from about 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulae (VI) and (VII) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68 and L71, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is among the positions in SEQ ID NO: 3. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position K34. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position F41. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position F43. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position K42. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position E61. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position P64. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position R37. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position T40. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position E67. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position Y44. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position V68. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VI), formula (VII), or a mixture of formulas (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position L71. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of the structure of formula (VI) to the amount of the structure of formula (VII) contained in the total amount of the IL-2 conjugate is about 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of the structure of formula (VI) to the amount of the structure of formula (VII) contained in the total amount of the IL-2 conjugate is greater than 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of the structure of formula (VI) to the amount of the structure of formula (VII) contained in the total amount of the IL-2 conjugate is less than 1:1.

[0020] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VI) or (VII) or a mixture of (VI) and (VII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and wherein n is an integer from 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or from about 341 to about 1136, or from about 341 to about 1023, or from about 341 to about 910, or from about 341 to about 796, or from about 341 to about 682, or from about 341 to about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of Formulas (VI) and (VII) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 45, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4545, and 4546.

[0021] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VI) or (VII), or a mixture of (VI) and (VII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (VI) and (VII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, and 1249.

[0022] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VI) or (VII), or a mixture of (VI) and (VII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (VI) and (VII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910.

[0023] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VI) or (VII), or a mixture of (VI) and (VII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (VI) and (VII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0024] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VI) or (VII), or a mixture of (VI) and (VII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (VI) and (VII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0025] Disclosed herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VI) or (VII) or a mixture of (VI) and (VII), wherein n is an integer such that the molecular weight of the PEG moiety is in the range of from about 1,000 Daltons to about 200,000 Daltons, or from about 2,000 Daltons to about 150,000 Daltons, or from about 3,000 Daltons to about 125,000 Daltons, or from about 4,000 Daltons to about 100,000 Daltons, or from about 5,000 Daltons to about 100,000 Daltons, or from about 6,000 Daltons to about 90,000 Daltons, or from about 7,000 daltons to about 80,000 daltons, or from about 8,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 65,000 daltons, or from about 5,000 daltons to about 60,000 daltons, or from about 5,000 daltons to about 50,000 daltons, or from about 6,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 45,000 daltons, or from about 7,000 daltons to about 40,000 daltons, or from about 8,000 daltons to about 40,000 daltons, or from about 8,500 daltons to about 40,000 daltons, or from about 8,500 daltons to about 35,000 daltons, or from about 9,000 daltons to about 50,000 daltons, or from about 9,000 daltons to about 45,000 daltons, or from about 9,000 daltons to about 40,000 daltons, or from about 9,000 daltons to about 35,000 daltons, or from about 9,000 daltons to about 30,000 daltons, or from about 9,500 daltons to about 35,000 daltons, or from about 9,500 daltons to about 30,000 daltons, or from about 10,000 daltons to about 50,000 daltons, or from about 10,000 daltons to about 45,000 daltons, or from about 10,000 daltons to about 40,000 daltons, or from about 10,000 daltons to about 35,000 daltons, or from about 10,000 daltons to about 30,000 daltons, or from about 15,000 daltons to about 50,000 daltons, or from about 15,000 daltons to about 45,000 daltons, or from about 15,000 daltons to about 40,000 daltons, or from about 15,000 daltons to about 35,000 daltons, or from about 15,000 daltons to about 30,000 daltons, or from about 20,000 daltons to about 50,000 daltons, or from about 20,000 daltons to about 45,000 daltons, or from about 20,000 daltons to about 40,000 daltons, or from about 20,000 daltons to about 35,000 daltons, or from about 20,000 daltons to about 30,000 daltons. Disclosed herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VI) or (VII) or a mixture of (VI) and (VII), wherein n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000 Daltons, about 60,000 Daltons, about 70,000 Daltons, about 80,000 Daltons, about 90,000 daltons, approximately 100,000 daltons, approximately 125,000 daltons, approximately 150,000 daltons, approximately 175,000 daltons, or approximately 200,000 daltons. Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (VI) or (VII) or a mixture of (VI) and (VII), wherein n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, or about 50,000 Daltons.

[0026] Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (VIII) or (IX) or a mixture of (VIII) and (IX),

formula (VIII),

formula (IX),

Where

n is an integer in the range from approximately 2 to approximately 5000; and

X has a structure , or a pharmaceutically acceptable salt, solvate or hydrate thereof. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formula (VIII) and (IX) is in the range of from about 5 to about 4600, or from about 10 to about 4000, or from about 20 to about 3000, or from about 100 to about 3000, or from about 100 to about 2900, or from about 150 to about 2900, or from about 125 to about 2900, or from about 100 to about 2500, or from about 100 to about 2000, or from about 100 to about 1900, or from about 100 to about 1850, or from about 100 to about 1750, or from about 100 to about 1650, or from about 100 to about 1500, or from about 100 to about 1400, or from about 100 to about 1300, or from about 100 to about 1250, or from about 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formula (VIII) and (IX) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546. In some embodiments, described herein is an IL-2 conjugate wherein a position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68 and L71, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate refers to the positions in SEQ ID NO: 3. In some embodiments, described herein is an IL-2 conjugate, wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68 and L71. In some embodiments, described herein is an IL-2 conjugate, wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position K34. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position F41. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position F43. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position K42. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position E61. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position P64. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position R37. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position T40. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position E67. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position Y44. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position V68. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formula (VIII), formula (IX), or a mixture of formulas (VIII) and (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position L71. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of the structure of formula (VIII) to the amount of the structure of formula (IX) contained in the total amount of the IL-2 conjugate is about 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of the structure of formula (VIII) to the amount of the structure of formula (IX) contained in the total amount of the IL-2 conjugate is greater than 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of the structure of formula (VIII) to the amount of the structure of formula (IX) contained in the total amount of the IL-2 conjugate is less than 1:1.

[0027] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VIII) or (IX), or a mixture of (VIII) and (IX), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and wherein n is an integer from 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or from about 341 to about 1136, or from about 341 to about 1023, or from about 341 to about 910, or from about 341 to about 796, or from about 341 to about 682, or from about 341 to about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formula (VIII) and (IX) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

[0028] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VIII) or (IX), or a mixture of (VIII) and (IX), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formula (VIII) and (IX) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137 and 1249.

[0029] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VIII) or (IX), or a mixture of (VIII) and (IX), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formula (VIII) and (IX) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910.

[0030] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VIII) or (IX), or a mixture of (VIII) and (IX), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formula (VIII) and (IX) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0031] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VIII) or (IX), or a mixture of (VIII) and (IX), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formula (VIII) and (IX) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0032] Disclosed herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VIII) or (IX) or a mixture of (VIII) and (IX), wherein n is an integer such that the molecular weight of the PEG moiety is in the range of from about 1,000 Daltons to about 200,000 Daltons, or from about 2,000 Daltons to about 150,000 Daltons, or from about 3,000 Daltons to about 125,000 Daltons, or from about 4,000 Daltons to about 100,000 Daltons, or from about 5,000 Daltons to about 100,000 Daltons, or from about 6,000 Daltons to about 90,000 Daltons, or from about 7,000 daltons to about 80,000 daltons, or from about 8,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 65,000 daltons, or from about 5,000 daltons to about 60,000 daltons, or from about 5,000 daltons to about 50,000 daltons, or from about 6,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 45,000 daltons, or from about 7,000 daltons to about 40,000 daltons, or from about 8,000 daltons to about 40,000 daltons, or from about 8,500 daltons to about 40,000 daltons, or from about 8,500 daltons to about 35,000 daltons, or from about 9,000 daltons to about 50,000 daltons, or from about 9,000 daltons to about 45,000 daltons, or from about 9,000 daltons to about 40,000 daltons, or from about 9,000 daltons to about 35,000 daltons, or from about 9,000 daltons to about 30,000 daltons, or from about 9,500 daltons to about 35,000 daltons, or from about 9,500 daltons to about 30,000 daltons, or from about 10,000 daltons to about 50,000 daltons, or from about 10,000 daltons to about 45,000 daltons, or from about 10,000 daltons to about 40,000 daltons, or from about 10,000 daltons to about 35,000 daltons, or from about 10,000 daltons to about 30,000 daltons, or from about 15,000 daltons to about 50,000 daltons, or from about 15,000 daltons to about 45,000 daltons, or from about 15,000 daltons to about 40,000 daltons, or from about 15,000 daltons to about 35,000 daltons, or from about 15,000 daltons to about 30,000 daltons, or from about 20,000 daltons to about 50,000 daltons, or from about 20,000 daltons to about 45,000 daltons, or from about 20,000 daltons to about 40,000 daltons, or from about 20,000 daltons to about 35,000 daltons, or from about 20,000 daltons to about 30,000 daltons. Disclosed herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (VIII) or (IX), or a mixture of (VIII) and (IX), wherein n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000 Daltons, about 60,000 Daltons, about 70,000 Daltons, about 80,000 Daltons, about 90,000 daltons, approximately 100,000 daltons, approximately 125,000 daltons, approximately 150,000 daltons, approximately 175,000 daltons, or approximately 200,000 daltons. Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (VIII) or (IX) or a mixture of (VIII) and (IX), wherein n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, or about 50,000 Daltons.

[0033] Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (X) or (XI) or a mixture of (X) and (XI),

formula (X),

formula (XI),

Where

n is an integer in the range from approximately 2 to approximately 5000; and

wavy lines indicate covalent bonds to amino acid residues in SEQ ID NO: 3 that are not substituted, or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0034] In some embodiments, the stereochemistry of the chiral center of formula (X) and formula (XI) provides a racemic mixture, provides a mixture enriched in (R), provides a mixture enriched in (S), provides substantially only (R), provides substantially only (S), provides only (R), or provides only (S). In some embodiments, the stereochemistry of the chiral center of formula (X) and formula (XI) provides a racemic mixture. In some embodiments, the stereochemistry of the chiral center of formula (X) and formula (XI) provides a mixture enriched in (R). In some embodiments, the stereochemistry of the chiral center of formula (X) and formula (XI) provides a mixture enriched in (S). In some embodiments, the stereochemistry of the chiral center of formula (X) and formula (XI) provides substantially only (R). In some embodiments, the stereochemistry of the chiral center of formula (X) and formula (XI) provides substantially only (S). In some embodiments, the stereochemistry of the chiral center in formula (X) and formula (XI) is only (R). In some embodiments, the stereochemistry of the chiral center in formula (X) and formula (XI) is only (S).

[0035] In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of Formulae (X) and (XI) is in the range of from about 5 to about 4600, or from about 10 to about 4000, or from about 20 to about 3000, or from about 100 to about 3000, or from about 100 to about 2900, or from about 150 to about 2900, or from about 125 to about 2900, or from about 100 to about 2500, or from about 100 to about 2000, or from about 100 to about 1900, or from about 100 to about 1850, or from about 100 to about 1750, or from about 100 to about 1650, or from about 100 to about 1500, or from about 100 to about 1400, or from about 100 to about 1300, or from about 100 to about 1250, or from about 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulae (X) and (XI) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 37, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (X) and (XI) or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68 and L71, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate refers to the positions in SEQ ID NO: 3. In some embodiments, described herein is an IL-2 conjugate, wherein the position of the structure of formulas (X) and (XI) or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68 and L71. In some embodiments, described herein is an IL-2 conjugate, wherein the position of the structure of formulas (X) and (XI) or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position K34. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (X) and (XI) or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position F41. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (X) and (XI) or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position F43. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (X) and (XI) or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position K42. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (X) and (XI) or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position E61. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (X) and (XI) or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position P64. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (X) and (XI) or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position R37. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (X) and (XI) or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position T40. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (X) and (XI) or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position E67. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (X) and (XI) or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position Y44. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (X) and (XI) or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position V68. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (X) and (XI) or a mixture of formulas (X) and (XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position L71. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of the structure of formula (X) to the amount of the structure of formula (XI) contained in the total amount of the IL-2 conjugate is about 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (X) to the amount of a structure of formula (XI) contained in the total amount of the IL-2 conjugate is greater than 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (X) to the amount of a structure of formula (XI) contained in the total amount of the IL-2 conjugate is less than 1:1.

[0036] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (X) or (XI), or a mixture of (X) and (XI), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and wherein n is an integer from 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or from about 341 to about 1136, or from about 341 to about 1023, or from about 341 to about 910, or from about 341 to about 796, or from about 341 to about 682, or from about 341 to about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of Formulas (VI) and (VII) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 45, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4545, and 4546.

[0037] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (X) or (XI), or a mixture of (X) and (XI), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (X) and (XI) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137 and 1249.

[0038] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (X) or (XI), or a mixture of (X) and (XI), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (X) and (XI) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910.

[0039] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (X) or (XI), or a mixture of (X) and (XI), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (X) and (XI) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0040] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (X) or (XI), or a mixture of (X) and (XI), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (X) and (XI) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0041] Disclosed herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (X) or (XI) or a mixture of (X) and (XI), wherein n is an integer such that the molecular weight of the PEG moiety is in the range of from about 1,000 Daltons to about 200,000 Daltons, or from about 2,000 Daltons to about 150,000 Daltons, or from about 3,000 Daltons to about 125,000 Daltons, or from about 4,000 Daltons to about 100,000 Daltons, or from about 5,000 Daltons to about 100,000 Daltons, or from about 6,000 Daltons to about 90,000 Daltons, or from about 7,000 daltons to about 80,000 daltons, or from about 8,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 65,000 daltons, or from about 5,000 daltons to about 60,000 daltons, or from about 5,000 daltons to about 50,000 daltons, or from about 6,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 45,000 daltons, or from about 7,000 daltons to about 40,000 daltons, or from about 8,000 daltons to about 40,000 daltons, or from about 8,500 daltons to about 40,000 daltons, or from about 8,500 daltons to about 35,000 daltons, or from about 9,000 daltons to about 50,000 daltons, or from about 9,000 daltons to about 45,000 daltons, or from about 9,000 daltons to about 40,000 daltons, or from about 9,000 daltons to about 35,000 daltons, or from about 9,000 daltons to about 30,000 daltons, or from about 9,500 daltons to about 35,000 daltons, or from about 9,500 daltons to about 30,000 daltons, or from about 10,000 daltons to about 50,000 daltons, or from about 10,000 daltons to about 45,000 daltons, or from about 10,000 daltons to about 40,000 daltons, or from about 10,000 daltons to about 35,000 daltons, or from about 10,000 daltons to about 30,000 daltons, or from about 15,000 daltons to about 50,000 daltons, or from about 15,000 daltons to about 45,000 daltons, or from about 15,000 daltons to about 40,000 daltons, or from about 15,000 daltons to about 35,000 daltons, or from about 15,000 daltons to about 30,000 daltons, or from about 20,000 daltons to about 50,000 daltons, or from about 20,000 daltons to about 45,000 daltons, or from about 20,000 daltons to about 40,000 daltons, or from about 20,000 daltons to about 35,000 daltons, or from about 20,000 daltons to about 30,000 daltons. Disclosed herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (X) or (XI), or a mixture of (X) and (XI), wherein n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000 Daltons, about 60,000 Daltons, about 70,000 Daltons, about 80,000 Daltons, about 90,000 daltons, approximately 100,000 daltons, approximately 125,000 daltons, approximately 150,000 daltons, approximately 175,000 daltons, or approximately 200,000 daltons. Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (X) or (XI) or a mixture of (X) and (XI), wherein n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, or about 50,000 Daltons.

[0042] Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (XII) or (XIII) or a mixture of (XII) and (XIII),

formula (XII),

formula (XIII),

Where

n is an integer in the range from approximately 2 to approximately 5000; and

The wavy lines indicate covalent bonds to the amino acid residues in SEQ ID NO: 3 that are not substituted, or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0043] In some embodiments, the stereochemistry of the chiral center of formula (XII) and formula (XIII) provides a racemic mixture, provides a mixture enriched in (R), provides a mixture enriched in (S), provides substantially only (R), provides substantially only (S), provides only (R), or provides only (S). In some embodiments, the stereochemistry of the chiral center of formula (XII) and formula (XIII) provides a racemic mixture. In some embodiments, the stereochemistry of the chiral center of formula (XII) and formula (XIII) provides a mixture enriched in (R). In some embodiments, the stereochemistry of the chiral center of formula (XII) and formula (XIII) provides a mixture enriched in (S). In some embodiments, the stereochemistry of the chiral center of formula (XII) and formula (XIII) provides substantially only (R). In some embodiments, the stereochemistry of the chiral center in formula (XII) and formula (XIII) is essentially only (S). In some embodiments, the stereochemistry of the chiral center in formula (XII) and formula (XIII) is essentially only (R). In some embodiments, the stereochemistry of the chiral center in formula (XII) and formula (XIII) is essentially only (S).

[0044] In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulae (XII) and (XIII) is in the range of from about 5 to about 4600, or from about 10 to about 4000, or from about 20 to about 3000, or from about 100 to about 3000, or from about 100 to about 2900, or from about 150 to about 2900, or from about 125 to about 2900, or from about 100 to about 2500, or from about 100 to about 2000, or from about 100 to about 1900, or from about 100 to about 1850, or from about 100 to about 1750, or from about 100 to about 1650, or from about 100 to about 1500, or from about 100 to about 1400, or from about 100 to about 1300, or from about 100 to about 1250, or from about 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or about 341 to about 1136, or about 341 to about 1023, or about 341 to about 910, or about 341 to about 796, or about 341 to about 682, or about 341 to about 568, or about 114 to about 1000, or about 114 to about 950, or about 114 to about 910, or about 114 to about 800, or about 114 to about 690, or about 114 to about 575. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulae (XII) and (XIII) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XII) and (XIII) or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68 and L71, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate refers to the positions in SEQ ID NO: 3. In some embodiments, described herein is an IL-2 conjugate, wherein the position of the structure of formulas (XII) and (XIII) or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68 and L71. In some embodiments, described herein is an IL-2 conjugate, wherein the position of the structure of formulas (XII) and (XIII) or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position K34. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XII) and (XIII) or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position F41. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XII) and (XIII) or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position F43. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XII) and (XIII) or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position K42. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XII) and (XIII) or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position E61. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XII) and (XIII) or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position P64. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XII) and (XIII) or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position R37. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XII) and (XIII) or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position T40. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XII) and (XIII) or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position E67. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XII) and (XIII) or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position Y44. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XII) and (XIII) or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position V68. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XII) and (XIII) or a mixture of formulas (XII) and (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position L71. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of the structure of formula (XII) to the amount of the structure of formula (XIII) contained in the total amount of the IL-2 conjugate is about 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of the structure of formula (XII) to the amount of the structure of formula (XIII) contained in the total amount of the IL-2 conjugate is greater than 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of the structure of formula (XII) to the amount of the structure of formula (XIII) contained in the total amount of the IL-2 conjugate is less than 1:1.

[0045] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XII) or (XIII), or a mixture of (XII) and (XIII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and wherein n is an integer from 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or from about 341 to about 1136, or from about 341 to about 1023, or from about 341 to about 910, or from about 341 to about 796, or from about 341 to about 682, or from about 341 to about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulae (XII) and (XIII) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

[0046] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XII) or (XIII), or a mixture of (XII) and (XIII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (XII) and (XIII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137 and 1249.

[0047] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XII) or (XIII), or a mixture of (XII) and (XIII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (XII) and (XIII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910.

[0048] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XII) or (XIII), or a mixture of (XII) and (XIII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (XII) and (XIII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0049] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XII) or (XIII), or a mixture of (XII) and (XIII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (XII) and (XIII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0050] Disclosed herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XII) or (XIII) or a mixture of (XII) and (XIII), wherein n is an integer such that the molecular weight of the PEG moiety is in the range of from about 1,000 Daltons to about 200,000 Daltons, or from about 2,000 Daltons to about 150,000 Daltons, or from about 3,000 Daltons to about 125,000 Daltons, or from about 4,000 Daltons to about 100,000 Daltons, or from about 5,000 Daltons to about 100,000 Daltons, or from about 6,000 Daltons to about 90,000 Daltons, or from about 7,000 daltons to about 80,000 daltons, or from about 8,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 65,000 daltons, or from about 5,000 daltons to about 60,000 daltons, or from about 5,000 daltons to about 50,000 daltons, or from about 6,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 45,000 daltons, or from about 7,000 daltons to about 40,000 daltons, or from about 8,000 daltons to about 40,000 daltons, or from about 8,500 daltons to about 40,000 daltons, or from about 8,500 daltons to about 35,000 daltons, or from about 9,000 daltons to about 50,000 daltons, or from about 9,000 daltons to about 45,000 daltons, or from about 9,000 daltons to about 40,000 daltons, or from about 9,000 daltons to about 35,000 daltons, or from about 9,000 daltons to about 30,000 daltons, or from about 9,500 daltons to about 35,000 daltons, or from about 9,500 daltons to about 30,000 daltons, or from about 10,000 daltons to about 50,000 daltons, or from about 10,000 daltons to about 45,000 daltons, or from about 10,000 daltons to about 40,000 daltons, or from about 10,000 daltons to about 35,000 daltons, or from about 10,000 daltons to about 30,000 daltons, or from about 15,000 daltons to about 50,000 daltons, or from about 15,000 daltons to about 45,000 daltons, or from about 15,000 daltons to about 40,000 daltons, or from about 15,000 daltons to about 35,000 daltons, or from about 15,000 daltons to about 30,000 daltons, or from about 20,000 daltons to about 50,000 daltons, or from about 20,000 daltons to about 45,000 daltons, or from about 20,000 daltons to about 40,000 daltons, or from about 20,000 daltons to about 35,000 daltons, or from about 20,000 daltons to about 30,000 daltons. Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XII) or (XIII), or a mixture of (XII) and (XIII), wherein n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000 Daltons, about 60,000 Daltons, about 70,000 Daltons, about 80,000 Daltons, approximately 90,000 daltons, approximately 100,000 daltons, approximately 125,000 daltons, approximately 150,000 daltons, approximately 175,000 daltons, or approximately 200,000 daltons. Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (XII) or (XIII) or a mixture of (XII) and (XIII), wherein n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, or about 50,000 Daltons.

[0051] Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (XIV) or (XV) or a mixture of (XIV) and (XV),

formula (XIV),

formula (XV),

Where

m is an integer between 0 and 20;

p is an integer between 0 and 20;

n is an integer in the range from approximately 2 to approximately 5000; and

wavy lines indicate covalent bonds to amino acid residues in SEQ ID NO: 3 that are not substituted, or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0052] In some embodiments, the stereochemistry of the chiral center of formula (XIV) and formula (XV) provides a racemic mixture, provides a mixture enriched in (R), provides a mixture enriched in (S), provides substantially only (R), provides substantially only (S), provides only (R), or provides only (S). In some embodiments, the stereochemistry of the chiral center of formula (XIV) and formula (XV) provides a racemic mixture. In some embodiments, the stereochemistry of the chiral center of formula (XIV) and formula (XV) provides a mixture enriched in (R). In some embodiments, the stereochemistry of the chiral center of formula (XIV) and formula (XV) provides a mixture enriched in (S). In some embodiments, the stereochemistry of the chiral center of formula (XIV) and formula (XV) provides substantially only (R). In some embodiments, the stereochemistry of the chiral center in formula (XIV) and formula (XV) is essentially only (S). In some embodiments, the stereochemistry of the chiral center in formula (XIV) and formula (XV) is essentially only (R). In some embodiments, the stereochemistry of the chiral center in formula (XIV) and formula (XV) is essentially only (S).

[0053] In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of Formulae (XIV) and (XV) is from 0 to 20, or 0 to 18, or 0 to 16, or 0 to 14, or 0 to 12, or 0 to 10, or 0 to 9, or 0 to 8, or 0 to 7, or 0 to 6, or 0 to 5, or 0 to 4, or 0 to 3, or 0 to 2. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of Formulae (XIV) and (XV) is 0. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of Formulae (XIV) and (XV) is 1. In some embodiments, described herein is The document describes an IL-2 conjugate wherein m in the compounds of formulae (XIV) and (XV) is 2. In some embodiments, the document describes an IL-2 conjugate wherein m in the compounds of formulae (XIV) and (XV) is 3. In some embodiments, the document describes an IL-2 conjugate wherein m in the compounds of formulae (XIV) and (XV) is 4. In some embodiments, the document describes an IL-2 conjugate wherein m in the compounds of formulae (XIV) and (XV) is 5. In some embodiments, the document describes an IL-2 conjugate wherein m in the compounds of formulae (XIV) and (XV) is 6. In some embodiments, the document describes an IL-2 conjugate wherein m in the compounds of formulae (XIV) and (XV) is 7. In some embodiments, the document describes a conjugate IL-2, wherein m in the compounds of formulas (XIV) and (XV) is 8. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XIV) and (XV) is 9. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XIV) and (XV) is 10. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XIV) and (XV) is 11. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XIV) and (XV) is 12. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XIV) and (XV) is 13. In some embodiments, described herein is a conjugate IL-2, wherein m in the compounds of formulas (XIV) and (XV) is 14. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XIV) and (XV) is 15. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XIV) and (XV) is 16. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XIV) and (XV) is 17. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XIV) and (XV) is 18. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XIV) and (XV) is 19. In some embodiments, described herein is a conjugate IL-2, in which m in compounds of formulas (XIV) and (XV) is equal to 20.

[0054] In some embodiments, described herein is an IL-2 conjugate wherein p in the compounds of Formulae (XIV) and (XV) is from 0 to 20, or 0 to 18, or 0 to 16, or 0 to 14, or 0 to 12, or 0 to 10, or 0 to 9, or 0 to 8, or 0 to 7, or 0 to 6, or 0 to 5, or 0 to 4, or 0 to 3, or 0 to 2. In some embodiments, described herein is an IL-2 conjugate wherein p in the compounds of Formulae (XIV) and (XV) is 0. In some embodiments, described herein is an IL-2 conjugate wherein p in the compounds of Formulae (XIV) and (XV) is 1. In some embodiments, described herein is The document describes an IL-2 conjugate wherein p in the compounds of formulae (XIV) and (XV) is 2. In some embodiments, the document describes an IL-2 conjugate wherein p in the compounds of formulae (XIV) and (XV) is 3. In some embodiments, the document describes an IL-2 conjugate wherein p in the compounds of formulae (XIV) and (XV) is 4. In some embodiments, the document describes an IL-2 conjugate wherein p in the compounds of formulae (XIV) and (XV) is 5. In some embodiments, the document describes an IL-2 conjugate wherein p in the compounds of formulae (XIV) and (XV) is 6. In some embodiments, the document describes an IL-2 conjugate wherein p in the compounds of formulae (XIV) and (XV) is 7. In some embodiments, the document describes a conjugate IL-2, wherein p in the compounds of formulas (XIV) and (XV) is 8. In some embodiments, described herein is an IL-2 conjugate wherein p in the compounds of formulas (XIV) and (XV) is 9. In some embodiments, described herein is an IL-2 conjugate wherein p in the compounds of formulas (XIV) and (XV) is 10. In some embodiments, described herein is an IL-2 conjugate wherein p in the compounds of formulas (XIV) and (XV) is 11. In some embodiments, described herein is an IL-2 conjugate wherein p in the compounds of formulas (XIV) and (XV) is 12. In some embodiments, described herein is an IL-2 conjugate wherein p in the compounds of formulas (XIV) and (XV) is 13. In some embodiments, described herein is a conjugate IL-2, wherein p in the compounds of formulas (XIV) and (XV) is 14. In some embodiments, described herein is an IL-2 conjugate wherein p in the compounds of formulas (XIV) and (XV) is 15. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XIV) and (XV) is 16. In some embodiments, described herein is an IL-2 conjugate wherein p in the compounds of formulas (XIV) and (XV) is 17. In some embodiments, described herein is an IL-2 conjugate wherein p in the compounds of formulas (XIV) and (XV) is 18. In some embodiments, described herein is an IL-2 conjugate wherein p in the compounds of formulas (XIV) and (XV) is 19. In some embodiments, described herein is a conjugate IL-2, in which p in the compounds of formulas (XIV) and (XV) is equal to 20.

[0055] In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulae (XIV) and (XV) is in the range of from about 5 to about 4600, or from about 10 to about 4000, or from about 20 to about 3000, or from about 100 to about 3000, or from about 100 to about 2900, or from about 150 to about 2900, or from about 125 to about 2900, or from about 100 to about 2500, or from about 100 to about 2000, or from about 100 to about 1900, or from about 100 to about 1850, or from about 100 to about 1750, or from about 100 to about 1650, or from about 100 to about 1500, or from about 100 to about 1400, or from about 100 to about 1300, or from about 100 to about 1250, or from about 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or from about 341 to about 1136, or from about 341 to about 1023, or from about 341 to about 910, or from about 341 to about 796, or from about 341 to about 682, or from about 341 to about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575.

[0056] In some embodiments, described herein is an IL-2 conjugate wherein, in the compounds of formulas (XIV) and (XV), m is an integer from 0 to 6, p is an integer from 0 to 6, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein, in the compounds of formulas (XIV) and (XV), m is an integer from 1 to 6, p is an integer from 1 to 6, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of formulae (XIV) and (XV), m is an integer from 2 to 6, p is an integer from 2 to 6, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulae (XIV) and (XV), m is an integer from 2 to 4, p is an integer from 2 to 4, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulae (XIV) and (XV), m is 1, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136 and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of formulae (XIV) and (XV), m is 2, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of formulae (XIV) and (XV), m is 3, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulas (XIV) and (XV), m is 4, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulas (XIV) and (XV), m is 5, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulae (XIV) and (XV), m is 6, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in In compounds of formulas (XIV) and (XV), m is 7, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in compounds of formulas (XIV) and (XV), m is 8, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulas (XIV) and (XV), m is 9, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulae (XIV) and (XV), m is 10, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein, in the compounds of Formulae (XIV) and (XV), m is 11, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein, in the compounds of Formulae (XIV) and (XV), m is 11, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulas (XIV) and (XV), m is 2, p is 2, and n is an integer selected from 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136 and 1137.

[0057] In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of Formulas (XIV) and (XV) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546. In some embodiments, described herein is an IL-2 conjugate, wherein the position of the structure of formulas (XIV) and (XV) or a mixture of formulas (XIV) and (XV) in the amino acid sequence of the IL-2 conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68 and L71, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate refers to the positions in SEQ ID NO: 3. In some embodiments, described herein is an IL-2 conjugate, wherein the position of the structure of formulas (XIV) and (XV) or a mixture of formulas (XIV) and (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68 and L71. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XIV) and (XV) or a mixture of formulas (XIV) and (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position K34. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XIV) and (XV) or a mixture of formulas (XIV) and (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position F41. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XIV) and (XV) or a mixture of formulas (XIV) and (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position F43. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XIV) and (XV) or a mixture of formulas (XIV) and (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position K42. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XIV) and (XV) or a mixture of formulas (XIV) and (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position E61. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XIV) and (XV) or a mixture of formulas (XIV) and (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position P64. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XIV) and (XV) or a mixture of formulas (XIV) and (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position R37. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XIV) and (XV) or a mixture of formulas (XIV) and (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position T40. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XIV) and (XV) or a mixture of formulas (XIV) and (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position E67. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XIV) and (XV) or a mixture of formulas (XIV) and (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position Y44. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XIV) and (XV) or a mixture of formulas (XIV) and (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position V68. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XIV) and (XV) or a mixture of formulas (XIV) and (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position L71. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (XIV) to the amount of a structure of formula (XV) contained in the total amount of the IL-2 conjugate is approximately 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (XIV) to the amount of a structure of formula (XV) contained in the total amount of the IL-2 conjugate is greater than 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (XIV) to the amount of a structure of formula (XV) contained in the total amount of the IL-2 conjugate is less than 1:1.

[0058] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or (XV) or a mixture of (XIV) and (XV), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and wherein n is an integer from 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or from about 341 to about 1136, or from about 341 to about 1023, or from about 341 to about 910, or from about 341 to about 796, or from about 341 to about 682, or from about 341 to about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulae (XIV) and (XV) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.

[0059] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or (XV), or a mixture of (XIV) and (XV), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (XIV) and (XV) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137 and 1249.

[0060] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or (XV), or a mixture of (XIV) and (XV), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (XIV) and (XV) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910.

[0061] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or (XV), or a mixture of (XIV) and (XV), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (XIV) and (XV) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0062] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or (XV), or a mixture of (XIV) and (XV), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (XIV) and (XV) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0063] Disclosed herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or (XV) or a mixture of (XIV) and (XV), wherein n is an integer such that the molecular weight of the PEG moiety is in the range of from about 1,000 Daltons to about 200,000 Daltons, or from about 2,000 Daltons to about 150,000 Daltons, or from about 3,000 Daltons to about 125,000 Daltons, or from about 4,000 Daltons to about 100,000 Daltons, or from about 5,000 Daltons to about 100,000 Daltons, or from about 6,000 Daltons to about 90,000 Daltons, or from about 7,000 daltons to about 80,000 daltons, or from about 8,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 65,000 daltons, or from about 5,000 daltons to about 60,000 daltons, or from about 5,000 daltons to about 50,000 daltons, or from about 6,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 45,000 daltons, or from about 7,000 daltons to about 40,000 daltons, or from about 8,000 daltons to about 40,000 daltons, or from about 8,500 daltons to about 40,000 daltons, or from about 8,500 daltons to about 35,000 daltons, or from about 9,000 daltons to about 50,000 daltons, or from about 9,000 daltons to about 45,000 daltons, or from about 9,000 daltons to about 40,000 daltons, or from about 9,000 daltons to about 35,000 daltons, or from about 9,000 daltons to about 30,000 daltons, or from about 9,500 daltons to about 35,000 daltons, or from about 9,500 daltons to about 30,000 daltons, or from about 10,000 daltons to about 50,000 daltons, or from about 10,000 daltons to about 45,000 daltons, or from about 10,000 daltons to about 40,000 daltons, or from about 10,000 daltons to about 35,000 daltons, or from about 10,000 daltons to about 30,000 daltons, or from about 15,000 daltons to about 50,000 daltons, or from about 15,000 daltons to about 45,000 daltons, or from about 15,000 daltons to about 40,000 daltons, or from about 15,000 daltons to about 35,000 daltons, or from about 15,000 daltons to about 30,000 daltons, or from about 20,000 daltons to about 50,000 daltons, or from about 20,000 daltons to about 45,000 daltons, or from about 20,000 daltons to about 40,000 daltons, or from about 20,000 daltons to about 35,000 daltons, or from about 20,000 daltons to about 30,000 daltons. Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or (XV) or a mixture of (XIV) and (XV), wherein n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000 Daltons, about 60,000 Daltons, about 70,000 Daltons, about 80,000 Daltons, approximately 90,000 daltons, approximately 100,000 daltons, approximately 125,000 daltons, approximately 150,000 daltons, approximately 175,000 daltons, or approximately 200,000 daltons. Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (XIV) or (XV) or a mixture of (XIV) and (XV), wherein n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, or about 50,000 Daltons.

[0064] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or (XV), or a mixture of (XIV) and (XV), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, m is an integer from 0 to 6, p is an integer from 0 to 6, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of formulas (XIV) and (XV), m is 2, p is 2, and n is an integer chosen from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, and 1249.

[0065] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or (XV) or a mixture of (XIV) and (XV), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and wherein m is an integer from 0 to 6, p is an integer from 0 to 6, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of formulas (XIV) and (XV), m is 2, p is 2, and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.

[0066] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or (XV), or a mixture of (XIV) and (XV), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein m is an integer from 0 to 6, p is an integer from 0 to 6, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of formulas (XIV) and (XV), m is 2, p is 2, and n is is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0067] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XIV) or (XV), or a mixture of (XIV) and (XV), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein m is an integer from 0 to 6, p is an integer from 0 to 6, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of formulas (XIV) and (XV), m is 2, p is 2, and n is is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0068] Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (XVI) or (XVII) or a mixture of (XVI) and (XVII),

formula (XVI),

formula (XVII),

Where

m is an integer between 0 and 20;

n is an integer in the range from approximately 2 to approximately 5000; and

wavy lines indicate covalent bonds to amino acid residues in SEQ ID NO: 3 that are not substituted, or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[0069] In some embodiments, the stereochemistry of the chiral center of formula (XVI) and formula (XVII) provides a racemic mixture, provides a mixture enriched in (R), provides a mixture enriched in (S), provides substantially only (R), provides substantially only (S), provides only (R), or provides only (S). In some embodiments, the stereochemistry of the chiral center of formula (XVI) and formula (XVII) provides a racemic mixture. In some embodiments, the stereochemistry of the chiral center of formula (XVI) and formula (XVII) provides a mixture enriched in (R). In some embodiments, the stereochemistry of the chiral center of formula (XVI) and formula (XVII) provides a mixture enriched in (S). In some embodiments, the stereochemistry of the chiral center of formula (XVI) and formula (XVII) provides substantially only (R). In some embodiments, the stereochemistry of the chiral center of formula (XVI) and formula (XVII) provides substantially only (S). In some embodiments, the stereochemistry of the chiral center in formula (XVI) and formula (XVII) includes only (R). In some embodiments, the stereochemistry of the chiral center in formula (XVI) and formula (XVII) includes only (S).

[0070] In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of Formulae (XVI) and (XVII) is from 0 to 20, or from 0 to 18, or from 0 to 16, or from 0 to 14, or from 0 to 12, or from 0 to 10, or from 0 to 9, or from 0 to 8, or from 0 to 7, or from 0 to 6, or from 0 to 5, or from 0 to 4, or from 0 to 3, or from 0 to 2. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of Formulae (XVI) and (XVII) is 0. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of Formulae (XVI) and (XVII) is 1. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulae (XVI) and (XVII) is 2. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulae (XVI) and (XVII) is 3. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulae (XVI) and (XVII) is 4. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulae (XVI) and (XVII) is 5. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulae (XVI) and (XVII) is 6. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulae (XVI) and (XVII) is 7. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XVI) and (XVII) is 8. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XVI) and (XVII) is 9. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XVI) and (XVII) is 10. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XVI) and (XVII) is 11. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XVI) and (XVII) is 12. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XVI) and (XVII) is 13. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XVI) and (XVII) is 14. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XVI) and (XVII) is 15. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XVI) and (XVII) is 16. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XVI) and (XVII) is 17. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XVI) and (XVII) is 18. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XVI) and (XVII) is 19. In some embodiments, described herein is an IL-2 conjugate wherein m in the compounds of formulas (XVI) and (XVII) equals 20.

[0071] In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of Formulae (XVI) and (XVII) is in the range of from about 5 to about 4600, or from about 10 to about 4000, or from about 20 to about 3000, or from about 100 to about 3000, or from about 100 to about 2900, or from about 150 to about 2900, or from about 125 to about 2900, or from about 100 to about 2500, or from about 100 to about 2000, or from about 100 to about 1900, or from about 100 to about 1850, or from about 100 to about 1750, or from about 100 to about 1650, or from about 100 to about 1500, or from about 100 to about 1400, or from about 100 to about 1300, or from about 100 to about 1250, or from about 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or from about 341 to about 1136, or from about 341 to about 1023, or from about 341 to about 910, or from about 341 to about 796, or from about 341 to about 682, or from about 341 to about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575.

[0072] In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulas (XVI) and (XVII), m is an integer from 0 to 6 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulas (XVI) and (XVII), m is an integer from 1 to 6 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of formulae (XVI) and (XVII), m is an integer from 2 to 6 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulas (XVI) and (XVII), m is an integer from 2 to 4, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136 and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulas (XVI) and (XVII), m is 1 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulas (XVI) and (XVII), m is 2 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulae (XVI) and (XVII), m is 3 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulas (XVI) and (XVII), m is 4 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, in Described herein is an IL-2 conjugate wherein in the compounds of Formulae (XVI) and (XVII), m is 5 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulae (XVI) and (XVII), m is 6 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulae (XVI) and (XVII), m is 7 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulae (XVI) and (XVII), m is 8 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in In compounds of formulas (XVI) and (XVII), m is 9 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in compounds of formulas (XVI) and (XVII), m is 10 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulae (XVI) and (XVII), m is 11 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulas (XVI) and (XVII), m is 12 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of Formulas (XVI) and (XVII) m is equal to 2, and n is an integer chosen from 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137.

[0073] In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of Formulas (XVI) and (XVII) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546. In some embodiments, described herein is an IL-2 conjugate, wherein the position of the structure of formulas (XVI) and (XVII) or a mixture of formulas (XVI) and (XVII) in the amino acid sequence of the IL-2 conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68 and L71, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate refers to the positions in SEQ ID NO: 3. In some embodiments, described herein is an IL-2 conjugate, wherein the position of the structure of formulas (XVI) and (XVII) or a mixture of formulas (XVI) and (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68 and L71. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XVI) and (XVII) or a mixture of formulas (XVI) and (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position K34. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XVI) and (XVII) or a mixture of formulas (XVI) and (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position F41. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XVI) and (XVII) or a mixture of formulas (XVI) and (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position F43. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XVI) and (XVII) or a mixture of formulas (XVI) and (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position K42. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XVI) and (XVII) or a mixture of formulas (XVI) and (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position E61. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XVI) and (XVII) or a mixture of formulas (XVI) and (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position P64. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XVI) and (XVII) or a mixture of formulas (XVI) and (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position R37. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XVI) and (XVII) or a mixture of formulas (XVI) and (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position T40. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XVI) and (XVII) or a mixture of formulas (XVI) and (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position E67. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XVI) and (XVII) or a mixture of formulas (XVI) and (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position Y44. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XVI) and (XVII) or a mixture of formulas (XVI) and (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position V68. In some embodiments, described herein is an IL-2 conjugate wherein the position of the structure of formulas (XVI) and (XVII) or a mixture of formulas (XVI) and (XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is position L71. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (XVI) to the amount of a structure of formula (XVII) contained in the total amount of the IL-2 conjugate is approximately 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (XVI) to the amount of a structure of formula (XVII) contained in the total amount of the IL-2 conjugate is greater than 1:1. In some embodiments, described herein is an IL-2 conjugate wherein the ratio of the amount of a structure of formula (XVI) to the amount of a structure of formula (XVII) contained in the total amount of the IL-2 conjugate is less than 1:1.

[0074] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XVI) or (XVII) or a mixture of (XVI) and (XVII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and wherein n is an integer from 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or from about 341 to about 1136, or from about 341 to about 1023, or from about 341 to about 910, or from about 341 to about 796, or from about 341 to about 682, or from about 341 to about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulae (XVI) and (XVII) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 45, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4545, and 4546.

[0075] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XVI) or (XVII), or a mixture of (XVI) and (XVII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (XVI) and (XVII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137 and 1249.

[0076] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XVI) or (XVII), or a mixture of (XVI) and (XVII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (XVI) and (XVII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910.

[0077] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XVI) or (XVII), or a mixture of (XVI) and (XVII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (XVI) and (XVII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0078] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XVI) or (XVII), or a mixture of (XVI) and (XVII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein n in the compounds of formulas (XVI) and (XVII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0079] Disclosed herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XVI) or (XVII) or a mixture of (XVI) and (XVII), wherein n is an integer such that the molecular weight of the PEG moiety is in the range of from about 1,000 Daltons to about 200,000 Daltons, or from about 2,000 Daltons to about 150,000 Daltons, or from about 3,000 Daltons to about 125,000 Daltons, or from about 4,000 Daltons to about 100,000 Daltons, or from about 5,000 Daltons to about 100,000 Daltons, or from about 6,000 Daltons to about 90,000 Daltons, or from about 7,000 daltons to about 80,000 daltons, or from about 8,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 70,000 daltons, or from about 5,000 daltons to about 65,000 daltons, or from about 5,000 daltons to about 60,000 daltons, or from about 5,000 daltons to about 50,000 daltons, or from about 6,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 50,000 daltons, or from about 7,000 daltons to about 45,000 daltons, or from about 7,000 daltons to about 40,000 daltons, or from about 8,000 daltons to about 40,000 daltons, or from about 8,500 daltons to about 40,000 daltons, or from about 8,500 daltons to about 35,000 daltons, or from about 9,000 daltons to about 50,000 daltons, or from about 9,000 daltons to about 45,000 daltons, or from about 9,000 daltons to about 40,000 daltons, or from about 9,000 daltons to about 35,000 daltons, or from about 9,000 daltons to about 30,000 daltons, or from about 9,500 daltons to about 35,000 daltons, or from about 9,500 daltons to about 30,000 daltons, or from about 10,000 daltons to about 50,000 daltons, or from about 10,000 daltons to about 45,000 daltons, or from about 10,000 daltons to about 40,000 daltons, or from about 10,000 daltons to about 35,000 daltons, or from about 10,000 daltons to about 30,000 daltons, or from about 15,000 daltons to about 50,000 daltons, or from about 15,000 daltons to about 45,000 daltons, or from about 15,000 daltons to about 40,000 daltons, or from about 15,000 daltons to about 35,000 daltons, or from about 15,000 daltons to about 30,000 daltons, or from about 20,000 daltons to about 50,000 daltons, or from about 20,000 daltons to about 45,000 daltons, or from about 20,000 daltons to about 40,000 daltons, or from about 20,000 daltons to about 35,000 daltons, or from about 20,000 daltons to about 30,000 daltons. Disclosed herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XVI) or (XVII) or a mixture of (XVI) and (XVII), wherein n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000 Daltons, about 60,000 Daltons, about 70,000 Daltons, about 80,000 Daltons, about 90,000 daltons, approximately 100,000 daltons, approximately 125,000 daltons, approximately 150,000 daltons, approximately 175,000 daltons, or approximately 200,000 daltons. Described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (XVI) or (XVII) or a mixture of (XVI) and (XVII), wherein n is an integer such that the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, or about 50,000 Daltons.

[0080] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XVI) or (XVII), or a mixture of (XVI) and (XVII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, m is an integer from 0 to 6, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of formulas (XVI) and (XVII), m is 2 and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137 and 1249.

[0081] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XVI) or (XVII), or a mixture of (XVI) and (XVII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and wherein m is an integer from 0 to 6, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of formulas (XVI) and (XVII), m is 2 and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910.

[0082] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XVI) or (XVII), or a mixture of (XVI) and (XVII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein m is an integer from 0 to 6, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of formulas (XVI) and (XVII), m is 2 and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0083] In some embodiments, described herein are IL-2 conjugates comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced with a structure of formula (XVI) or (XVII), or a mixture of (XVI) and (XVII), wherein the amino acid residue in SEQ ID NO: 3 that is replaced is P64, and wherein m is an integer from 0 to 6, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments, described herein is an IL-2 conjugate wherein in the compounds of formulas (XVI) and (XVII), m is 2 and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[0084] Described herein are pharmaceutical compositions comprising an effective amount of an IL conjugate described herein and one or more pharmaceutically acceptable excipients.

[0085] Described herein are methods of treating cancer in a subject comprising administering to the subject in need thereof an effective amount of an IL-2 conjugate described herein. In some embodiments of the method of treating cancer described herein, the cancer in the subject is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin's lymphoma (cHL), primary mediastinal large B cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability cancer, microsatellite stable cancer, gastric cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), melanoma, small cell lung cancer (SCLC), esophageal, glioblastoma, mesothelioma, breast cancer, triple-negative breast cancer, prostate cancer, castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, metastatic castration-resistant prostate cancer with defects in DNA damage response (DDR), bladder cancer, ovarian cancer, tumors with intermediate or low mutational burden, cutaneous squamous cell carcinoma (CSCC), cutaneous squamous cell carcinoma (SCSC), tumors with low or no PD-L1 expression, tumors that have spread systemically to the liver and CNS beyond their primary anatomic site of origin, and diffuse large B-cell lymphoma. Described herein are methods of treating a cancer in a subject comprising administering to a subject in need thereof an effective amount of an IL-2 conjugate described herein. In some embodiments of the method of treating a cancer described herein, the cancer in the subject is cholangiocarcinoma. In some embodiments of the method of treating cancer described herein, the cancer in the subject is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), urothelial carcinoma, and melanoma. In some embodiments of the method of treating cancer described herein, the IL-2 conjugate is administered to the subject in need thereof once every two weeks, once every three weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, or once every 8 weeks. In some embodiments of the method of treating cancer described herein, the IL-2 conjugate is administered to the subject in need thereof once a week or once every two weeks. In some embodiments of the method of treating cancer described herein, the IL-2 conjugate is administered to the subject in need thereof once a week. In some embodiments of the method of treating cancer described herein, the IL-2 conjugate is administered to a subject in need thereof once every two weeks. In some embodiments of the method of treating cancer described herein, administering an effective amount of the IL-2 conjugate to the subject does not result in vascular leak syndrome in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of the IL-2 conjugate to the subject does not result in vascular leak syndrome grade 2, grade 3, or grade 4 in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of the IL-2 conjugate to the subject does not result in vascular leak syndrome grade 2 in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of the IL-2 conjugate to the subject does not result in vascular leak syndrome grade 3 in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not result in grade 4 vascular leak syndrome in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not result in loss of vascular tone in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not result in extravasation of plasma proteins and fluid into the extravascular space in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not result in hypotension and decreased organ perfusion in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not result in impairment of neutrophil function in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not result in a decrease in chemotaxis in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject is not associated with an increased risk of disseminated infection in the subject. In some embodiments of the method of treating cancer described herein, the disseminated infection is sepsis or bacterial endocarditis. In some embodiments of the method of treating cancer described herein, the disseminated infection is sepsis. In some embodiments of the method of treating cancer described herein, the disseminated infection is bacterial endocarditis. In some embodiments of the method of treating cancer described herein, the subject is treated for any prior bacterial infections prior to administration of the IL-2 conjugate. In some embodiments of the method of treating cancer described herein, the subject is treated with an antibacterial agent selected from oxacillin, nafcillin, ciprofloxacin, and vancomycin prior to administration of the IL-2 conjugate. In some embodiments of the method of treating cancer described herein, administration of an effective amount of the IL-2 conjugate to the subject does not worsen manifestations of a pre-existing or new-onset autoimmune disease or inflammatory disorder in the subject. In some embodiments of the method of treating cancer described herein, administration of an effective amount of the IL-2 conjugate to the subject does not worsen manifestations of a pre-existing or new-onset autoimmune disease autoimmune disease in the subject. In some embodiments of the method of treating cancer described herein, administration of an effective amount of the IL-2 conjugate to the subject does not worsen manifestations of a pre-existing or new-onset inflammatory disorder in the subject. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is selected from Crohn's disease, scleroderma, thyroiditis, inflammatory arthritis, diabetes mellitus, oculobulbar myasthenia gravis, crescentic IgA glomerulonephritis, cholecystitis, cerebral vasculitis, Stevens-Johnson syndrome, and bullous pemphigoid. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is Crohn's disease. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is scleroderma. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is thyroiditis. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is inflammatory arthritis. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is diabetes mellitus. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is oculobulbar myasthenia gravis. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is crescentic IgA glomerulonephritis. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is cholecystitis. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is cerebral vasculitis. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is Stevens-Johnson syndrome. In some embodiments of the method of treating cancer described herein, the autoimmune disease or inflammatory disorder in the subject is bullous pemphigoid. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not result in mental status changes, speech impediment, cortical blindness, limb ataxia or uncoordinated gait, hallucinations, agitation, lethargy, or coma in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not cause the subject to have seizures. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject is not contraindicated in subjects who have a confirmed seizure disorder. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not cause the subject to have capillary leak syndrome. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not cause the subject to have grade 2, grade 3, or grade 4 capillary leak syndrome. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not cause the subject to have grade 2 capillary leak syndrome. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not result in capillary leak syndrome grade 3 in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not result in capillary leak syndrome grade 4 in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not result in a decrease in mean arterial blood pressure in the subject following administration of the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not result in hypotension in the subject following administration of the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not result in the subject having a decrease in systolic blood pressure below 90 mmHg or a decrease of 20 mmHg from the baseline systolic pressure after administering the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not result in edema in the subject after administering the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not result in renal or hepatic dysfunction in the subject after administering the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not result in eosinophilia in the subject after administering the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not result in an increase in the number of eosinophils in the subject's peripheral blood to a value greater than 500 per μL after administering the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not result in an increase in the number of eosinophils in the subject's peripheral blood to a value greater than 500 per μL to 1500 per μL after administering the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not increase the subject's peripheral blood eosinophil count to a value greater than 1500 per μL to 5000 per μL after administering the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not increase the subject's peripheral blood eosinophil count to a value greater than 5000 per μL after administering the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject is not contraindicated in subjects who are already undergoing treatment with psychotropic drugs.

[0086] In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject is not contraindicated in subjects who are already undergoing treatment with nephrotoxic, myelotoxic, cardiotoxic, or hepatotoxic drugs. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject is not contraindicated in subjects who are already undergoing treatment with aminoglycosides, cytotoxic chemotherapy, doxorubicin, methotrexate, or asparaginase. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject is not contraindicated in subjects who are undergoing treatment with combination therapies containing antineoplastic agents. In some embodiments of the method of treating cancer described herein, the antineoplastic agent is selected from dacarbazine, cisplatin, tamoxifen, and interferon-alpha. In some embodiments of the method of treating cancer described herein, administering an effective amount of the IL-2 conjugate to the subject does not result in one or more grade 4 adverse events in the subject after administering the IL-2 conjugate to the subject. In some embodiments of the method of treating cancer described herein, the one or more grade 4 adverse events are selected from hypothermia, shock, bradycardia, ventricular extrasystole, myocardial ischemia, syncope, hemorrhage, atrial arrhythmia, phlebitis, second degree atrioventricular block, endocarditis, pericardial effusion, peripheral gangrene, thrombosis, coronary artery disorder, stomatitis, nausea and vomiting, liver function tests abnormal, gastrointestinal hemorrhage, hematemesis, hemorrhagic diarrhea, gastrointestinal disorder, intestinal perforation, pancreatitis, anemia, leukopenia, leukocytosis, hypocalcemia, alkaline phosphatase increased, blood urea nitrogen (BUN) increased, hyperuricemia, non-protein nitrogen (NPN) increased, respiratory acidosis, somnolence, agitation, neuropathy, paranoid reaction, seizure, grand mal seizure, delirium, asthma, pulmonary edema, hyperventilation, hypoxia, hemoptysis, hypoventilation, pneumothorax, mydriasis, pupillary dysfunction, renal dysfunction, renal failure, and acute tubular necrosis. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a group of subjects does not result in one or more grade 4 adverse events occurring in more than 1% of the subjects following administration of the IL-2 conjugate to the subjects. In some embodiments of the method of treating cancer described herein, the one or more grade 4 adverse events are selected from hypothermia, shock, bradycardia, ventricular extrasystole, myocardial ischemia, syncope, hemorrhage, atrial arrhythmia, phlebitis, second degree atrioventricular block, endocarditis, pericardial effusion, peripheral gangrene, thrombosis, coronary artery disorder, stomatitis, nausea and vomiting, liver function tests abnormal, gastrointestinal hemorrhage, hematemesis, hemorrhagic diarrhea, gastrointestinal disorder, intestinal perforation, pancreatitis, anemia, leukopenia, leukocytosis, hypocalcemia, alkaline phosphatase increased, blood urea nitrogen (BUN) increased, hyperuricemia, non-protein nitrogen (NPN) increased, respiratory acidosis, somnolence, agitation, neuropathy, paranoid reaction, seizure, grand mal seizure, delirium, asthma, pulmonary edema, hyperventilation, hypoxia, hemoptysis, hypoventilation, pneumothorax, mydriasis, pupillary dysfunction, renal dysfunction, renal failure and acute tubular necrosis. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a group of subjects does not result in one or more adverse events occurring in more than 1% of the subjects following administration of the IL-2 conjugate to the subjects, wherein the one or more adverse events are selected from duodenal ulcer, intestinal necrosis, myocarditis, supraventricular tachycardia, permanent or transient blindness secondary to optic neuritis, transient ischemic attack, meningitis, cerebral edema, pericarditis, allergic interstitial nephritis, and tracheoesophageal fistula. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a group of subjects does not result in one or more adverse events occurring in more than 1% of the subjects after administering the IL-2 conjugate to the subjects, wherein the one or more adverse events are selected from malignant hyperthermia, cardiac arrest, myocardial infarction, pulmonary embolism, stroke, intestinal perforation, liver or kidney failure, major depression leading to suicide, pulmonary edema, respiratory arrest, respiratory failure. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not result in the production of neutralizing antibodies to the IL-2 conjugate. In some embodiments of the method of treating cancer described herein, administering an IL-2 conjugate to a subject increases the number of peripheral CD8+ T and NK cells in the subject without increasing the number of peripheral CD4+ regulatory T cells in the subject. In some embodiments of the method of treating cancer described herein, administering an IL-2 conjugate to a subject increases the number of peripheral CD8+ T and NK cells in the subject without increasing the number of peripheral eosinophils in the subject. In some embodiments of the method of treating cancer described herein, administering an IL-2 conjugate to a subject increases the number of intratumoral CD8+ T and NK cells in the subject without increasing the number of intratumoral CD4+ regulatory T cells in the subject. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to a subject does not require an intensive care unit or skilled cardiopulmonary or critical care professionals. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not require an intensive care unit. In some embodiments of the method of treating cancer described herein, administering an effective amount of an IL-2 conjugate to the subject does not require skilled cardiopulmonary or intensive care personnel.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0087] The novel features of the present invention are particularly set forth in the appended claims. The features and advantages of the present invention will become more apparent from the following detailed description, which sets forth illustrative embodiments in which the principles of the present invention are utilized, and the accompanying drawings.

[0088] Fig. 1 shows illustrative unnatural amino acids. This figure is adapted from Fig. 2 of Young et al., "Beyond the canonical 20 amino acids: expanding the genetic lexicon," J. of Biological Chemistry 285 (15): 11039–11044 (2010).

[0089] Fig. 2A - Fig. 2B show exemplary unnatural amino acids. Fig. 2A shows exemplary lysine derivatives. Fig. 2B shows exemplary phenylalanine derivatives.

[0090] Fig. 3A-Fig. 3D show exemplary unnatural amino acids. These unnatural amino acids (UAAs) are genetically encoded in proteins ( Fig. 3A - UAA#1-42; Fig. 3B - UAA#43-89; Fig. 3C - UAA#90-128; Fig. 3D - UAA#129-167). Figs. 3A-3D are adapted from Table 1 of Dumas et al ., Chemical Science 2015, 6, 50-69.

[0091] In FIG. 4A-Fig. 4C shows surface plasmon resonance (SPR) analysis of native IL-2, P65_30 kDa, P65_5 kDa, E62_30 kDa, E62_5 kDa, and F42_30 kDa PEG conjugates. In fig. 4A shows an analysis of SPR variants of IL-2 binding to immobilized IL-2 Rα. In fig. 4B shows an analysis of SPR variants of IL-2 binding to immobilized IL-2 Rβ. In fig. 4C shows SPR analysis of recombinant IL-2 and the F42_30 kDa IL-2 variant binding to immobilized IL-2 Rα and IL-2 Rβ.

[0092] Fig. 5A-Fig. 5F show an exemplary dose response curve for an IL-2 variant for pSTAT5 signaling in primary human LRS cell populations. Fig. 5A : native IL-2; Fig. 5B : P65_30 kDa; Fig. 5C : K64_30 kDa; Fig. 5D : K43_30 kDa; Fig. 5E : K35_30 kDa, and Fig. 5F : F42_30 kDa.

[0093] Figures 6A-C show that PEG and residue substitution contribute to the pharmacology of IL-2 variants lacking the alpha chain. Figure 6A : native IL-2; Figure 6B : E62K; Figure 6C : E62_30 kDa.

[0094] Figure 7 shows that the pharmacology of IL-2 variants lacking the alpha chain is independent of PEG size.

[0095] Figure 8 shows the mean (±SD) plasma concentration-time profiles following administration of a single intravenous bolus dose of aldesleukin (IL-2), E62_5, E62_30, and P65_30 to C57BL/6 mice.

[0096] Fig. 9 shows the change in the percentage of pSTAT5+ CD8+ T cells over time among peripheral blood cells following treatment with a single intravenous bolus dose of P65_30 or aldesleukin in C57BL/6 mice.

[0097] Figures 10A-10C show the percentage of CD8+ T cells ( Figure 10A ), NK cells ( Figure 10B ), and CD4+ Treg cells ( Figure 10C ) in the PBMC population following treatment with a single intravenous bolus dose of P65_30 or aldesleukin (IL-2). Blood was collected by cardiac puncture at the indicated time points and the immune cell population was assessed by flow cytometry. Each data point represents the mean of 3 replicates at each time point ± SEM.

[0098] Figures 11A-11B show the differences between P65_30 and IL-2 (aldesleukin) in stimulating the proliferation of memory CD8+CD44+ T cells in the CD3+ cell population following treatment with a single intravenous bolus dose of P65_30 or aldesleukin (IL-2). Blood was collected by cardiac puncture at the indicated time points and the immune cell population was assessed by flow cytometry. Data were analyzed using an unpaired Student's t test *** Indicates P -values < 0.001. Figure 11A shows the proliferation of memory CD8+CD44+ T cells at 72, 96, and 120 hours. Figure 11B shows the flow cytometric analysis of these cells at the 120 hour time point.

[0099] Fig. 12A - Fig. 12B show the time-dependent increase in tumor infiltrating lymphocytes (TIL) in C57Bl6 mice bearing syngeneic B16F10 tumors following treatment with a single intravenous bolus dose of P65_30. Fig. 12A shows the percentage of NK, CD8+ T, and CD4+ Treg cells in animals treated with P65_30 compared to untreated animals (vehicle) on day 5 of treatment. Fig. 12B shows the ratio of CD8+/CD4+ Treg cells in animals treated with P65_30 and the control (vehicle). Data were analyzed using an unpaired Student's t-test . *** Indicates P -values < 0.001.

[00100] Figures 13A-13B show the levels of IL-2, TNF-α, IFNγ, IL-5, and IL-6 in mouse plasma following treatment with a single intravenous bolus dose of P65_30 or aldesleukin (IL-2) at increasing levels (0.01-5 mg/kg). The plasma concentration of each cytokine was determined by ELISA (Abcam, Cambridge, UK). Mice were grouped in N=3 for each dose and samples were collected at 4, 34, and 72 hours post-dose. Figure 13A shows the cytokine levels for aldesleukin-dosed animals and Figure 13B shows the levels for P65_30-dosed animals.

[00101] Fig. 14 shows white blood cell, lymphocyte, and eosinophil counts (mean ± SD) following a single intravenous dose of P65_30 kDa in male cynomolgus monkeys.

[00102] Fig. 15A - Fig. 15B show dose response curves of an exemplary IL-2 embodiment for pSTAT5 signaling in primary human cells generated by LRS ( Fig. 15A ) and the proliferation response in mouse CTLL-2 populations ( Fig. 15B ).

[00103] Fig. 16A - Fig. 16B show that PEG IL-2 compounds can specifically expand immune cell populations ex vivo in primary lymphocytes compared to normal IL-2 control. Fig. 16A shows the expansion of immune cells following treatment with IL-2 (control). Fig. 16B shows the expansion of immune cells following treatment with P65_30 kDa.

[00104] Fig. 17A - Fig. 17B show sensorgrams of the binding responses for rhIL-2 (recombinant interleukin-2, Fig. 17A ) and the synthetic IL-2_P65[AzK_L1_PEG 30 kDa]-1 conjugate ( Fig. 17B ) on IL-2R alpha chain surfaces. No significant binding response was detected for IL-2_P65[AzK_L1_PEG 30 kDa]-1 under these conditions.

[00105] Fig. 17C - Fig. 17D show sensorgrams of binding of rhIL-2 (recombinant human interleukin-2, Fig. 17C ) and synthetic IL-2_P65[AzK_L1_PEG 30 kDa]-1 conjugate ( Fig. 17D ) samples to IL-2R beta chain surfaces.

[00106] Figure 18 shows the gating strategy for sorting cells for Tregs using flow cytometry. Cells were first gated on singlets using FSC-A through FSC-H to exclude any aggregates or doublets (singlet gate, panel ¹ ). Within this gate, cells were gated on mid-high forward scatter (FSC-A) and side scatter (SSC-A) to exclude red blood cells, debris, and granulocytes (lymphocyte gate, panel ² ). T cells were then gated as the CD3+, CD56/16-negative population, panel ^3. NK cells were identified as the CD3-negative, CD56/16-high population, panel ³ . T cells were then sorted into CD4+ T cells and CD8+ T cells (panel ⁴ ). Tregs were then gated from CD4+ T cells as a CD25 ^high x C127 ^low population, panel ⁵ .

[00107] Fig. 19 shows the stability of the compound IL-2_P65 [AzK_L1_PEG 30 kDa]-1 in human serum at three concentrations for up to 168 hours, as described in Example 15.

DETAILED DESCRIPTION OF THE INVENTION

[00108] Cytokines include a family of cell signaling proteins such as chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, and other growth factors that are involved in the homeostasis of innate and adaptive immune cells. Cytokines are produced by cells of the immune system such as macrophages, B lymphocytes, T lymphocytes, and mast cells, endothelial cells, fibroblasts, and various stromal cells. In some cases, cytokines modulate the balance between humoral and cellular immune responses.

[00109] Interleukins are signaling proteins that modulate the development and differentiation of T and B lymphocytes, cells of the monocytic lineage, neutrophils, basophils, eosinophils, megakaryocytes, and hematopoietic cells. Interleukins are produced by CD4 helper T and B lymphocytes, monocytes, macrophages, and endothelial cells and other tissue residents.

[00110] Interleukin 2 (IL-2) is a type 1 pleiotropic cytokine whose structure comprises a bundle of four α-helices with a molecular weight of 15.5 kDa. The precursor form of IL-2 is 153 amino acid residues long, with the first 20 amino acids forming the signal peptide and residues 21-153 forming the mature form. IL-2 is produced primarily by CD4+ T cells following antigen stimulation and to a lesser extent by CD8+ cells, natural killer (NK) cells and natural killer (NKT) T cells, activated dendritic cells (DC), and mast cells. IL-2 signaling occurs through interaction with specific combinations of IL-2 receptor (IL-2R) subunits, IL-2Rα (also known as CD25), IL-2Rβ (also known as CD122), and IL-2Rγ (also known as CD132). The interaction of IL-2 with IL-2Rα forms a "low affinity" IL-2 receptor complex with a K _d of approximately 10 ^-8 M. The interaction of IL-2 with IL-2Rβ and IL-2Rγ forms an "intermediate affinity" IL-2 receptor complex with a K _d of approximately 10 ^-9 M. The interaction of IL-2 with all three subunits of IL-2Rα, IL-2Rβ, and IL-2Rγ forms a "high affinity" IL-2 receptor complex with a K _d of approximately >10 ^-11 M.

[00111] In some cases, IL-2 signaling via the "high affinity" IL-2Rαβγ complex modulates the activation and proliferation of regulatory T cells. Regulatory T cells or CD4 ⁺ CD25 ⁺ Foxp3 ⁺ regulatory T cells (Treg) mediate the maintenance of immune homeostasis by suppressing effector cells such as CD4 ⁺ T cells, CD8 ⁺ T cells, B cells, NK cells, and NKT cells. In some cases, Treg cells are generated in the thymus (tTreg cells) or are induced to generate from naive T cells in the periphery (pTreg cells). In some cases, Treg cells are considered a mediator of peripheral tolerance. Indeed, in one study, transfer of CD25-depleted peripheral CD4 ⁺ T cells induced a variety of autoimmune diseases in nude mice, whereas co-transfer of CD4 ⁺ CD25 ⁺ T cells suppressed the development of autoimmunity (Sakaguchi, et al ., "Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25)," J. Immunol . 155(3): 1151–1164 (1995)). Expansion of the Treg population suppresses the proliferation of effector T cells and inhibits autoimmune and antitumor T cell responses.

[00112] IL-2 signaling via the IL-2Rβγ intermediate-affinity complex modulates the activation and proliferation of CD8 ⁺ effector T cells (Teff), NK cells, and NKT cells. CD8 ⁺ Teff cells (also known as cytotoxic T cells, Tc cells, cytotoxic T lymphocytes, CTLs, killer T cells, cytolytic T cells, Tcon, or killer T cells) are T lymphocytes that recognize and destroy damaged cells, cancer cells, and pathogen-infected cells. NK and NKT cells are types of lymphocytes that, like CD8 ⁺ Teff cells, target cancer cells and pathogen-infected cells.

[00113] In some cases, IL-2 signaling is used to modulate T cell responses and, therefore, to treat cancer. For example, IL-2 is administered at high doses to induce expansion of Teff cell populations for the treatment of cancer. However, high doses of IL2 also result in concomitant stimulation of Treg cells, which attenuate anti-tumor immune responses. High doses of IL-2 also cause toxic adverse events mediated by the recruitment of IL-2R alpha chain-expressing cells into the vasculature, including innate immune cells type 2 (ILC-2), eosinophils, and endothelial cells. This results in eosinophilia, increased capillary permeability, and vascular leak syndrome (VLS).

[00114] Adoptive cell therapy allows doctors to effectively use a patient's own immune cells to combat diseases such as proliferative diseases (e.g., cancer), as well as an infectious disease. In one non-limiting example, T lymphocytes can be taken from a patient, reengineered to target a specific antigen on the surface of malignant cells, and reintroduced into the patient to specifically target the malignant cells. In addition, adoptive cell-based therapies provide a durable response in the body by signaling immune cells to grow and divide long after the reengineered cells have been reintroduced into the patient's immune system.

[00115] In some embodiments, disclosed herein is a method for selectively activating a distinct population(s) of lymphocytes (e.g., CD4+ helper cells, CD8+ effector naive and memory cells, NK cells, or NKT cells) via cytokine/cytokine receptor signaling. In some cases, the cytokine comprises an interleukin, an interferon, or a tumor necrosis factor. In some cases, the cytokine is a cytokine conjugate, such as an interleukin conjugate, an interferon conjugate, or a tumor necrosis factor conjugate. In additional cases, disclosed herein are pharmaceutical compositions and kits comprising one or more cytokine conjugates as described herein.

[00116] In some embodiments, described herein are also a method for selectively activating CD4+ helper cell populations, CD8+ effector naive and memory cells, NK cells, and/or NKT cells via IL-2/IL-2R signaling. In some cases, the IL-2 is an IL-2 conjugate that interacts with an IL-2Rβγ complex "with intermediate affinity," optionally with the same potency as an IL-2Rαβγ complex and with reduced IL-2Rα interaction compared to wild-type IL-2. In some embodiments, described herein are further methods of treating cancer using an IL-2 conjugate described herein. In additional embodiments, described herein are pharmaceutical compositions and kits that comprise one or more IL-2 conjugates described herein. In some embodiments, IL-2 conjugates comprise conjugating moieties (e.g., PEG) that promote an increase or decrease in the "clearance rate" or plasma half-life in a subject without affecting pharmacokinetics, including desired cytokine-receptor interactions and immune cell expansion.

[00117] In some embodiments, disclosed herein are reagents that can be used to develop adoptive cell-based therapeutics comprising cells engineered to express modified cytokines that result in selective cytokine-receptor interactions and immune cell expansion. In some embodiments, the reagents comprise a nucleic acid construct encoding the IL-2 conjugates described above. Also disclosed are adoptive cell-based therapeutics comprising the IL-2 conjugates described above, which can be useful for treating a proliferative or infectious disease described herein.

[00118] In some embodiments, compositions that result in selective cytokine-receptor interactions and immune cell expansion are disclosed herein. In some embodiments, reagents comprise a nucleic acid construct encoding the IL-2 conjugates described above. Also disclosed are pharmaceutical compositions comprising the IL-2 conjugates described above, which may be useful for treating a proliferative or infectious disease described herein.

Cytokine conjugates

[00119] In some embodiments, cytokine conjugates are described herein. In some cases, the cytokine comprises interleukins, tumor necrosis factor, interferon, chemokine, lymphokine, or growth factor. In some cases, the cytokine is an interleukin. In some cases, the cytokine is an interferon. In additional cases, the cytokine is a tumor necrosis factor. In other cases, the cytokine is a growth factor.

[00120] In some embodiments, an interleukin conjugate is described herein. Exemplary interleukins include, but are not limited to, interleukin 2 (IL-2).

IL-2 conjugates

[00121] Described herein are the polypeptides shown in Table 20. In some embodiments, the IL-2 conjugates described herein are exemplified in Table 20 .

[00122] Table 20

Name Subsequence SEQ ID NO: IL-2
(Homo sapiens)
(mature form) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 1 IL-2
(Homo sapiens)
(predecessor)
NCBI Accession No: AAB46883.1 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 2 aldesleukin PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 3 IL-2_C125S APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 4 IL-2_P65X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK X LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 5 IL-2_E62X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE X LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 6 IL-2_F42X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT X KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 7 IL-2_K43X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF X FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 8 IL-2_K35X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP X LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 9 IL-2_P65[AzK] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 10 IL-2_E62[AzK] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE[ AzK] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 11 IL-2_F42[AzK] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT[ AzK] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 12 IL-2_K43[AzK] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF[ AzK] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 13 IL-2_K35[AzK] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP[ AzK] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 14 IL-2_P65[AzK_PEG] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_PEG] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 15 IL-2_E62[AzK_PEG] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_PEG] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 16 IL-2_F42[AzK_PEG] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_PEG] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 17 IL-2_K43[AzK_PEG] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_PEG] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 18 IL-2_K35[AzK_PEG] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_PEG] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 19 IL-2_P65[AzK_PEG5kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_PEG5kD] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 20 IL-2_E62[AzK_PEG5kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_PEG5kD] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 21 IL-2_F42[AzK_PEG5kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_PEG5kD] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 22 IL-2_K43[AzK_PEG5kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_PEG5kD] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 23 IL-2_K35[AzK_PEG5kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_PEG5kD] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 24 IL-2_P65[AzK_PEG30kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_PEG30kD] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 25 IL-2_E62[AzK_PEG30kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_PEG30kD] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 26 IL-2_F42[AzK_PEG30kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_PEG30kD] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 27 IL-2_K43[AzK_PEG30kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_PEG30kD] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 28 IL-2_K35[AzK_PEG30kD] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_PEG30kD] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 29 IL-2_P65X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK X LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 30 IL-2_E62X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE X LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 31 IL-2_F42X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT X KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 32 IL-2_K43X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF X FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 33 IL-2_K35X-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP X LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 34 IL-2_P65[AzK]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 35 IL-2_E62[AzK]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE[ AzK] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 36 IL-2_F42[AzK]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT[ AzK] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 37 IL-2_K43[AzK]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF[ AzK] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 38 IL-2_K35[AzK]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP[ AzK] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 39 IL-2_P65[AzK_L1_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_L1_PEG] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 40 IL-2_E62[AzK_ L1_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_L1_PEG] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 41 IL-2_F42[AzK_ L1_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_L1_PEG] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 42 IL-2_K43[AzK_ L1_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_L1_PEG] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 43 IL-2_K35[AzK_ L1_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_L1_PEG] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 44 IL-2_P65[AzK_ L1_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_L1_PEG5kD] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 45 IL-2_E62[AzK_ L1_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_L1_PEG5kD] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 46 IL-2_F42[AzK_ L1_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_L1_PEG5kD] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 47 IL-2_K43[AzK_ L1_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_L1_PEG5kD] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 48 IL-2_K35[AzK_ L1_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_L1_PEG5kD] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 49 IL-2_P65[AzK_ L1_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_L1_PEG30kD] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 50 IL-2_E62[AzK_ L1_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_L1_PEG30kD] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 51 IL-2_F42[AzK_ L1_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_L1_PEG30kD] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 52 IL-2_K43[AzK_ L1_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_L1_PEG30kD] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 53 IL-2_K35[AzK_ L1_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_L1_PEG30kD] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 54 IL-2_P65[AzK_L1_PEG]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_L1_PEG] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 55 IL-2_E62[AzK_ L1_PEG]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_L1_PEG] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 56 IL-2_F42[AzK_ L1_PEG]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_L1_PEG] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 57 IL-2_K43[AzK_ L1_PEG]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_L1_PEG] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 58 IL-2_K35[AzK_ L1_PEG]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_L1_PEG] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 59 IL-2_P65[AzK_ L1_PEG5kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_L1_PEG5kD] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 60 IL-2_E62[AzK_ L1_PEG5kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_L1_PEG5kD] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 61 IL-2_F42[AzK_ L1_PEG5kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_L1_PEG5kD] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 62 IL-2_K43[AzK_ L1_PEG5kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_L1_PEG5kD] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 63 IL-2_K35[AzK_ L1_PEG5kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_L1_PEG5kD] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 64 IL-2_P65[AzK_ L1_PEG30kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_L1_PEG30kD] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 65 IL-2_E62[AzK_ L1_PEG30kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_L1_PEG30kD] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 66 IL-2_F42[AzK_ L1_PEG30kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_L1_PEG30kD] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 67 IL-2_K43[AzK_ L1_PEG30kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_L1_PEG30kD] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 68 IL-2_K35[AzK_ L1_PEG30kD]-2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_L1_PEG30kD] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 69 IL-2_P65[AzK_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_PEG] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 70 IL-2_E62[AzK_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_PEG] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 71 IL-2_F42[AzK_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_PEG] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 72 IL-2_K43[AzK_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_PEG] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 73 IL-2_K35[AzK_PEG]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_PEG] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 74 IL-2_P65[AzK_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_PEG5kD] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 75 IL-2_E62[AzK_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_PEG5kD] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 76 IL-2_F42[AzK_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_PEG5kD] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 77 IL-2_K43[AzK_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_PEG5kD] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 78 IL-2_K35[AzK_PEG5kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_PEG5kD] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 79 IL-2_P65[AzK_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELK [AzK_PEG30kD] LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 80 IL-2_E62[AzK_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEE [ AzK_PEG30kD] LKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 81 IL-2_F42[AzK_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLT [ AzK_PEG30kD] KFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 82 IL-2_K43[AzK_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTF [ AzK_PEG30kD] FYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 83 IL-2_K35[AzK_PEG30kD]-1 PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNP [ AzK_PEG30kD] LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLT 84

X= site containing unnatural amino acid.

[AzK] = N6-((2-azidoethoxy)-carbonyl)-L-lysine (whose structure is revealed as compound 90 in Fig. 3C). The compound has Chemical Abstracts accession number 1167421-25-1.

[AzK_PEG]= N6-((2-azidoethoxy)-carbonyl)-L-lysine stably conjugated to PEG by DBCO-mediated click chemistry to form a compound comprising a structure of formula (II) or formula (III). For example, when indicated, PEG 5 kDa refers to a linear polyethyleneglycol chain with an average molecular weight of 5 kilodaltons capped with a methoxy group. The ratio of regioisomers formed by the click reaction is approximately 1:1 or greater than 1:1. The term "DBCO" refers to a chemical moiety containing a dibenzocyclooctyne group, such as that containing the mPEG-DBCO compound shown in Scheme 1 of Example 2. An exemplary structure of a methoxy-PEG moiety is shown in the mPEG-DBCO structure in Scheme 1 of Example 2

[AzK_L1_PEG]= N6-((2-azidoethoxy)-carbonyl)-L-lysine stably conjugated to PEG by DBCO-mediated click chemistry to form a compound comprising a structure of formula (IV) or formula (V). For example, when indicated, PEG 5 kDa refers to a linear polyethyleneglycol chain with an average molecular weight of 5 kilodaltons, capped with a methoxy group. The ratio of regioisomers formed by the click reaction is approximately 1:1 or greater than 1:1. The term "DBCO" refers to a chemical moiety containing a dibenzocyclooctyne group, such as the mPEG-DBCO containing compound shown in Scheme 1 of Example 2.

[00123] In some embodiments, described herein are IL-2 conjugates that are modified at an amino acid position. In some cases, the modification is to a naturally occurring amino acid. In some cases, the modification is to a non-naturally occurring amino acid. In some cases, described herein is an isolated and modified IL-2 polypeptide that comprises at least one non-naturally occurring amino acid. In some cases, the IL-2 polypeptide is an isolated and purified mammalian IL-2, such as a rodent IL-2 protein or a human IL-2 protein. In some cases, the IL-2 polypeptide is a human IL-2 protein. In some instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 1. In some instances, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 1. In some instances, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 1. In further instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 2. In further instances, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 2. In further instances, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 2. In some instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 3. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 3. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 3. In further cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 4. In further cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 4. In further cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 4. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 5. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 5. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 5. In further cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 6. In further cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 6. In further cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 6. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 7. In some cases, the polypeptide IL-2 comprises the sequence of SEQ ID NO: 7. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 7. In further cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 8. In further cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 8. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 9. In some cases, the IL-2 polypeptide consists of sequence of SEQ ID NO: 9. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 10. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 10. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 10. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 11. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 11. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 11. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 12. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 12. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 12. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 13. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 13. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 13. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 14. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 14. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 14. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 15. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 15. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 15. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to sequence with the sequence of SEQ ID NO: 16. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 16. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 16. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 17. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 17. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 17. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 18. In some cases, the polypeptide IL-2 comprises the sequence of SEQ ID NO: 18. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 18. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 19. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 19. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 20. In some cases, the IL-2 polypeptide IL-2 consists of the sequence of SEQ ID NO: 20. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 21. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 21. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 21. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 22. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 22. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 22. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 23. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 23. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 23. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 24. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 24. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 24. In further cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 25. In further cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 25. In further cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 25. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 26. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 26. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 26. In further instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 27. In further instances, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 27. In further instances, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 27. In some instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 28. In some instances, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 28. In some instances, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 28. In further instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 29. In further instances, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 29. In further instances, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 29. In some instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 30. In some instances, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 30. In some instances, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 30. In some instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 31. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 31. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 31. In further cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 32. In further cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 32. In further cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 32. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 33. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 33. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 33. In further cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 34. In further cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 34. In further cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 34. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 35. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 35. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 35. In further cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 36. In further cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 36. In further cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 36. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 37. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 37. In some cases, the IL-2 polypeptide consists of the sequence SEQ ID NO: 37. In further instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 38. In further instances, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 38. In further instances, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 38. In some instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 39. In some instances, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 39. In some instances, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 39. In some instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 40. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 40. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 40. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 41. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 41. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 41. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 42. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 42. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 42. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 43. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 43. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 43. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to sequence of SEQ ID NO: 44. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 44. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 44. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 45. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 45. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 45. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 46. In some cases, the polypeptide IL-2 comprises the sequence of SEQ ID NO: 46. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 46. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 47. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 47. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 48. In some cases, the IL-2 polypeptide IL-2 consists of the sequence of SEQ ID NO: 48. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 49. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 49. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 49. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 50. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 50. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 50. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 51. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 51. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 51. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 52. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 52. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 52. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 53. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 53. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 53. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 54. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 54. In some instances, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 54. In further instances, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 55. In further instances, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 55. In further instances, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 55. In some instances, the IL-2 polypeptide comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 56. In some instances, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 56. In some instances, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 56. In further instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 57. In further instances, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 57. In further instances, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 57. In some instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 58. In some instances, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 58. In some instances, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 58. In further instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 59. In further instances, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 59. In further instances, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 59. In some instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 60. In some instances, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 60. In some instances, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 60. In some instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 61. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 61. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 61. In further cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 62. In further cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 62. In further cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 62. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 63. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 63. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 63. In further cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 64. In further cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 64. In further cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 64. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 65. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 65. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 65. In further cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 66. In further cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 66. In further cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 66. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 67. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 67. In some cases, the IL-2 polypeptide consists of the sequence SEQ ID NO: 67. In further instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 68. In further instances, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 68. In further instances, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 68. In some instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 69. In some instances, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 69. In some instances, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 69. In some instances, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 70. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 70. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 70. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 71. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 71. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 71. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 72. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 72. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 72. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 73. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 73. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 73. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to sequence with the sequence of SEQ ID NO: 74. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 74. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 74. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 75. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 75. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 75. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 76. In some cases, the polypeptide IL-2 comprises the sequence of SEQ ID NO: 76. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 76. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 77. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 77. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 77. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 78. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 78. In some cases, the polypeptide IL-2 consists of the sequence of SEQ ID NO: 78. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 79. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 79. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 79. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 80. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 80. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 80. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 81. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 81. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 81. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 82. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 82. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 82. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 83. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 83. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 83. In some cases, the IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 84. In some cases, the IL-2 polypeptide comprises the sequence of SEQ ID NO: 84. In some cases, the IL-2 polypeptide consists of the sequence of SEQ ID NO: 84.

[00124] In some instances, the IL-2 polypeptide is a truncated variant. In some instances, the truncation is an N-terminal deletion. In other instances, the truncation is a C-terminal deletion. In further instances, the truncation includes both an N-terminal and a C-terminal deletion. For example, the truncation can be a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or more residues from either the N-terminus, the C-terminus, or both. In some cases, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more residues. In some cases, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues. In some cases, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 2 residues. In some cases, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 3 residues. In some cases, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 4 residues. In some cases, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 5 residues. In some cases, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 6 residues. In some cases, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 7 residues. In some cases, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 8 residues. In some cases, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 9 residues. In some cases, the IL-2 polypeptide comprises an N-terminal deletion of at least or about 10 residues.

[00125] In some embodiments, the IL-2 polypeptide is a functionally active fragment. In some cases, the functionally active fragment comprises a region of IL-2 10-133, 20-133, 30-133, 10-130, 20-130, 30-130, 10-125, 20-125, 30-125, 1-130, or 1-125, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some cases, the functionally active fragment comprises a region of IL-2 10-133, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some cases, the functionally active fragment comprises a region of IL-2 20-133, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some cases, the functionally active fragment comprises a region of IL-2 30-133, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some cases, the functionally active fragment comprises a region of IL-2 10-125, wherein the residue positions are related to the positions in SEQ ID NO: 1. In some cases, the functionally active fragment comprises the region of IL-2 20-125, wherein the residue positions are related to the positions in SEQ ID NO: 1. In some cases, the functionally active fragment comprises the region of IL-2 1-130, wherein the residue positions are related to the positions in SEQ ID NO: 1. In some cases, the functionally active fragment comprises the region of IL-2 1-125, wherein the residue positions are related to the positions in SEQ ID NO: 1.

[00126] In some embodiments, described herein is an IL-2 conjugate that comprises an isolated, purified, and modified IL-2 polypeptide and a conjugate moiety. In some cases, the IL-2 conjugate has a reduced affinity for the IL-2 receptor α subunit (IL-2Rα) compared to a wild-type IL-2 polypeptide. In some cases, the conjugate moiety is linked to an amino acid residue that interacts with IL-2Rα (e.g., at an IL-2/IL-2Rα interface). In some cases, the conjugate moiety is linked to an amino acid residue that is proximal to the IL-2/IL-2Rα interface (e.g., about 5 Å, about 10 Å, about 15 Å, or about 20 Å from the IL-2/IL-2Rα interface). In the context of this document, residues forming the IL-2/IL-2Rα interface include residues of IL-2 that mediate hydrophobic interactions, hydrogen bonds, or ionic interactions with residues from the IL-2Rα subunit.

[00127] In some instances, the conjugate moiety is linked to an amino acid residue selected from amino acid position Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, F44, Y45, P47, K48, Q57, E60, E61, E62, L63, K64, P65, E68, V69, N71, L72, Q74, S75, K76, N77, M104, C105, E106, Y107, A108, D109, E110, T111, or A112, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some instances, the amino acid position is selected from Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, F44, Y45, P47, K48, E61, E62, E68, K64, P65, V69, L72, Q74, S75, K76, N77, M104, C105, E106, Y107, A108, D109, E110, T111, and A1 12. In some cases, the amino acid position is selected from N33, P34, K35, T37, R38, M39, T41, F42, K43, F44, Y45, Q57, E60, E61, E62, L63, K64, P65, E68, V69, N71, L72, M104, C105, E106, Y107, A108, D109, E110, T111, and A112. In some cases, the amino acid position is selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some cases, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some cases, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some cases, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some cases, the amino acid position is selected from R38 and K64. In some cases, the amino acid position is selected from E61, E62, and E68. In some cases, the amino acid position is K35. In some cases, the amino acid position is T37. In some cases, the amino acid position is R38. In some cases, the amino acid position is T41. In some cases, the amino acid position is F42. In some cases, the amino acid position is K43. In some cases, the amino acid position is F44. In some cases, the amino acid position is Y45. In some cases, the amino acid position is E61. In some cases, the amino acid position is E62. In some cases, the amino acid position is K64. In some cases, the amino acid position is E68. In some cases, the amino acid position is P65. In some cases, the amino acid position is V69. In some cases, the amino acid position is L72. In some cases, the amino acid position is Y107. In some cases, the amino acid position is L72. In some cases, the amino acid position is D109.

[00128] In some cases, the IL-2 conjugate further comprises an additional mutation. In some cases, the additional mutation is at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In such cases, the amino acid is conjugated to an additional conjugation moiety to increase serum half-life, stability, or a combination thereof. Alternatively, the amino acid is first mutated to a naturally occurring amino acid, such as lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine, or to a non-naturally occurring amino acid, prior to linkage to the additional conjugation moiety.

[00129] In some embodiments, the decrease in affinity of the modified IL-2 polypeptide for the IL-2 receptor α subunit (IL-2Rα) compared to a wild-type IL-2 polypeptide without the unnatural amino acid modification (e.g., a wild-type IL-2 polypeptide) is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or greater than 99%. In some cases, the decrease in affinity is about 10%. In some cases, the decrease in affinity is about 20%. In some cases, the decrease in affinity is about 40%. In some cases, the decrease in affinity is about 50%. In some cases, the decrease in affinity is about 60%. In some cases, the decrease in affinity is about 80%. In some cases, the decrease in affinity is approximately 90%. In some cases, the decrease in affinity is approximately 99%. In some cases, the decrease in affinity is greater than 99%. In some cases, the decrease in affinity is approximately 80%. In some cases, the decrease in affinity is approximately 100%.

[00130] In some embodiments, the decrease in affinity of the modified IL-2 polypeptide for the IL-2 receptor α subunit (IL-2Rα) compared to an equivalent IL-2 polypeptide without the unnatural amino acid modification (e.g., a wild-type IL-2 polypeptide) is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more. In some cases, the decrease in affinity is about 1-fold. In some cases, the decrease in affinity is approximately 2-fold. In some cases, the decrease in affinity is approximately 4-fold. In some cases, the decrease in affinity is approximately 5-fold. In some cases, the decrease in affinity is approximately 6-fold. In some cases, the decrease in affinity is approximately 8-fold. In some cases, the decrease in affinity is approximately 10-fold. In some cases, the decrease in affinity is approximately 30-fold. In some cases, the decrease in affinity is approximately 50-fold. In some cases, the decrease in affinity is approximately 100-fold. In some cases, the decrease in affinity is approximately 300-fold. In some cases, the decrease in affinity is approximately 500-fold. In some cases, the decrease in affinity is approximately 1000-fold. In some cases, the decrease in affinity is greater than 1000-fold.

[00131] In some cases, the modified IL-2 polypeptide does not interact with IL-2Rα. In some cases, the modified IL-2 polypeptide is further conjugated to a conjugating moiety. In some cases, the IL-2 conjugate does not interact with IL-2Rα.

[00132] In some embodiments, the modified IL-2 polypeptide exhibits efficacy in transducing a first receptor signal to an IL-2βγ signaling complex and efficacy in transducing a second receptor signal to an IL-2αβγ signaling complex, and wherein the difference between the efficacy in transducing the first receptor signal and the efficacy in transducing the second receptor signal is less than 10-fold. In some embodiments, the modified IL-2 polypeptide exhibits efficacy in transducing a first receptor signal to an IL-2βγ signaling complex and efficacy in transducing a second receptor signal to an IL-2αβγ signaling complex, and wherein the difference between the efficacy in transducing the first receptor signal and the efficacy in transducing the second receptor signal is less than 5-fold. In some cases, the difference is less than 9-fold, less than 8-fold, less than 7-fold, less than 6-fold, less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold. In some cases, the modified IL-2 polypeptide is a partial agonist, such as an agonist that activates a receptor (e.g., an IL-2βγ signaling complex or an IL-2αβγ signaling complex), but is only partially potent at the receptor compared to a full agonist. In some cases, the modified IL-2 polypeptide is a full agonist, such as an agonist that activates a receptor (e.g., an IL-2βγ signaling complex or an IL-2αβγ signaling complex) with a maximal response.

[00133] In some cases, the efficiency of receptor signaling is measured using an EC50 value. In some cases, the modified IL-2 polypeptide provides a first EC50 value for activating the IL-2βγ signaling complex and a second EC50 value for activating the IL-2αβγ signaling complex, and wherein the difference between the first EC50 value and the second EC50 value is less than 10-fold. In some cases, the modified IL-2 polypeptide provides a first EC50 value for activating the IL-2βγ signaling complex and a second EC50 value for activating the IL-2αβγ signaling complex, and wherein the difference between the first EC50 value and the second EC50 value is less than 5-fold. In some cases the difference is less than 9-fold, less than 8-fold, less than 7-fold, less than 6-fold, less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold.

[00134] In some cases, the efficiency of receptor signaling is measured using an ED50 value. In some cases, the modified IL-2 polypeptide provides a first ED50 value for activating the IL-2βγ signaling complex and a second ED50 value for activating the IL-2αβγ signaling complex, and wherein the difference between the first ED50 value and the second ED50 value is less than 10-fold. In some cases, the modified IL-2 polypeptide provides a first ED50 value for activating the IL-2βγ signaling complex and a second ED50 value for activating the IL-2αβγ signaling complex, and wherein the difference between the first ED50 value and the second ED50 value is less than 5-fold. In some cases the difference is less than 9-fold, less than 8-fold, less than 7-fold, less than 6-fold, less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold.

[00135] In some embodiments, the conjugation moiety is linked to the N-terminus or C-terminus of the IL-2 polypeptide, either directly or indirectly through a linker peptide. In some cases, the conjugation moiety (e.g., a polymer, protein, or peptide) is genetically fused to IL-2 at the N-terminus or C-terminus of IL-2, either directly or indirectly through a linker peptide. In some cases, the conjugation moiety is linked to an N-terminal or C-terminal amino acid residue. In some cases, the conjugation moiety is linked to a reactive group that is linked to an N-terminal or C-terminal amino acid residue.

[00136] In some embodiments, an IL-2 conjugate with reduced binding affinity for IL-2Rα is capable of promoting expansion of a population of CD4+ helper cells, CD8+ effector naive and memory T cells, natural killer (NK) cells, or natural killer T (NKT) cells. In some cases, the conjugation moiety disrupts or blocks the binding of IL-2 to IL-2Rα.

[00137] In some cases, activation of a population of CD4+ helper cells, CD8+ effector naive and memory cells, natural killer (NK) cells, or natural killer T (NKT) cells by the IL-2Rβγ complex with a modified IL-2 polypeptide maintains significant efficiency of activation of said cell population compared to wild-type IL-2 polypeptide. In some cases, activation by the modified IL-2 polypeptide is equivalent to activation by wild-type IL-2 polypeptide. In other cases, activation by the modified IL-2 polypeptide is greater than activation by wild-type IL-2 polypeptide. In some cases, the efficiency of receptor signaling of the modified IL-2 polypeptide to the IL-2Rβγ complex is greater than the efficiency of receptor signaling of the wild-type IL-2 polypeptide to the IL-2Rβγ complex. In some cases, the receptor signaling efficiency of the modified IL-2 polypeptide is at least 1-fold higher than the corresponding efficiency of the wild-type IL-2 polypeptide. In some cases, the receptor signaling efficiency of the modified IL-2 polypeptide is about or at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold or more higher than the corresponding efficiency of the wild-type IL-2 polypeptide. In such cases, the dose or concentration of the modified IL-2 polypeptide used to achieve a level of activation of the CD4+ helper cell population, CD8+ effector naive and memory cells, natural killer (NK) cells, or natural killer T (NKT) cells similar to the level of the wild-type IL-2 polypeptide is lower than the dose or concentration used for the wild-type IL-2 polypeptide.

[00138] In some embodiments, activation of a population of CD4+ helper cells, CD8+ effector naive and memory cells, natural killer (NK) cells, or natural killer T (NKT) cells by an IL-2Rβγ complex with a modified IL-2 polypeptide allows for significant maintenance of the efficiency of activation of said cell population by a wild-type IL-2 polypeptide. In some cases, the efficiency of receptor signaling of the modified IL-2 polypeptide to the IL-2Rβγ complex is lower than the efficiency of receptor signaling of the wild-type IL-2 polypeptide to the IL-2Rβγ complex. In some cases, the efficiency of receptor signaling of the modified IL-2 polypeptide is lower by about or at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, or 50-fold than the corresponding efficiency of the wild-type IL-2 polypeptide.

[00139] In some embodiments, the modified IL-2 polypeptide exhibits efficacy in transducing a first receptor to IL-2Rβγ and efficacy in transducing a second receptor to IL-2Rαβγ. In some cases, the efficacy in transducing a first receptor to IL-2Rβγ is an improved efficacy compared to a wild-type IL-2 polypeptide. In some cases, the efficacy in transducing a second receptor to IL-2Rαβγ is an impaired efficacy compared to a wild-type IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide exhibits efficacy in transducing a first receptor signal to IL-2Rβγ and efficacy in transducing a second receptor signal to IL-2Rαβγ, and wherein the efficacy in transducing the first receptor signal is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 500-fold, 1000-fold or more higher than the efficacy in transducing the second receptor signal. In some cases, the efficacy in transducing the first receptor signal is at least 1-fold or more higher than the efficacy in transducing the second receptor signal. In some cases, the efficacy in transducing the first receptor signal is at least 2-fold or more higher than the efficacy in transducing the second receptor signal. In some cases, the efficiency in transducing the signal of the first receptor is at least 5 times or more higher than the efficiency in transducing the signal of the second receptor. In some cases, the efficiency in transducing the signal of the first receptor is at least 10 times or more higher than the efficiency in transducing the signal of the second receptor. In some cases, the efficiency in transducing the signal of the first receptor is at least 20 times or more higher than the efficiency in transducing the signal of the second receptor. In some cases, the efficiency in transducing the signal of the first receptor is at least 50 times or more higher than the efficiency in transducing the signal of the second receptor. In some cases, the efficiency in transducing the signal of the first receptor is at least 100 times or more higher than the efficiency in transducing the signal of the second receptor. In some cases, the efficiency in transducing the signal of the first receptor is at least 500 times or more higher than the efficiency in transducing the signal of the second receptor. In some cases, the efficiency in transducing the signal of the first receptor is at least 1000 times or more higher than the efficiency in transducing the signal of the second receptor. In some cases, the signaling efficiency of the first receptor of the modified IL-2 polypeptide is higher than the signaling efficiency of the wild-type IL-2 polypeptide to IL-2Rβγ, and the signaling efficiency of the second receptor of the modified IL-2 polypeptide is lower than the signaling efficiency of the wild-type IL-2 polypeptide to IL-2Rαβγ. In some cases, the signaling efficiencies of both receptors are lower than their corresponding efficiencies of the wild-type IL-2 polypeptide. In other cases, the signaling efficiencies of both receptors are higher than their corresponding efficiencies of the wild-type IL-2 polypeptide.

[00140] In some embodiments, the IL-2 conjugate reduces a toxic adverse event in a subject administered the IL-2 conjugate. Exemplary toxic adverse events include eosinophilia, increased capillary permeability, and vascular leak syndrome (VLS). In some cases, the IL-2 conjugate reduces the incidence of a toxic adverse event in a subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% compared to a second subject administered wild-type IL-2 or aldesleukin. In some cases, the IL-2 conjugate reduces the severity of the toxic adverse event in a subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% compared to a second subject administered wild-type IL-2 or aldesleukin.

[00141] In some cases, the toxic adverse event is eosinophilia. In some cases, the IL-2 conjugate reduces the incidence of eosinophilia in a subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% compared to a second subject administered wild-type IL-2 or aldesleukin. In some cases, the IL-2 conjugate reduces the severity of eosinophilia in a subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% compared to a second subject administered wild-type IL-2 or aldesleukin.

[00142] In some cases, the toxic adverse event is increased capillary permeability. In some cases, the IL-2 conjugate reduces the incidence of increased capillary permeability in a subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% compared to a second subject administered wild-type IL-2 or aldesleukin. In some cases, the IL-2 conjugate reduces the severity of increased capillary permeability in a subject by approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or approximately 100% compared to a second subject administered wild-type IL-2 or aldesleukin.

[00143] In some cases, the toxic adverse event is VLS. In some cases, the IL-2 conjugate reduces the incidence of VLS in a subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% compared to a second subject administered wild-type IL-2 or aldesleukin. In some cases, the IL-2 conjugate reduces the severity of VLS in a subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% compared to a second subject administered wild-type IL-2 or aldesleukin.

[00144] In some embodiments, the IL-2 conjugate has a plasma half-life of greater than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more. In some embodiments, the IL-2 conjugate has a plasma half-life of greater than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, or more. In some embodiments, the IL-2 conjugate has a plasma half-life of greater than 1 hour. In some embodiments, the IL-2 conjugate has a plasma half-life of greater than 2 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of greater than 3 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of greater than 4 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of greater than 5 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of greater than 6 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of greater than 7 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of greater than 8 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of greater than 9 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of greater than 10 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of greater than 12 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of greater than 18 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of greater than 24 hours.

[00145] In some embodiments, the IL-2 conjugate has a plasma half-life of at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more. In some embodiments, the IL-2 conjugate has a plasma half-life of at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, or more. In some embodiments, the IL-2 conjugate has a plasma half-life of at least 1 hour. In some embodiments, the IL-2 conjugate has a plasma half-life of at least 2 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of at least 3 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of at least 4 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of at least 5 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of at least 6 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of at least 7 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of at least 8 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of at least 9 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of at least 10 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of at least 12 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of at least 18 hours. In some embodiments, the IL-2 conjugate has a plasma half-life of at least 24 hours.

[00146] In some embodiments, the IL-2 conjugate has a plasma half-life of from about 1 hour to about 7 days, from about 12 hours to about 7 days, from about 18 hours to about 7 days, from about 24 hours to about 7 days, from about 1 hour to about 5 days, from about 12 hours to about 5 days, from about 24 hours to about 5 days, from about 2 days to about 5 days, or from about 2 days to about 3 days.

[00147] In some embodiments, the IL-2 conjugate has a plasma half-life of from about 1 hour to about 18 hours, from about 1 hour to about 12 hours, from about 2 hours to about 10 hours, from about 2 hours to about 8 hours, from about 4 hours to about 18 hours, from about 4 hours to about 12 hours, from about 4 hours to about 10 hours, from about 4 hours to about 8 hours, from about 6 hours to about 18 hours, from about 6 hours to about 12 hours, from about 6 hours to about 10 hours, from about 6 hours to about 8 hours, from about 8 hours to about 18 hours, from about 8 hours to about 12 hours, or from about 8 hours to about 10 hours.

[00148] In some embodiments, the IL-2 conjugate has a plasma half-life that is capable of promoting proliferation and/or expansion of a CD4+ helper cell, a CD8+ effector naive and memory T cell, an NK cell, an NKT cell, or a combination thereof, but does not cause detrimental effects such as apoptosis.

[00149] In some embodiments, the IL-2 conjugate has an increased plasma half-life, such as at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more compared to wild-type IL-2. In some embodiments, the IL-2 conjugate has an increased plasma half-life, such as at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, or more compared to wild-type IL-2.

[00150] In some embodiments, the IL-2 conjugate has an increased plasma half-life, such as from about 1 hour to about 18 hours, from about 1 hour to about 12 hours, from about 2 hours to about 10 hours, from about 2 hours to about 8 hours, from about 4 hours to about 18 hours, from about 4 hours to about 12 hours, from about 4 hours to about 10 hours, from about 4 hours to about 8 hours, from about 6 hours to about 18 hours, from about 6 hours to about 12 hours, from about 6 hours to about 10 hours, from about 6 hours to about 8 hours, from about 8 hours to about 18 hours, from about 8 hours to about 12 hours, or from about 8 hours to about 10 hours, compared to wild-type IL-2.

[00151] In some embodiments, the IL-2 conjugate has an increased plasma half-life, such as at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more, compared to aldesleukin. In some embodiments, the IL-2 conjugate has an increased plasma half-life, such as at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, or more, compared to aldesleukin.

[00152] In some embodiments, the IL-2 conjugate has an increased plasma half-life, such as from about 1 hour to about 18 hours, from about 1 hour to about 12 hours, from about 2 hours to about 10 hours, from about 2 hours to about 8 hours, from about 4 hours to about 18 hours, from about 4 hours to about 12 hours, from about 4 hours to about 10 hours, from about 4 hours to about 8 hours, from about 6 hours to about 18 hours, from about 6 hours to about 12 hours, from about 6 hours to about 10 hours, from about 6 hours to about 8 hours, from about 8 hours to about 18 hours, from about 8 hours to about 12 hours, or from about 8 hours to about 10 hours, compared to aldesleukin.

[00153] In some embodiments, the IL-2 conjugate has an increased plasma half-life with reduced toxicity. In some cases, the IL-2 conjugate has an increased plasma half-life of at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more, with reduced toxicity. In some cases, the IL-2 conjugate is characterized by an increased plasma half-life of at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours or more, with reduced toxicity. In some cases, the IL-2 conjugate is characterized by an increased half-life in blood plasma from about 1 hour to about 18 hours, from about 1 hour to about 12 hours, from about 2 hours to about 10 hours, from about 2 hours to about 8 hours, from about 4 hours to about 18 hours, from about 4 hours to about 12 hours, from about 4 hours to about 10 hours, from about 4 hours to about 8 hours, from about 6 hours to about 18 hours, from about 6 hours to about 12 hours, from about 6 hours to about 10 hours, from about 6 hours to about 8 hours, from about 8 hours to about 18 hours, from about 8 hours to about 12 hours, or from about 8 hours to about 10 hours with reduced toxicity. In some cases, the reduced toxicity is reduced by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, or more compared to wild-type IL-2. In some cases, the reduced toxicity is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or more compared to wild-type IL-2.

[00154] In some embodiments, the IL-2 conjugate has an increased plasma half-life with reduced toxicity. In some cases, the IL-2 conjugate has an increased plasma half-life of at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more, with reduced toxicity. In some cases, the IL-2 conjugate is characterized by an increased plasma half-life of at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours or more, with reduced toxicity. In some cases, the IL-2 conjugate is characterized by an increased half-life in blood plasma from about 1 hour to about 18 hours, from about 1 hour to about 12 hours, from about 2 hours to about 10 hours, from about 2 hours to about 8 hours, from about 4 hours to about 18 hours, from about 4 hours to about 12 hours, from about 4 hours to about 10 hours, from about 4 hours to about 8 hours, from about 6 hours to about 18 hours, from about 6 hours to about 12 hours, from about 6 hours to about 10 hours, from about 6 hours to about 8 hours, from about 8 hours to about 18 hours, from about 8 hours to about 12 hours, or from about 8 hours to about 10 hours with reduced toxicity. In some cases, the reduced toxicity is reduced by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold or more compared to aldesleukin. In some cases, the reduced toxicity is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more compared to aldesleukin.

[00155] In some embodiments, the IL-2 conjugate comprises a conjugation moiety, wherein the size (e.g., volume or length) of the conjugation moiety provides increased stability in blood plasma, but does not reduce potency. In some cases, the size of the conjugation moiety increases the plasma half-life by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more. In some cases, the size of the conjugating moiety increases the plasma half-life by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours or more. In some cases, the size of the conjugating moiety provides an increase in plasma half-life from about 1 hour to about 18 hours, from about 1 hour to about 12 hours, from about 2 hours to about 10 hours, from about 2 hours to about 8 hours, from about 4 hours to about 18 hours, from about 4 hours to about 12 hours, from about 4 hours to about 10 hours, from about 4 hours to about 8 hours, from about 6 hours to about 18 hours, from about 6 hours to about 12 hours, from about 6 hours to about 10 hours, from about 6 hours to about 8 hours, from about 8 hours to about 18 hours, from about 8 hours to about 12 hours, or from about 8 hours to about 10 hours. In some cases, the size of the conjugating moiety reduces the potency by less than 5%, 4%, 3%, 2%, 1%, or less compared to aldesleukin.

[00156] In some embodiments, the IL-2 conjugate comprises a conjugation moiety, wherein the size (e.g., volume or length) of the conjugation moiety provides increased plasma stability and efficacy. In some cases, the size of the conjugation moiety increases plasma half-life by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more. In some cases, the size of the conjugating moiety increases the plasma half-life by at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours or more. In some cases, the size of the conjugating moiety provides an increase in plasma half-life from about 1 hour to about 18 hours, from about 1 hour to about 12 hours, from about 2 hours to about 10 hours, from about 2 hours to about 8 hours, from about 4 hours to about 18 hours, from about 4 hours to about 12 hours, from about 4 hours to about 10 hours, from about 4 hours to about 8 hours, from about 6 hours to about 18 hours, from about 6 hours to about 12 hours, from about 6 hours to about 10 hours, from about 6 hours to about 8 hours, from about 8 hours to about 18 hours, from about 8 hours to about 12 hours, or from about 8 hours to about 10 hours. In some cases, the size of the conjugating moiety further increases the potency by more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more compared to aldesleukin.

[00157] In some embodiments, described herein is an IL-2 conjugate comprising a non-natural amino acid covalently attached to a conjugation moiety, wherein the non-natural amino acid is located in region 35-107, and wherein region 35-107 corresponds to residues K35-Y107 in the sequence of SEQ ID NO: 1.

[00158] In some embodiments, described herein is an interleukin 2 receptor βγ (IL-2Rβγ) binding protein, wherein the binding affinity for interleukin 2 receptor α (IL-2Rα) of said binding protein is less than that of wild-type human IL-2 (hIL-2), wherein the binding affinity for interleukin 2 receptor α (IL-2Rα) of said binding protein is less than that of wild-type human IL-2 (hIL-2). In some embodiments, described herein is an interleukin 2 receptor βγ (IL-2Rβγ) binding protein, wherein the binding affinity for interleukin 2 receptor α (IL-2Rα) of said binding protein is less than that of wild-type human IL-2 (hIL-2), and wherein said binding protein comprises at least one unnatural amino acid. In some cases, said binding protein is a modified IL-2 polypeptide or a functionally active fragment thereof, wherein the modified IL-2 polypeptide comprises at least one unnatural amino acid. In some cases, the at least one unnatural amino acid is located in region 35-107, and wherein region 35-107 corresponds to residues K35-Y107 of SEQ ID NO: 1.

[00159] In some embodiments, described herein is an IL-2/IL-2Rβγ complex comprising a modified IL-2 polypeptide comprising a mutation and IL-2Rβγ, wherein the modified IL-2 polypeptide has a reduced binding affinity for IL-2Rα, and wherein the reduced binding affinity is compared to the binding affinity between a wild-type IL-2 polypeptide and IL-2Rα. In some cases, the modified IL-2 polypeptide further comprises a conjugation moiety covalently attached to a mutation site. In some cases, the mutation site comprises an amino acid mutated to a naturally occurring amino acid. In some cases, the mutation site comprises an amino acid mutated to a cysteine residue. In other cases, the mutation site comprises an amino acid mutated to a lysine residue.

[00160] In some embodiments, described herein is an IL-2/IL-2Rβγ complex comprising a modified IL-2 polypeptide comprising a non-natural amino acid and IL-2Rβγ, wherein the modified IL-2 polypeptide has a reduced binding affinity for IL-2Rα, and wherein the reduced binding affinity is compared to the binding affinity between a wild-type IL-2 polypeptide and IL-2Rα. In some cases, the modified IL-2 polypeptide further comprises a conjugation moiety covalently attached to the non-natural amino acid.

[00161] In some embodiments, described herein is an IL-2/IL-2Rβγ complex comprising a modified IL-2 polypeptide comprising a non-natural amino acid and IL-2Rβγ, wherein the modified IL-2 polypeptide has reduced efficiency of receptor signaling to IL-2Rα, and wherein the reduced efficiency of receptor signaling is compared to the efficiency of receptor signaling between a wild-type IL-2 polypeptide and IL-2Rα. In some cases, the modified IL-2 polypeptide further comprises a conjugation moiety covalently attached to the non-natural amino acid.

[00162] In some embodiments, described herein is a CD4+ helper cell, CD8+ effector naive and memory T cell, natural killer (NK) cell, or natural killer T cell (NKT) activator that selectively allows for the expansion of CD4+ helper cells, CD8+ effector naive and memory T cells, NK cells, NKT cells, or a combination thereof in a cell population, wherein said activator comprises a modified interleukin 2 (IL-2) polypeptide comprising at least one mutation. In some cases, the mutation is a mutation to a naturally occurring amino acid. In other cases, the mutation is a mutation to a non-natural amino acid. In some embodiments, described herein is a CD4+ helper cell, CD8+ effector naive and memory T cell, natural killer (NK) cell, or natural killer T cell (NKT) activator that selectively promotes expansion of CD4+ helper cells, CD8+ effector naive and memory T cells, NK cells, NKT cells, or a combination thereof in a cell population, wherein said activator comprises a modified interleukin 2 (IL-2) polypeptide comprising at least one unnatural amino acid. In some cases, said activator provides for an expansion of CD4+ regulatory T cells (Treg) by less than 20%, 15%, 10%, 5%, 1%, or less than 0.1% when said activator is contacted with said CD3+ cell population, compared to the expansion of CD4+ Treg cells in a CD3+ cell population when contacted with a wild-type IL-2 polypeptide. In some cases, said activator does not provide for the expansion of Treg cells in said cell population. In some cases, said cell population is an in vivo cell population. In some cases, said cell population is an in vitro cell population. In some cases, said cell population is an ex vivo cell population.

[00163] In some cases, the document also describes a method for expanding a population of CD4+ helper cells, CD8+ effector naive and memory T cells, natural killer (NK) cells, or natural killer T cells (NKT cells), comprising contacting said cell population with a therapeutically effective amount of an activator of a CD4+ helper cell, CD8+ effector naive and memory T cell, natural killer (NK) cell, or natural killer T cell (NKT), wherein said activator comprises a modified interleukin 2 (IL-2) polypeptide comprising at least one mutation, thereby expanding a population of CD4+ helper cells, CD8+ effector naive and memory T cells, natural killer (NK) cells, or natural killer T cells (NKT). In some cases, the mutation is a mutation into a naturally occurring amino acid. In other cases, the mutation is a mutation into a non-natural amino acid. In some cases, the present document also describes a method for expanding a population of CD4+ helper cells, CD8+ effector naive and memory T cells, natural killer (NK) cells, or natural killer T cells (NKT) comprising contacting said cell population with a therapeutically effective amount of an activator of a CD4+ helper cell, CD8+ effector naive and memory T cell, natural killer (NK) cell, or natural killer T cell (NKT), wherein said activator comprises a modified interleukin 2 (IL-2) polypeptide comprising at least one non-natural amino acid, thereby expanding the population of CD4+ helper cells, CD8+ effector naive and memory T cells, natural killer (NK) cells, or natural killer T cells (NKT).

[00164] In some embodiments, a modified IL-2 polypeptide comprising a mutation at position K35, corresponding to residue position 35 of SEQ ID NO: 1, comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide to IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide to IL-2Rα.

[00165] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue T37 corresponding to position 37 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00166] In some cases, the conjugate moiety is linked to an amino acid residue selected from amino acid position P2, T3, S4, S5, S6, T7, K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, R81, P82, R83, D84, S87, N88, N89, V91, I92, L94, E95, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, T113, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some cases, the amino acid position is selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, I92, L94, E95, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133. In some cases, the amino acid position is selected from P2, T3, S4, S5, S6, T7, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, P82, R83, N89, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, and T113. In some cases, the amino acid position is selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95, and Q126. In some cases, the amino acid position is selected from K8, K9, and H16. In some cases, the amino acid position is selected from Q22, N26, N88, and Q126. In some cases, the amino acid position is selected from E15, D20, D84, and E95. In some cases, the amino acid position is selected from L12, L19, and M23. In some cases, the amino acid position is selected from Q22 and N26. In some cases, the amino acid position is K8. In some cases, the amino acid position is K9. In some cases, the amino acid position is Q11. In some cases, the amino acid position is L12. In some cases, the amino acid position is E15. In some cases, the amino acid position is H16. In some cases, the amino acid position is L18. In some cases, the amino acid position is L19. In some cases, the amino acid position is D20. In some cases, the amino acid position is Q22. In some cases, the amino acid position is M23. In some cases, the amino acid position is N26. In some cases, the amino acid position is R81. In some cases, the amino acid position is D84. In some cases, the amino acid position is S87. In some cases, the amino acid position is N88. In some cases, the amino acid position is V91. In some cases, the amino acid position is I92. In some cases, the amino acid position is L94. In some cases, the amino acid position is E95. In some cases, the amino acid position is E116. In some cases, the amino acid position is N119. In some cases, the amino acid position is R120. In some cases, the amino acid position is T123. In some cases, the amino acid position is A125. In some cases, the amino acid position is Q126. In some cases, the amino acid position is S127. In some cases, the amino acid position is S130. In some cases, the amino acid position is T131. In some cases, the amino acid position is L132. In some cases, the amino acid position is T133.

[00167] In some cases, the IL-2 conjugate further comprises an additional mutation. In such cases, the amino acid is conjugated to an additional conjugation moiety to increase serum half-life, stability, or a combination thereof. Alternatively, the amino acid is first mutated to a natural amino acid such as lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine, or to a non-natural amino acid, prior to coupling to the additional conjugation moiety.

[00168] In some embodiments, the IL-2 conjugate has a reduced binding affinity for the IL-2 receptor β subunit (IL-2Rβ), the IL-2 receptor γ subunit (IL-2Rγ), or a combination thereof of the IL-2Rαβγ complex compared to a wild-type IL-2 polypeptide. In some cases, the reduced affinity of the IL-2 conjugate for the IL-2 receptor β subunit (IL-2Rβ), the IL-2 receptor γ subunit (IL-2Rγ), or a combination thereof, compared to a wild-type IL-2 polypeptide, is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99%. In some cases, the reduction in affinity is about 10%. In some cases, the reduction in affinity is about 20%. In some cases, the reduction in affinity is about 40%. In some cases, the reduction in affinity is about 50%. In some cases, the reduction in affinity is about 60%. In some cases, the reduction in affinity is about 80%. In some cases, the reduction in affinity is about 90%. In some cases, the reduction in affinity is approximately 99%. In some cases, the reduction in affinity is greater than 99%. In some cases, the reduction in affinity is approximately 80%. In some cases, the reduction in affinity is approximately 100%.

[00169] In some embodiments, the decrease in binding affinity of the IL-2 conjugate to the IL-2 receptor β subunit (IL-2Rβ), the IL-2 receptor γ subunit (IL-2Rγ), or a combination thereof compared to a wild-type IL-2 polypeptide is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or greater. In some cases, the decrease in affinity is about 1-fold. In some cases, the decrease in affinity is approximately 2-fold. In some cases, the decrease in affinity is approximately 4-fold. In some cases, the decrease in affinity is approximately 5-fold. In some cases, the decrease in affinity is approximately 6-fold. In some cases, the decrease in affinity is approximately 8-fold. In some cases, the decrease in affinity is approximately 10-fold. In some cases, the decrease in affinity is approximately 30-fold. In some cases, the decrease in affinity is approximately 50-fold. In some cases, the decrease in affinity is approximately 100-fold. In some cases, the decrease in affinity is approximately 300-fold. In some cases, the decrease in affinity is approximately 500-fold. In some cases, the decrease in affinity is approximately 1000-fold. In some cases, the decrease in affinity is greater than 1000-fold.

[00170] In some embodiments, the IL-2 conjugate has reduced recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex. In some cases, the reduced recruitment is compared to the recruitment of the IL-2Rγ subunit by an equivalent IL-2 polypeptide lacking the unnatural amino acid (e.g., a wild-type IL-2 polypeptide). In some cases, the reduction in IL-2Rγ subunit recruitment is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% compared to an equivalent IL-2 polypeptide without the unnatural amino acid modification. In some cases, the reduction in IL-2Rγ subunit recruitment is about 10%. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 20%. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 40%. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 50%. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 60%. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 70%. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 80%. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 90%. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 99%. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 99%. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 100%. In some cases, the IL-2 conjugate is further characterized by increased recruitment of the IL-2Rα subunit.

[00171] In some embodiments, the reduction in IL-2Rγ subunit recruitment is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more compared to an equivalent IL-2 polypeptide without a non-natural amino acid modification (e.g., a wild-type IL-2 polypeptide). In some cases, the reduction in IL-2Rγ subunit recruitment is about 1-fold. In some cases, the reduction in IL-2Rγ subunit recruitment is about 2-fold. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 4-fold. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 5-fold. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 6-fold. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 8-fold. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 10-fold. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 30-fold. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 50-fold. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 100-fold. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 300-fold. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 500-fold. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 1000-fold. In some cases, the reduction in IL-2Rγ subunit recruitment is approximately 1000-fold. In some cases, the IL-2 conjugate is further characterized by an increase in IL-2Rα subunit recruitment.

[00172] In some embodiments, the IL-2 conjugate is characterized by an increase in recruitment of the IL-2Rα subunit to the IL-2 polypeptide. In some cases, the decreased recruitment is compared to the recruitment of the IL-2Rα subunit by an equivalent IL-2 polypeptide lacking the unnatural amino acid (e.g., a wild-type IL-2 polypeptide). In some cases, the increase in recruitment of the IL-2Rα subunit is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or greater than 99% compared to an equivalent IL-2 polypeptide without the unnatural amino acid modification. In some cases, the increase in recruitment of the IL-2Rα subunit is about 10%. In some cases, the increase in IL-2Rα subunit recruitment is approximately 20%. In some cases, the increase in IL-2Rα subunit recruitment is approximately 40%. In some cases, the increase in IL-2Rα subunit recruitment is approximately 50%. In some cases, the increase in IL-2Rα subunit recruitment is approximately 60%. In some cases, the increase in IL-2Rα subunit recruitment is approximately 70%. In some cases, the increase in IL-2Rα subunit recruitment is approximately 80%. In some cases, the increase in IL-2Rα subunit recruitment is approximately 90%. In some cases, the increase in IL-2Rα subunit recruitment is approximately 99%. In some cases, the increase in IL-2Rα subunit recruitment is greater than 99%. In some cases, the increase in IL-2Rα subunit recruitment is approximately 100%. In some cases, the IL-2 conjugate is further characterized by a decrease in recruitment of the IL-2Rβ subunit and/or the IL-2Rγ subunit.

[00173] In some embodiments, the increase in IL-2Rα subunit recruitment is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more compared to an equivalent IL-2 polypeptide without a non-natural amino acid modification (e.g., a wild-type IL-2 polypeptide). In some cases, the increase in IL-2Rα subunit recruitment is about 1-fold. In some cases, the increase in IL-2Rα subunit recruitment is about 2-fold. In some cases, the increase in IL-2Rα subunit recruitment is approximately 4-fold. In some cases, the increase in IL-2Rα subunit recruitment is approximately 5-fold. In some cases, the increase in IL-2Rα subunit recruitment is approximately 6-fold. In some cases, the increase in IL-2Rα subunit recruitment is approximately 8-fold. In some cases, the increase in IL-2Rα subunit recruitment is approximately 10-fold. In some cases, the increase in IL-2Rα subunit recruitment is approximately 30-fold. In some cases, the increase in IL-2Rα subunit recruitment is approximately 50-fold. In some cases, the increase in IL-2Rα subunit recruitment is approximately 100-fold. In some cases, the increase in IL-2Rα subunit recruitment is approximately 300-fold. In some cases, the increase in IL-2Rα subunit recruitment is approximately 500-fold. In some cases, the increase in IL-2Rα subunit recruitment is approximately 1000-fold. In some cases, the increase in IL-2Rα subunit recruitment is greater than 1000-fold. In some cases, the IL-2 conjugate is further characterized by a decrease in IL-2Rβ subunit and/or IL-2Rγ subunit recruitment.

[00174] In some embodiments, the IL-2 polypeptide described herein has a decrease in the efficiency of receptor signaling to IL-2Rβγ. In some cases, the decrease in the efficiency of receptor signaling is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more to IL-2Rβγ compared to a wild-type IL-2 polypeptide. In some cases, the decrease in the efficiency of receptor signaling is about 2-fold. In some cases, the decrease in the efficiency of receptor signaling is about 5-fold. In some cases, the decrease in the efficiency of receptor signaling is approximately 10-fold. In some cases, the decrease in the efficiency of receptor signaling is approximately 20-fold. In some cases, the decrease in the efficiency of receptor signaling is approximately 30-fold. In some cases, the decrease in the efficiency of receptor signaling is approximately 40-fold. In some cases, the decrease in the efficiency of receptor signaling is approximately 50-fold. In some cases, the decrease in the efficiency of receptor signaling is approximately 100-fold. In some cases, the decrease in the efficiency of receptor signaling is approximately 200-fold. In some cases, the decrease in the efficiency of receptor signaling is approximately 300-fold. In some cases, the decrease in the efficiency of receptor signaling is approximately 400-fold. In some cases, the decrease in the efficiency of receptor signaling is approximately 500-fold. In some cases, the decrease in the efficiency of receptor signaling is approximately 1000-fold.

[00175] In some cases, the efficiency of receptor signaling is measured using an EC50 value. In some cases, a decrease in the efficiency of receptor signaling results in an increase in EC50. In some cases, the increase in EC50 is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more compared to a wild-type IL-2 polypeptide.

[00176] In some cases, the efficiency of receptor signaling is measured using an ED50 value. In some cases, a decrease in the efficiency of receptor signaling results in an increase in ED50. In some cases, the increase in ED50 is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more compared to a wild-type IL-2 polypeptide.

[00177] In some embodiments, an IL-2 polypeptide described herein has an expanded therapeutic window compared to the therapeutic window of a wild-type IL-2 polypeptide. In some cases, the expanded therapeutic window is due to decreased binding between the IL-2 polypeptide and interleukin 2 receptor βγ (IL-2Rβγ), decreased efficiency of receptor signaling to IL-2Rβγ, decreased recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex, or increased recruitment of the IL-2Rα subunit to the IL-2 polypeptide. In some cases, the IL-2 polypeptide does not have impaired activation of the interleukin 2 receptor αβγ (IL-2Rαβγ).

[00178] In some embodiments, the modified IL-2 polypeptide exhibits efficacy in transducing a first receptor signal to an IL-2βγ signaling complex and efficacy in transducing a second receptor signal to an IL-2αβγ signaling complex, and wherein the difference between the efficacy in transducing the first receptor signal and the efficacy in transducing the second receptor signal is at least 1-fold. In some cases, the difference is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold or greater. In some cases, the efficiency of the first receptor in signaling is less than the efficiency of the second receptor in signaling. In some cases, the efficiency in transmitting the first receptor signal is less than the efficiency in transmitting the second receptor signal by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 500-fold, 1000-fold or more. In some cases, the modified IL-2 polypeptide is characterized by a lower efficiency of receptor signaling to the IL-2βγ signaling complex than the efficiency in transmitting the second receptor signal to the IL-2αβγ signaling complex. In some cases, the efficiency in transmitting the first receptor signal of the modified IL-2 polypeptide is at least 1-fold lower than the efficiency of receptor signaling of the wild-type IL-2 polypeptide. In some cases, the first receptor signaling efficiency of the modified IL-2 polypeptide is at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, or 500-fold lower than the receptor signaling efficiency of the wild-type IL-2 polypeptide. In some cases, both the first receptor signaling efficiency and the second receptor signaling efficiency are lower than the corresponding efficiencies of the wild-type IL-2 polypeptide, but the first receptor signaling efficiency is lower than the second receptor signaling efficiency. In some cases, the difference between the first receptor signaling efficiency and the second receptor signaling efficiency increases the therapeutic window for the modified IL-2 polypeptide.

[00179] In some cases, the conjugating moiety impairs or blocks the efficiency of IL-2 receptor signaling with IL-2Rβγ or reduces the recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex.

[00180] In some cases, a modified IL-2 polypeptide with reduced efficiency of receptor signaling to IL-2Rβγ is able to promote the expansion of CD4+ regulatory T cells (Treg).

[00181] In some embodiments, the proliferation of CD4+ Treg cells by the modified IL-2/IL-2Rαβγ complex is equivalent to or greater than that of a wild-type IL-2 polypeptide.

[00182] In some embodiments, the IL-2/IL-2Rαβγ complex induces proliferation of CD4+ Treg cells into the population, which is sufficient to modulate the course of the disease in an animal model.

[00183] In some embodiments, described herein is an interleukin 2 receptor αβγ (IL-2Rαβγ) binding protein, wherein the efficiency of interleukin 2 receptor βγ (IL-2Rβγ) receptor signaling is less than that of wild-type human IL-2 (hIL-2), and wherein the binding protein comprises at least one unnatural amino acid. In some cases, the binding protein is a modified IL-2 polypeptide or a functionally active fragment thereof, wherein the modified IL-2 polypeptide comprises at least one unnatural amino acid.

[00184] In some embodiments, described herein is an interleukin 2 receptor αβγ (IL-2Rαβγ) binding protein, wherein recruitment of an IL-2Rγ subunit to the IL-2/IL-2Rβ complex by said binding protein is less than that of wild-type human IL-2 (hIL-2), and wherein said binding protein comprises at least one unnatural amino acid. In some cases, said binding protein is a modified IL-2 polypeptide or a functionally active fragment thereof, wherein the modified IL-2 polypeptide comprises at least one unnatural amino acid.

[00185] In some embodiments, described herein is an interleukin 2 receptor αβγ (IL-2Rαβγ) binding protein, wherein the binding affinity for interleukin 2 receptor βγ (IL-2Rβγ) of said binding protein is less than that of wild-type human IL-2 (hIL-2), and wherein said binding protein comprises at least one unnatural amino acid. In such cases, said binding protein is a modified IL-2 polypeptide or a functionally active fragment thereof, wherein the modified IL-2 polypeptide comprises at least one unnatural amino acid.

[00186] In some embodiments, described herein is an IL-2/IL-2Rαβγ complex comprising a modified IL-2 polypeptide comprising a non-natural amino acid and IL-2Rαβγ, wherein the modified IL-2 polypeptide is characterized by reduced efficiency of receptor signaling to IL-2Rβγ, and wherein the reduced efficiency of receptor signaling is compared to the binding affinity between a wild-type IL-2 polypeptide and IL-2Rβγ. In some cases, the modified IL-2 polypeptide further comprises a conjugation moiety covalently attached to the non-natural amino acid.

[00187] In some embodiments, described herein is an IL-2/IL-2Rαβγ complex comprising a modified IL-2 polypeptide comprising a non-natural amino acid and IL-2Rαβγ, wherein the recruitment of an IL-2Rγ subunit to the IL-2/IL-2Rβ complex by said modified IL-2 polypeptide is less than that of a wild-type IL-2 polypeptide. In some cases, the modified IL-2 polypeptide further comprises a conjugation moiety covalently attached to the non-natural amino acid.

[00188] In some embodiments, described herein is an IL-2/IL-2Rαβγ complex comprising a modified IL-2 polypeptide comprising a non-natural amino acid and IL-2Rαβγ, wherein the modified IL-2 polypeptide has a reduced binding affinity for IL-2Rβγ, and wherein the reduced binding affinity is compared to the binding affinity between a wild-type IL-2 polypeptide and IL-2Rβγ. In some embodiments, described herein is an IL-2/IL-2Rαβγ complex comprising a modified IL-2 polypeptide comprising a non-natural amino acid and IL-2Rαβγ, wherein the recruitment of an IL-2Rγ subunit to the IL-2/IL-2Rβ complex by said modified IL-2 polypeptide is less than that of a wild-type IL-2 polypeptide. In some cases, the modified IL-2 polypeptide further comprises a conjugation moiety covalently attached to a non-natural amino acid.

[00189] In some embodiments, described herein is a CD4+ Treg cell activator that selectively causes expansion of CD4+ Treg cells in a cell population, wherein said activator comprises a modified IL-2 polypeptide comprising at least one unnatural amino acid. In some cases, said activator causes expansion of a CD8+ effector T cell and/or natural killer cell by less than 20%, 15%, 10%, 5%, 1%, or 0.1% in a CD3+ cell population when said activator is contacted with said CD3+ cell population, compared to expansion of a CD8+ effector T cell and/or natural killer cell in a CD3+ cell population when contacted with a wild-type IL-2 polypeptide. In some cases, said cell population is an in vivo cell population. In some cases, said cell population is an in vitro cell population. In some cases, the cell population in question represents an ex vivo cell population.

[00190] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue R38, corresponding to position 38 of SEQ ID NO: 1, comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00191] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue T41 corresponding to position 41 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00192] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue F42 corresponding to position 42 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00193] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue K43 corresponding to position 43 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00194] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue F44 corresponding to position 44 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has a minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00195] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue Y45 corresponding to position 45 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00196] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue E60 corresponding to position 60 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00197] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue E61 corresponding to position 61 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00198] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue E62 corresponding to position 62 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00199] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue K64, corresponding to position 64 of SEQ ID NO: 1, comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00200] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue P65 corresponding to position 65 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00201] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue E68 corresponding to position 68 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has a minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00202] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue V69 corresponding to position 69 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00203] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue N71, corresponding to position 71 of SEQ ID NO: 1, comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00204] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue L72 corresponding to position 72 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00205] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue M104 corresponding to position 104 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00206] In some embodiments, a modified IL-2 polypeptide comprising a mutation at C105 corresponding to position 105 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has a minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00207] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue Y107, corresponding to position 107 of SEQ ID NO: 1, comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has a minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

Cytokine conjugate precursors

[00208] Described herein are cytokine conjugate precursors comprising a mutant cytokine (such as IL-2) in which one or more amino acids have been mutated from a wild-type amino acid. Such precursors are often used in the methods disclosed herein for treating diseases or conditions. In some embodiments, the cytokine precursor is not conjugated. Such mutations variously include additions, deletions, or substitutions. In some embodiments, the mutation includes substitution with another naturally occurring amino acid. In some cases, the mutant cytokine comprises a mutation at amino acid position Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, F44, Y45, P47, K48, Q57, E60, E61, E62, L63, K64, P65, E68, V69, N71, L72, Q74, S75, K76, N77, M104, C105, E106, Y107, A108, D109, E110, T111, or A112, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some cases, the amino acid position is selected from Y31, K32, N33, P34, K35, T37, R38, T41, F42, K43, F44, Y45, P47, K48, E61, E62, E68, K64, P65, V69, L72, Q74, S75, K76, N77, M104, C105, E106, Y107, A108, D109, E110, T111, and A112. In some cases, the amino acid position is selected from N33, P34, K35, T37, R38, M39, T41, F42, K43, F44, Y45, Q57, E60, E61, E62, L63, K64, P65, E68, V69, N71, L72, M104, C105, E106, Y107, A108, D109, E110, T111, and A112. In some cases, the amino acid position is selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some cases, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some cases, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some cases, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some cases, the amino acid position is selected from R38 and K64. In some cases, the amino acid position is selected from E61, E62, and E68. In some cases, the amino acid position is K35. In some cases, the amino acid position is T37. In some cases, the amino acid position is R38. In some cases, the amino acid position is T41. In some cases, the amino acid position is F42. In some cases, the amino acid position is K43. In some cases, the amino acid position is F44. In some cases, the amino acid position is Y45. In some cases, the amino acid position is E61. In some cases, the amino acid position is E62. In some cases, the amino acid position is K64. In some cases, the amino acid position is E68. In some cases, the amino acid position is P65. In some cases, the amino acid position is V69. In some cases, the amino acid position is L72. In some cases, the amino acid position is Y107. In some cases, the amino acid position is L72. In some cases, the amino acid position is D109. In some embodiments, the mutant cytokine comprises a conjugating moiety, wherein the conjugating moiety is attached to a mutant site in the mutant cytokine.

[00209] The mutant cytokines described herein often comprise one or more mutations to naturally occurring amino acids. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and at least one mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the E62K mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the E62C mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the E62A mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the E62I mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the E62L mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the E62Y mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the E62W mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the E62N mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the E62R mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the E62D mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the E62Q mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the E62G mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the E62H mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the E62M mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the E62F mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the E62P mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the E62S mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the E62T mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the E62V mutation.

[00210] In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and at least one mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the P65K mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the P65C mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the P65A mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the P65I mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the P65L mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the P65Y mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the P65W mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the P65N mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the P65R mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the P65D mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the P65Q mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the P65G mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the P65H mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the P65M mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the P65F mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the P65E mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the P65S mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the P65T mutation. In some embodiments, the mutant cytokine comprises SEQ ID NO:1 and the P65V mutation.

Fusion proteins or peptides

[00211] In some embodiments, a cytokine conjugate described herein comprises a cytokine (e.g., IL-2 or another cytokine) that is fused to a peptide or protein (fusion). In some embodiments, the peptide or protein is an antibody or antibody fragment. In some embodiments, a cytokine conjugate described herein comprises a cytokine (e.g., IL-2 or another cytokine) that is fused to an antibody or binding fragments thereof. In some embodiments, a cytokine described herein is fused to more than one protein or peptide. In some embodiments, a cytokine conjugate comprises a fusion construct of a cytokine and a protein or peptide and at least one conjugating moiety. In some cases, the antibody or binding fragments thereof comprises a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab ₂ , F(ab)' ₃ fragments, a single-chain variable fragment (scFv), a bis-scFv, (scFv) ₂ , a diabody, a minibody, a nanobody, a triantibody, a tetraantibody, a humanibody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camel antibody or binding fragment thereof, a bispecific antibody or binding fragment thereof, or a chemically modified derivative thereof. Such fusion proteins are in some cases produced directly by translation. In some embodiments, the fusion constructs are prepared using a chemical or other enzymatic ligation method. In some embodiments, the cytokine conjugate comprises a fusion peptide or protein attached via a linker. In some embodiments, the linker is a peptide. In some embodiments, the cytokine conjugate comprises a peptide or protein fused at the N-terminus. In some embodiments, the cytokine conjugate comprises a peptide or protein fused at the C-terminus. In some cases, the cytokine fused to the peptide or protein is further conjugated to one or more conjugating moieties described below.

[00212] In some cases, the cytokine conjugate comprises a fusion to an scFv, a bis-scFv, (scFv) ₂ , dsFv, or a fusion to an sdAb. In some cases, the fusion comprises an scFv. In some cases, the cytokine conjugate comprises a fusion to a bis-scFv. In some cases, the cytokine conjugate comprises a fusion to (scFv) ₂ . In some cases, the cytokine conjugate comprises a fusion to a dsFv. In some cases, the cytokine conjugate comprises a fusion to an sdAb. In some cases, the cytokine fused to an scFv, bis-scFv, (scFv) ₂ , dsFv, or sdAb is further conjugated to one or more conjugating moieties described below.

[00213] In some cases, the cytokine conjugate comprises a fusion with the Fc portion of an antibody, such as IgG, IgA, IgM, IgE, or IgD. In some cases, the cytokine conjugate comprises a fusion with the Fc portion of an IgG (e.g., IgG ₁ , IgG ₃ , or IgG ₄ ). In some cases, the cytokine fused to the Fc portion is further conjugated to one or more conjugating moieties described below.

[00214] In some cases, a cytokine polypeptide (e.g., interleukin, IFN, or TNF) is fused to an antibody or binding fragments thereof. In some cases, the cytokine polypeptide is fused to a humanized antibody or a binding fragment thereof, a murine antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, a monoclonal antibody or a binding fragment thereof, a monovalent Fab', a bivalent Fab ₂ , a F(ab)' ₃ fragment, a single-chain variable fragment (scFv), a bis-scFv, (scFv) ₂ , a diabody, a minibody, a nanobody, a tribody, a tetrabody, a humanibody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camel antibody or a binding fragment thereof, a bispecific antibody or a binding fragment thereof, or a chemically modified derivative thereof. In additional cases, the cytokine polypeptide is fused to the Fc portion of an antibody. In additional cases, the cytokine polypeptide is fused to the Fc portion of an IgG (e.g., IgG ₁ , IgG ₃ , or IgG ₄ ). In some cases, the cytokine fused to the antibody or binding fragments thereof is further conjugated to one or more conjugating moieties described below.

[00215] In some cases, the IL-2 polypeptide is fused to an antibody or binding fragments thereof. In some cases, the IL-2 polypeptide is fused to a humanized antibody or a binding fragment thereof, a murine antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, a monoclonal antibody or a binding fragment thereof, a monovalent Fab', a bivalent Fab ₂ , a F(ab)' ₃ fragment, a single-chain variable fragment (scFv), a bis-scFv, (scFv) ₂ , a diabody, a minibody, a nanobody, a tribody, a tetrabody, a humanibody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camel antibody or a binding fragment thereof, a bispecific antibody or a binding fragment thereof, or a chemically modified derivative thereof. In additional cases, the IL-2 polypeptide is fused to the Fc portion of an antibody. In additional cases, the IL-2 polypeptide is fused to the Fc portion of an IgG (e.g., IgG ₁ , IgG ₃ , or IgG ₄ ). In some cases, the IL-2 polypeptide fused to the antibody or binding fragments thereof is further conjugated to one or more conjugating moieties described below.

Natural and unnatural amino acids

[00216] In some embodiments, an amino acid residue described herein (e.g., in a cytokine such as IL-2) is mutated to lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine prior to binding (or reacting) with a conjugating moiety. For example, a side chain of lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine can bind to a conjugating moiety described herein. In some cases, the amino acid residue is mutated to cysteine, lysine, or histidine. In some cases, the amino acid residue is mutated to cysteine. In some cases, the amino acid residue is mutated to lysine. In some cases, the amino acid residue is mutated to histidine. In some cases, the amino acid residue is mutated to tyrosine. In some cases, the amino acid residue is mutated to tryptophan. In some embodiments, the unnatural amino acid is not conjugated to the conjugation moiety. In some embodiments, a cytokine described herein comprises a unnatural amino acid, wherein the cytokine is conjugated to a protein, wherein the attachment point is not a unnatural amino acid.

[00217] In some embodiments, an amino acid residue described herein (e.g., in a cytokine such as IL-2) is mutated to a non-natural amino acid prior to binding to a conjugating moiety. In some cases, the mutation to a non-natural amino acid prevents or minimizes the immune system's response to the self-antigen. As used herein, the term "non-natural amino acid" or "non-canonical amino acid" refers to an amino acid other than the 20 amino acids that naturally occur in a protein. Non-limiting examples of unnatural amino acids include p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, p-methoxyphenylalanine, O-methyl-L-tyrosine, p-propargyloxyphenylalanine, p-propargylphenylalanine, L-3-(2-naphthyl)alanine, 3-methylphenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p -boronphenylalanine, O -propargyltyrosine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, selenocysteine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, N6-(2-azidoethoxy)-carbonyl-L-lysine (AzK, the chemical structure of which is shown as compound 90 in Figure 3C), an unnatural analog of the amino acid tyrosine; an unnatural analog of the amino acid glutamine; an unnatural analog of the amino acid phenylalanine; an unnatural analog of the amino acid serine; an unnatural analog of the amino acid threonine; an amino acid substituted with alkyl, aryl, acyl, azido, cyano, halogen, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocycle, enone, imine, aldehyde, hydroxylamine, keto or amino, or a combination thereof; an amino acid with a photoactivatable crosslinker; a spin-labeled amino acid; a fluorescent amino acid; a metal-binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photoshielded and/or photoisomerizable amino acid; an amino acid containing biotin or a biotin analog; a keto-containing amino acid; an amino acid containing polyethyleneglycol or a polyether; an amino acid substituted with a heavy atom; a chemically cleavable or photocleavable amino acid; an amino acid with an extended side chain; an amino acid containing a toxic group; an amino acid substituted with a sugar; an amino acid containing a carbon-linked sugar; a redox-active amino acid; an a-hydroxy-containing acid; an amino thio acid; an α,α-disubstituted amino acid; a β-amino acid; a cyclic amino acid other than proline or histidine, and an aromatic amino acid other than phenylalanine, tyrosine, or tryptophan.

[00218] In some embodiments, the unnatural amino acid comprises a selective reactive group or a reactive group for site-selective labeling of a target polypeptide. In some cases, the chemistry is a biorthogonal reaction (e.g., biocompatible and selective reactions). In some cases, the chemistry is a Cu(I)-catalyzed or "copper-free" alkyne-azide triazole formation reaction, Staudinger ligation, inverse electron demand Diels-Alder (IEDDA) reaction, "photo-click" chemistry, or a metal-mediated process such as olefin metathesis and Suzuki-Miyaura or Sonogashira cross-coupling.

[00219] In some embodiments, the unnatural amino acid comprises a photoreactive group that crosslinks upon irradiation, such as UV.

[00220] In some embodiments, the unnatural amino acid comprises a photoshielded amino acid.

[00221] In some cases, the unnatural amino acid is para-substituted, meta-substituted or ortho-substituted amino acid derivative.

[00222] In some cases, the unnatural amino acid includes p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, O-methyl-L-tyrosine, p-methoxyphenylalanine, p-propargyloxyphenylalanine, p-propargylphenylalanine, L-3-(2-naphthyl)alanine, 3-methylphenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, p-amino-L-phenylalanine or isopropyl-L-phenylalanine.

[00223] In some cases, the unnatural amino acid is 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, or 3-iodotyrosine.

[00224] In some cases, the unnatural amino acid is phenylselenocysteine.

[00225] In some cases, the unnatural amino acid is a benzophenone-, ketone-, iodide-, methoxy-, acetyl-, benzoyl-, or azide-containing derivative of phenylalanine.

[00226] In some cases, the unnatural amino acid is a benzophenone-, ketone-, iodide-, methoxy-, acetyl-, benzoyl-, or azide-containing derivative of lysine.

[00227] In some cases, the unnatural amino acid contains an aromatic side chain.

[00228] In some cases, the unnatural amino acid does not contain an aromatic side chain.

[00229] In some cases, the unnatural amino acid contains an azido group.

[00230] In some cases, the unnatural amino acid comprises a Michael acceptor group. In some cases, the Michael acceptor groups comprise an unsaturated moiety capable of forming a covalent bond in a 1,2-addition reaction. In some cases, the Michael acceptor groups comprise electron-deficient alkenes or alkynes. In some cases, the Michael acceptor groups include, but are not limited to, alpha-, beta-unsaturated ketones, aldehydes, sulfoxides, sulfones, nitriles, imines, or aromatic compounds.

[00231] In some cases, the unnatural amino acid is dehydroalanine.

[00232] In some cases, the unnatural amino acid contains an aldehyde or ketone group.

[00233] In some cases, the unnatural amino acid is a derivative of lysine containing an aldehyde or ketone group.

[00234] In some cases, the unnatural amino acid is a derivative of lysine containing one or more O, N, Se, or S atoms at the beta, gamma, or delta position. In some cases, the unnatural amino acid is a derivative of lysine containing O, N, Se, or S atoms at the gamma position.

[00235] In some cases, the unnatural amino acid is a derivative of lysine in which the epsilon N atom is replaced by an oxygen atom.

[00236] In some cases, the unnatural amino acid is a lysine derivative that is not a naturally occurring post-translationally modified lysine.

[00237] In some cases, the unnatural amino acid is an amino acid having a side chain in which the sixth atom from the alpha position contains a carbonyl group. In some cases, the unnatural amino acid is an amino acid having a side chain in which the sixth atom from the alpha position contains a carbonyl group and the fifth atom from the alpha position is nitrogen. In some cases, the unnatural amino acid is an amino acid having a side chain in which the seventh atom from the alpha position is an oxygen atom.

[00238] In some cases, the unnatural amino acid is a serine derivative containing selenium. In some cases, the unnatural amino acid is selenoserine (2-amino-3-hydroselenopropanoic acid). In some cases, the unnatural amino acid is 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid. In some cases, the unnatural amino acid is 2-amino-3-(phenylselanyl)propanoic acid. In some cases, the unnatural amino acid contains selenium, where oxidation of the selenium results in the formation of an unnatural amino acid containing an alkene.

[00239] In some cases, the unnatural amino acid contains a cyclooctinyl group.

[00240] In some cases, the unnatural amino acid contains a transcyclooctenyl group.

[00241] In some cases, the unnatural amino acid contains a norbornenyl group.

[00242] In some cases, the unnatural amino acid contains a cyclopropenyl group.

[00243] In some cases, the unnatural amino acid contains a diazirine group.

[00244] In some cases, the unnatural amino acid contains a tetrazine group.

[00245] In some cases, the unnatural amino acid is a lysine derivative in which the side chain nitrogen is carbamylated. In some cases, the unnatural amino acid is a lysine derivative in which the side chain nitrogen is acylated. In some cases, the unnatural amino acid is 2-amino-6-{[( tert -butoxy)carbonyl]amino}hexanoic acid. In some cases, the unnatural amino acid is 2-amino-6-{[( tert -butoxy)carbonyl]amino}hexanoic acid. In some cases, the unnatural amino acid is N6-Boc-N6-methyllysine. In some cases, the unnatural amino acid is N6-acetyl lysine. In some cases, the unnatural amino acid is pyrrolysine. In some cases, the unnatural amino acid is N6-trifluoroacetyl lysine. In some cases, the unnatural amino acid is 2-amino-6-{[(benzyloxy)carbonyl]amino}hexanoic acid. In some cases, the unnatural amino acid is 2-amino-6-{[(p-iodobenzyloxy)carbonyl]amino}hexanoic acid. In some cases, the unnatural amino acid is 2-amino-6-{[(p-nitrobenzyloxy)carbonyl]amino}hexanoic acid. In some cases, the unnatural amino acid is N6-prolyl lysine. In some cases, the unnatural amino acid is 2-amino-6-{[(cyclopentyloxy)carbonyl]amino}hexanoic acid. In some cases, the unnatural amino acid is N6-(cyclopentanecarbonyl)lysine. In some cases, the unnatural amino acid is N6-(tetrahydrofuran-2-carbonyl)lysine. In some cases, the unnatural amino acid is N6-(3-ethynyltetrahydrofuran-2-carbonyl)lysine. In some cases, the unnatural amino acid is N6-((prop-2-yn-1-yloxy)carbonyl)lysine. In some cases, the unnatural amino acid is 2-amino-6-{[(2-azidocyclopentyloxy)carbonyl]amino}hexanoic acid. In some cases, the unnatural amino acid is N6-(2-azidoethoxy)-carbonyl lysine. In some cases, the unnatural amino acid is 2-amino-6-{[(2-nitrobenzyloxy)carbonyl]amino}hexanoic acid. In some cases, the unnatural amino acid is 2-amino-6-{[(2-cyclooctynyloxy)carbonyl]amino}hexanoic acid. In some cases, the unnatural amino acid is N6-(2-aminobut-3-ynoyl)lysine. In some cases, the unnatural amino acid is 2-amino-6-((2-aminobut-3-ynoyl)oxy)hexanoic acid. In some cases, the unnatural amino acid is N6-(allyloxycarbonyl)lysine. In some cases, the unnatural amino acid is N6-(butenyl-4-oxycarbonyl)lysine. In some cases, the unnatural amino acid is N6-(pentenyl-5-oxycarbonyl)lysine. In some cases, the unnatural amino acid is N6-((but-3-yn-1-yloxy)carbonyl)lysine. In some cases, the unnatural amino acid is N6-(pent-4-yn-1-yloxy)carbonyl)lysine. In some cases, the unnatural amino acid is N6-(thiazolidine-4-carbonyl)lysine. In some cases, the unnatural amino acid is 2-amino-8-oxononanoic acid. In some cases, the unnatural amino acid is 2-amino-8-oxooctanoic acid. In some cases, the unnatural amino acid is N6-(2-oxoacetyl)lysine.

[00246] In some instances, the unnatural amino acid is N6-propionyl lysine. In some instances, the unnatural amino acid is N6-butyryllysine. In some instances, the unnatural amino acid is N6-(but-2-enoyl)lysine. In some instances, the unnatural amino acid is N6-((bicyclo[2.2.1]hept-5-en-2-yloxy)carbonyl)lysine. In some instances, the unnatural amino acid is N6-((spiro[2.3]hex-1-en-5-ylmethoxy)carbonyl)lysine. In some instances, the unnatural amino acid is N6-(((4-(1-(trifluoromethyl)cycloprop-2-en-1-yl)benzyl)oxy)carbonyl)lysine. In some cases, the unnatural amino acid is N6-((bicyclo[2.2.1]hept-5-en-2-ylmethoxy)carbonyl)lysine. In some cases, the unnatural amino acid is cysteinyl lysine. In some cases, the unnatural amino acid is N6-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)lysine. In some cases, the unnatural amino acid is N6-((2-(3-methyl-3H-diazirin-3-yl)ethoxy)carbonyl)lysine. In some cases, the unnatural amino acid is N6-((3-(3-methyl-3H-diazirin-3-yl)propoxy)carbonyl)lysine. In some cases, the unnatural amino acid is N6-((meta-nitrobenzyloxy)-N6-methylcarbonyl)lysine. In some cases, the unnatural amino acid is N6-((bicyclo[6.1.0]non-4-yn-9-ylmethoxy)carbonyl)lysine. In some cases, the unnatural amino acid is N6-((cyclohept-3-en-1-yloxy)carbonyl)-L-lysine.

[00247] In some cases, the unnatural amino acid is 2-amino-3-(((((benzyloxy)carbonyl)amino)methyl)selanyl)propanoic acid.

[00248] In some embodiments, an unnatural amino acid is inserted into a cytokine (e.g., an IL polypeptide) via a repurposed amber stop codon, opal stop codon, or ochre stop codon.

[00249] In some embodiments, the unnatural amino acid is incorporated into the cytokine (e.g., IL polypeptide) using a 4-nucleotide codon.

[00250] In some embodiments, an unnatural amino acid is inserted into a cytokine (e.g., an IL polypeptide) using a repurposed rare sense codon.

[00251] In some embodiments, a non-natural amino acid is incorporated into a cytokine (e.g., an IL polypeptide) using a synthetic codon containing a non-natural nucleic acid.

In some cases, the unnatural amino acid is incorporated into the cytokine using an orthogonal modified synthetase/tRNA pair. Such orthogonal pairs comprise a unnatural synthetase that is capable of loading a unnatural tRNA with the unnatural amino acid while minimizing the loading of a) other endogenous amino acids onto the unnatural tRNA and b) unnatural amino acids onto other endogenous tRNAs. Such orthogonal pairs comprise tRNAs that are capable of being loaded by the unnatural synthetase while avoiding the loading of a) other endogenous amino acids by endogenous synthetases. In some embodiments, such pairs have been identified in different organisms, such as bacteria, yeast, archaea, or humans. In some embodiments, an orthogonal synthetase/tRNA pair comprises components from a single organism. In some embodiments, an orthogonal synthetase/tRNA pair comprises components from two different organisms. In some embodiments, the orthogonal synthetase/tRNA pair comprises components that, prior to modification, facilitate the translation of two different amino acids. In some embodiments, the orthogonal synthetase is a modified alanine synthetase. In some embodiments, the orthogonal synthetase is a modified arginine synthetase. In some embodiments, the orthogonal synthetase is a modified asparagine synthetase. In some embodiments, the orthogonal synthetase is a modified aspartic acid synthetase. In some embodiments, the orthogonal synthetase is a modified cysteine synthetase. In some embodiments, the orthogonal synthetase is a modified glutamine synthetase. In some embodiments, the orthogonal synthetase is a modified glutamic acid synthetase. In some embodiments, the orthogonal synthetase is a modified alanine glycine. In some embodiments, the orthogonal synthetase is a modified histidine synthetase. In some embodiments, the orthogonal synthetase is a modified leucine synthetase. In some embodiments, the orthogonal synthetase is a modified isoleucine synthetase. In some embodiments, the orthogonal synthetase is a modified lysine synthetase. In some embodiments, the orthogonal synthetase is a modified methionine synthetase. In some embodiments, the orthogonal synthetase is a modified phenylalanine synthetase. In some embodiments, the orthogonal synthetase is a modified proline synthetase. In some embodiments, the orthogonal synthetase is a modified serine synthetase. In some embodiments, the orthogonal synthetase is a modified threonine synthetase. In some embodiments, the orthogonal synthetase is a modified tryptophan synthetase. In some embodiments, the orthogonal synthetase is a modified tyrosine synthetase. In some embodiments, the orthogonal synthetase is a modified valine synthetase. In some embodiments, the orthogonal synthetase is a modified phosphoserine synthetase. In some embodiments, the orthogonal tRNA is a modified alanine tRNA. In some embodiments, the orthogonal tRNA is a modified arginine tRNA. In some embodiments, the orthogonal tRNA is a modified asparagine tRNA. In some embodiments, the orthogonal tRNA is a modified aspartic acid tRNA. In some embodiments, the orthogonal tRNA is a modified cysteine tRNA. In some embodiments, the orthogonal tRNA is a modified glutamine tRNA. In some embodiments, the orthogonal tRNA is a modified glutamic acid tRNA. In some embodiments, the orthogonal tRNA is a modified alanine glycine. In some embodiments, the orthogonal tRNA is a modified histidine tRNA. In some embodiments, the orthogonal tRNA is a modified leucine tRNA. In some embodiments, the orthogonal tRNA is a modified isoleucine tRNA. In some embodiments, the orthogonal tRNA is a modified lysine tRNA. In some embodiments, the orthogonal tRNA is a modified methionine tRNA. In some embodiments, the orthogonal tRNA is a modified phenylalanine tRNA. In some embodiments, the orthogonal tRNA is a modified proline tRNA. In some embodiments, the orthogonal tRNA is a modified serine tRNA. In some embodiments, the orthogonal tRNA is a modified threonine tRNA. In some embodiments, the orthogonal tRNA is a modified tryptophan tRNA. In some embodiments, the orthogonal tRNA is a modified tyrosine tRNA. In some embodiments, the orthogonal tRNA is a modified valine tRNA. In some embodiments, the orthogonal tRNA is a modified phosphoserine tRNA.

[00252] In some embodiments, a non-natural amino acid is incorporated into a cytokine (e.g., an IL polypeptide) via an aminoacyl (aaRS or RS)-tRNA synthetase-tRNA pair. Exemplary aaRS-tRNA pairs include, but are not limited to, aaRS/tRNA pairsMethanococcus jannaschii(Mj-Tyr), TyrRS pairsE. coli(Ec-Tyr)/tRNA_CUA B. stearothermophilus, LeuRS pairs (Ec-Leu)E. coli/tRNA_CUA B. stearothermophilusand pyrrolizyl-tRNA pairs. In some cases, an unnatural amino acid is incorporated into a cytokine (e.g., the IL polypeptide) using an RS/tRNA pair.Mj-Tyr. Illustrative UAAs that can be inserted using an RS/tRNA pairMj-Tyr, include, but are not limited to, para-substituted phenylalanine derivatives such as p-aminophenylalanine and p-methoxyphenylalanine; meta-substituted tyrosine derivatives such as 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine and 3-iodotyrosine; phenylselenocysteine; p-boronphenylalanine and o-nitrobenzyltyrosine.

[00253] In some cases, an unnatural amino acid is incorporated into a cytokine (e.g., an IL polypeptide) via an Ec-Tyr /tRNA _CUA or an Ec-Leu /tRNA _CUA pair. Exemplary UAAs that can be incorporated via an Ec-Tyr /tRNA _CUA or an Ec-Leu /tRNA _CUA pair include, but are not limited to, phenylalanine derivatives containing benzophenone, ketone, iodide, or azide substituents; O -propargyl tyrosine; α-aminocaprylic acid, O-methyl tyrosine, O-nitrobenzyl cysteine, and 3-(naphthalen-2-ylamino)-2-aminopropanoic acid.

[00254] In some cases, an unnatural amino acid is inserted into a cytokine (e.g., an IL polypeptide) via a pyrrolyl-tRNA pair. In some cases, the PylRS is derived from an archaebacterium, such as a methanogenic archaebacterium. In some cases, the PylRS is derived from Methanosarcina barkeri , Methanosarcina mazei , or Methanosarcina acetivorans . Exemplary UAAs that can be inserted via a pyrrolyl-tRNA pair include, but are not limited to, amide- and carbamate-substituted lysines such as 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid, N -ε- _D -prolyl- _L -lysine, and N -ε-cyclopentyloxycarbonyl _-L -lysine; N -ε-acryloyl- _L -lysine; N -ε-[(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl] _-L- lysine and N -ε-(1-methylcyclopro-2-enecarboxamido)lysine. In some embodiments, the IL-2 conjugates disclosed herein can be prepared by using M. mazei Pyl tRNA that is selectively loaded with a non-natural amino acid, such as N 6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), using M. barkeri pyrrolizyl-tRNA synthetase ( Mb PylRS). Other methods are known to those skilled in the art, such as the methods disclosed in Zhang et al., Nature 2017, 551(7682): 644-647.

[00255] In some cases, a non-natural amino acid is incorporated into a cytokine described herein (e.g., an IL polypeptide) using a synthetase disclosed in U.S. Pat. No. 9,988,619 and U.S. Pat. No. 9,938,516. Exemplary UAAs that can be incorporated using such synthetases include para-methylazido-L-phenylalanine, aralkyl, heterocyclyl, heteroaralkyl substituted non-natural amino acids, and others. In some embodiments, such UAAs include pyridyl, pyrazinyl, pyrazolyl, triazolyl, oxazolyl, thiazolyl, thiophenyl, or another heterocycle. Such amino acids, in some embodiments, include azides, tetrazines, or another chemical moiety capable of conjugation to a coupling partner, such as a water-soluble moiety. In some embodiments, such synthetases are expressed and used to incorporate UAAs into cytokines in vivo. In some embodiments, such synthetases are used to incorporate UAA into cytokines using a cell-free translation system.

[00256] In some cases, an unnatural amino acid is incorporated into a cytokine described herein (e.g., an IL polypeptide) by a naturally occurring synthetase. In some embodiments, an unnatural amino acid is incorporated into a cytokine by an organism that is auxotrophic for one or more amino acids. In some embodiments, synthetases corresponding to the auxotrophic amino acid are capable of loading a corresponding tRNA with the unnatural amino acid. In some embodiments, the unnatural amino acid is selenocysteine or a derivative thereof. In some embodiments, the unnatural amino acid is selenomethionine or a derivative thereof. In some embodiments, the unnatural amino acid is an aromatic amino acid, wherein the aromatic amino acid includes an aryl halide, such as iodide. In embodiments, the unnatural amino acid is structurally similar to the auxotrophic amino acid.

[00257] In some cases, the unnatural amino acid includes the unnatural amino acid shown in Fig. 1.

[00258] In some cases, the unnatural amino acid includes a derivative or analog of lysine or phenylalanine. In some cases, the unnatural amino acid includes a derivative of lysine or an analog of lysine. In some cases, the unnatural amino acid includes pyrrolysine (Pyl). In some cases, the unnatural amino acid includes a derivative of phenylalanine or an analog of phenylalanine. In some cases, the unnatural amino acid is the unnatural amino acid described in Wan, et al., "Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool," Biochem Biophys Aceta 1844(6): 1059-4070 (2014). In some cases, the unnatural amino acid includes the unnatural amino acid shown in Fig. 2 (e.g., Fig. 2A and Fig. 2B).

[00259] In some embodiments, the unnatural amino acid comprises the unnatural amino acid shown in Fig. 3A - Fig. 3D (adapted from Table 1 of Dumas et al ., Chemical Science 2015, 6 , 50-69).

[00260] In some embodiments, a non-natural amino acid incorporated into a cytokine described herein (e.g., an IL polypeptide) is disclosed in US 9,840,493, US 9,682,934, US 2017/0260137, US 9,938,516, or US 2018/0086734. Exemplary UAAs that can be incorporated by such synthetases include para-methylazido-L-phenylalanine, aralkyl, heterocyclyl, and heteroaralkyl, as well as non-natural amino acids that are lysine derivatives. In some embodiments, such UAAs include pyridyl, pyrazinyl, pyrazolyl, triazolyl, oxazolyl, thiazolyl, thiophenyl, or another heterocycle. Such amino acids, in some embodiments, include azides, tetrazines, or another chemical moiety capable of conjugation with a coupling partner, such as a water-soluble moiety. In some embodiments, the UAA includes an azide attached to an aromatic moiety via an alkyl linker. In some embodiments, the alkyl linker is a C ₁ -C ₁₀ linker. In some embodiments, the UAA includes a tetrazine attached to an aromatic moiety via an alkyl linker. In some embodiments, the UAA includes a tetrazine attached to an aromatic moiety via an amino group. In some embodiments, the UAA includes a tetrazine attached to an aromatic moiety via an alkylamino group. In some embodiments, the UAA includes an azide attached to a terminal nitrogen (e.g., N6 from a lysine derivative, or N5, N4, or N3 from a derivative containing a shorter alkyl side chain) of an amino acid side chain via an alkyl chain. In some embodiments, the UAA comprises a tetrazine attached to the terminal nitrogen of an amino acid side chain via an alkyl chain. In some embodiments, the UAA comprises an azide or tetrazine attached to an amide via an alkyl linker. In some embodiments, the UAA is an azide- or tetrazine-containing carbamate or amide of 3-aminoalanine, serine, lysine, or a derivative thereof. In some embodiments, such UAAs are incorporated into cytokines in vivo. In some embodiments, such UAAs are incorporated into cytokines in a cell-free system.

Conjugating fragments

[00261] In certain embodiments, disclosed herein are conjugation moieties that are linked to one or more cytokines (e.g., interleukins, IFN, or TNF) described above. In some cases, the conjugating moiety is a molecule that disrupts the interaction of a cytokine with its receptor. In some cases, the conjugating moiety is any molecule that, when bound to a cytokine, allows the cytokine conjugate to modulate an immune response. In some cases, the conjugating moiety is linked to the cytokine via a covalent bond. In some cases, a cytokine described herein is attached to the conjugating moiety via a triazole group. In some cases, a cytokine described herein is attached to the conjugating moiety via a dihydropyridazine or pyridazine group. In some cases, the conjugating moiety includes a water-soluble polymer. In other cases, the conjugating moiety includes a protein or a binding fragment thereof. In further cases, the conjugating moiety includes a peptide. In further cases, the conjugating moiety includes a nucleic acid. In further cases, the conjugating moiety includes a small molecule. In additional cases, the conjugate moiety comprises a bioconjugate (e.g., a TLR agonist such as a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 agonist; or a synthetic ligand such as Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, poly I:C, poly A:U, AGP, MPL A, RC-529, MDF2β, CFA, or flagellin). In some cases, the conjugate moiety increases serum half-life and/or improves stability. In some cases, the conjugate moiety reduces the interaction of the cytokine with one or more domains or subunits of the cytokine receptor. In additional instances, the conjugate moiety blocks the interaction of the cytokine with one or more domains or subunits of its cognate cytokine receptor(s). In some embodiments, the cytokine conjugates described herein comprise multiple conjugate moieties. In some embodiments, the conjugate moiety is attached to a non-natural or natural amino acid in the cytokine peptide. In some embodiments, the cytokine conjugate comprises a conjugate moiety attached to a natural amino acid. In some embodiments, the cytokine conjugate is attached to a non-natural amino acid in the cytokine peptide. In some embodiments, the conjugate moiety is attached to the N- or C-terminal amino acid of the cytokine peptide. Various combinations of sites are disclosed herein, such as a first conjugation moiety attached to a non-natural or natural amino acid in a cytokine peptide, and a second conjugation moiety attached to the N- or C-terminal amino acid of the cytokine peptide. In some embodiments, one conjugation moiety is attached to multiple residues of the cytokine peptide (e.g., a loop). In some embodiments, the conjugation moiety is attached to both the N- and C-terminal amino acid of the cytokine peptide.

Water-soluble polymers

[00262] In some embodiments, the conjugate moiety described herein is a water-soluble polymer. In some cases, the water-soluble polymer is non-peptide, non-toxic, and biocompatible. As used herein, a substance is considered biocompatible if the beneficial effects associated with the use of the substance alone or with another substance (e.g., an active agent, such as a cytokine fragment) in combination with living tissue (e.g., when administered to a patient) outweigh any detrimental effects, as assessed by a clinician, such as a physician, toxicologist, or clinical development specialist. Additionally, in some cases, the water-soluble polymer is non-immunogenic. In some cases, a substance is considered non-immunogenic if the intended use of the substance in vivo does not elicit an adverse immune response (e.g., antibody formation), or, if an immune response occurs, such response is not considered clinically significant or important, as assessed by a clinician, such as a physician, toxicologist, or clinical development specialist.

[00263] In some cases, the water-soluble polymer is characterized by having from about 2 to about 300 ends. Illustrative water-soluble polymers include, but are not limited to, poly(alkylene glycols) such as polyethylene glycol ("PEG"), poly(propylene glycol) ("PPG"), copolymers of ethylene glycol and propylene glycol, and the like, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polymethylacrylamide (PDAAm), polyphosphazene, polyoxazolines ("POZ") (as described in WO 2008/106186), poly(N-acryloylmorpholine), and combinations of any of the foregoing.

[00264] In some cases, the water-soluble polymer is not limited to a specific structure. In some cases, the water-soluble polymer is linear (e.g., end-capped, such as alkoxy-PEG or bifunctional PEG), branched or multi-armed (e.g., bifurcated PEG or PEG attached to a polyol core), characterized by a dendritic (or star) structure, each with or without one or more degradable bonds. Moreover, the internal structure of the water-soluble polymer can be organized in any number of different repeating patterns and can be selected from the group consisting of a homopolymer, an alternating copolymer, a random copolymer, a block copolymer, an alternating tripolymer, a random tripolymer, and a block tripolymer.

[00265] In some cases, the water-soluble polymer is represented by the length of the repeating polymer units, for example, the number n of polyethylene glycol units. In some cases, the water-soluble polymer has the structure

, where the wavy line indicates attachment to a linker, reactive group, or unnatural amino acid, and n is 1-5000. In some cases, the water-soluble polymer has the structure

, where the wavy line indicates attachment to a linker, reactive group, or unnatural amino acid, "Cap" is a capping group (such as, for example, _-OCH3 , -O( _C1 - _C6 alkyl), -SMe, -S( _C1 - _C6 alkyl), -CO2H, _-CO2 ( _C1 - _C6 alkyl), _-CONH2 , _-CONH ( _C1 - _C6 alkyl), -CON( _C1 - _C6 alkyl) ₂ , _-NH2 , -SH, or OH), and n is 1-5000. In some embodiments, n is 100-2000, 200-1000, 300-750, 400-600, 450-550, 400-2000, 750-3000, or 100-750. In some embodiments, n is about 100, 200, 300, 400, 500, 600, 700, 800, 900, or about 1000. In some embodiments, n is at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or at least 1000. In some embodiments, n is no more than 100, 200, 300, 400, 500, 600, 700, 800, 900, or no more than 1000. In some embodiments, n is represented as the average length of the water-soluble polymer.

[00266] In some embodiments, the weight average molecular weight of the water-soluble polymer in the IL-2 conjugate is from about 100 Daltons to about 150,000 Daltons. Illustrative ranges include, for example, weight average molecular weights in the range of greater than 5,000 Daltons to about 100,000 Daltons, in the range of about 6,000 Daltons to about 90,000 Daltons, in the range of about 10,000 Daltons to about 85,000 Daltons, in the range of greater than 10,000 Daltons to about 85,000 Daltons, in the range of about 20,000 Daltons to about 85,000 Daltons, in the range of about 53,000 Daltons to about 85,000 Daltons, in the range of about 25,000 Daltons to about 120,000 Daltons, in the range of about 29,000 Daltons to about 120,000 Daltons, in the range of about 35,000 Daltons to about 120,000 daltons and in the range from approximately 40,000 daltons to approximately 120,000 daltons.

[00267] Exemplary weight average molecular weights of the water-soluble polymer include values of about 100 Daltons, about 200 Daltons, about 300 Daltons, about 400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons, about 900 Daltons, about 1000 Daltons, about 1500 Daltons, about 2000 Daltons, about 2200 Daltons, about 2500 Daltons, about 3000 Daltons, about 4000 Daltons, about 4400 Daltons, about 4500 Daltons, about 5000 Daltons, about 5500 Daltons, about 6000 Daltons, about 7,000 daltons, approximately 7,500 daltons, approximately 8,000 daltons, approximately 9,000 daltons, approximately 10,000 daltons, approximately 11,000 daltons, approximately 12,000 daltons, approximately 13,000 daltons, approximately 14,000 daltons, approximately 15,000 daltons, approximately 20,000 daltons, approximately 22,500 daltons, approximately 25,000 daltons, approximately 30,000 daltons, approximately 35,000 daltons, approximately 40,000 daltons, approximately 45,000 daltons, approximately 50,000 daltons, approximately 55,000 daltons, approximately 60,000 daltons, approximately 65,000 daltons, approximately 70,000 daltons and approximately 75,000 daltons. Branched versions of the water-soluble polymer (e.g., a branched water-soluble polymer with a molecular weight of 40,000 daltons, consisting of two polymers with a molecular weight of 20,000 daltons) having a combined molecular weight of any of the above can also be used. In one or more embodiments, the conjugate will not contain any PEG moieties attached, directly or indirectly, to the PEG having a weight average molecular weight of less than about 6,000 daltons.

[00268] Typically, PEGs will contain a number of ( _{OCH2CH2 )} monomers [or ( _CH2CH2O ) _monomers , depending on how the PEG is defined]. As used herein, the number of repeating units is denoted by the subscript " _n " in "( _OCH2CH2 ) _n ". Thus, the value (n) typically falls within one or more of the following ranges: from 2 to about 3400, from about 100 to about 2300, from about 100 to about 2270, from about 136 to about 2050, from about 225 to about 1930, from about 450 to about 1930, from about 1200 to about 1930, from about 568 to about 2727, from about 660 to about 2730, from about 795 to about 2730, from about 795 to about 2730, from about 909 to about 2730, and from about 1200 to about 1900. For any given polymer for which the molecular weight is known, the number of repeating units (i.e., "n") can be determined by dividing the total weight average molecular weight polymer per molecular weight of the repeating monomer.

[00269] In some embodiments, the water-soluble polymer is an end-capped polymer, i.e., a polymer having at least one end capped with a relatively inert group, such as a lower C _1-6 alkoxy group or a hydroxyl group. When the polymer is PEG, for example, methoxy-PEG (commonly referred to as mPEG), which is a linear form of PEG, wherein one end of the polymer is a methoxy(-OCH ₃ ) group, while the other end is a hydroxyl or other functional group, which may optionally be chemically modified, can be used.

[00270] In some embodiments, the PEG moiety contained in the IL-2 conjugates disclosed herein is a linear or branched PEG moiety. In some embodiments, the PEG moiety is a linear PEG moiety. In some embodiments, the PEG moiety is a branched PEG moiety. In some embodiments, the PEG moiety is a methoxy-PEG moiety. In some embodiments, the PEG moiety is a linear or branched methoxy-PEG moiety. In some embodiments, the PEG moiety is a linear methoxy-PEG moiety. In some embodiments, the PEG moiety is a branched methoxy-PEG moiety. In some embodiments, the PEG moiety is a linear or branched PEG moiety with an average molecular weight of from about 100 Daltons to about 150,000 Daltons. Illustrative ranges include, for example, weight average molecular weights in the range of greater than 5,000 Daltons to about 100,000 Daltons, in the range of about 6,000 Daltons to about 90,000 Daltons, in the range of about 10,000 Daltons to about 85,000 Daltons, in the range of greater than 10,000 Daltons to about 85,000 Daltons, in the range of about 20,000 Daltons to about 85,000 Daltons, in the range of about 53,000 Daltons to about 85,000 Daltons, in the range of about 25,000 Daltons to about 120,000 Daltons, in the range of about 29,000 Daltons to about 120,000 Daltons, in the range of about 35,000 Daltons to about 120,000 daltons and in the range from approximately 40,000 daltons to approximately 120,000 daltons. Illustrative weight average molecular weights for the PEG group include values of about 100 Daltons, about 200 Daltons, about 300 Daltons, about 400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons, about 900 Daltons, about 1000 Daltons, about 1500 Daltons, about 2000 Daltons, about 2200 Daltons, about 2500 Daltons, about 3000 Daltons, about 4000 Daltons, about 4400 Daltons, about 4500 Daltons, about 5000 Daltons, about 5500 Daltons, about 6000 Daltons, about 7000 dalton, approximately 7,500 dalton, approximately 8,000 dalton, approximately 9,000 dalton, approximately 10,000 dalton, approximately 11,000 dalton, approximately 12,000 dalton, approximately 13,000 dalton, approximately 14,000 dalton, approximately 15,000 dalton, approximately 20,000 dalton, approximately 22,500 dalton, approximately 25,000 dalton, approximately 30,000 dalton, approximately 35,000 dalton, approximately 40,000 dalton, approximately 45,000 dalton, approximately 50,000 dalton, approximately 55,000 dalton, approximately 60,000 dalton, approximately 65,000 dalton, approximately 70,000 dalton, approximately 75,000 dalton, approximately 80,000 daltons, about 90,000 daltons, about 95,000 daltons, and about 100,000 daltons. In some embodiments, the PEG moiety is a linear PEG moiety with an average molecular weight as disclosed above. In some embodiments, the PEG moiety is a branched PEG moiety with an average molecular weight as disclosed above. In some embodiments, the PEG moiety contained in the IL-2 conjugates disclosed herein is a linear or branched PEG moiety with a specific molecular weight of ±10%, or 15%, or 20%, or 25%. For example, included within the scope of the present invention are IL-2 conjugates comprising a PEG group having a molecular weight of 30,000 Da ± 3,000 Da, or 30,000 Da ± 4,500 Da, or 30,000 Da ± 6,000 Da.

[00271] In some embodiments, the PEG moiety contained in the IL-2 conjugates disclosed herein is a linear or branched PEG moiety with an average molecular weight of from about 5,000 Daltons to about 60,000 Daltons. In some embodiments, the PEG moiety is a linear or branched PEG moiety with an average molecular weight of about 5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000 daltons, about 55,000 daltons, about 60,000 daltons, about 65,000 daltons, about 70,000 daltons, about 75,000 daltons, about 80,000 daltons, about 90,000 daltons, about 95,000 daltons, and about 100,000 daltons. In some embodiments, the PEG moiety is a linear or branched PEG moiety with an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 20,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons. In some embodiments, the PEG group is a linear or branched PEG group with an average molecular weight of about 5,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is a linear PEG group with an average molecular weight of about 5,000 Daltons, about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is a branched PEG group with an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 20,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons.

[00272] In some embodiments, the PEG moiety contained in the IL-2 conjugates disclosed herein is a linear methoxy-PEG moiety with an average molecular weight of from about 5,000 Daltons to about 60,000 Daltons. In some embodiments, the PEG moiety is a linear methoxy-PEG moiety with an average molecular weight of about 5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000 daltons, about 55,000 daltons, about 60,000 daltons, about 65,000 daltons, about 70,000 daltons, about 75,000 daltons, about 80,000 daltons, about 90,000 daltons, about 95,000 daltons, and about 100,000 daltons. In some embodiments, the PEG group is a linear methoxy-PEG group with an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 20,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons. In some embodiments, the PEG group is a linear methoxy-PEG group with an average molecular weight of about 5,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is a linear methoxy-PEG group with an average molecular weight of about 5,000 Daltons, about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is a linear methoxy-PEG group with an average molecular weight of about 5,000 Daltons. In some embodiments, the PEG group is a linear methoxy-PEG group with an average molecular weight of about 10,000 Daltons. In some embodiments, the PEG moiety is a linear methoxy-PEG moiety with an average molecular weight of about 20,000 Daltons. In some embodiments, the PEG moiety is a linear methoxy-PEG moiety with an average molecular weight of about 30,000 Daltons. In some embodiments, the PEG moiety is a linear methoxy-PEG moiety with an average molecular weight of about 50,000 Daltons. In some embodiments, the PEG moiety is a linear methoxy-PEG moiety with an average molecular weight of about 60,000 Daltons. In some embodiments, the PEG moiety contained in the IL-2 conjugates disclosed herein is a linear methoxy-PEG moiety with a specific molecular weight of ±10%, or 15%, or 20%, or 25%. For example, included within the scope of the present invention are IL-2 conjugates comprising a linear methoxy-PEG group having a molecular weight of 30,000 Da ± 3,000 Da, or 30,000 Da ± 4,500 Da, or 30,000 Da ± 6,000 Da.

[00273] In some embodiments, the PEG moiety contained in the IL-2 conjugates disclosed herein is a branched methoxy-PEG moiety with an average molecular weight of from about 5,000 Daltons to about 60,000 Daltons. In some embodiments, the PEG moiety is a branched methoxy-PEG moiety with an average molecular weight of about 5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000 daltons, about 55,000 daltons, about 60,000 daltons, about 65,000 daltons, about 70,000 daltons, about 75,000 daltons, about 80,000 daltons, about 90,000 daltons, about 95,000 daltons, and about 100,000 daltons. In some embodiments, the PEG group is a branched methoxy-PEG group with an average molecular weight of about 5,000 daltons, about 10,000 daltons, about 20,000 daltons, about 30,000 daltons, about 50,000 daltons, or about 60,000 daltons. In some embodiments, the PEG group is a branched methoxy-PEG group with an average molecular weight of about 5,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is a branched methoxy-PEG group with an average molecular weight of about 5,000 Daltons, about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is a branched methoxy-PEG group with an average molecular weight of about 5,000 Daltons, about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is a branched methoxy-PEG group with an average molecular weight of about 5,000 Daltons. In some embodiments, the PEG group is a branched methoxy-PEG group with an average molecular weight of about 10,000 Daltons. In some embodiments, the PEG group is a branched methoxy-PEG group with an average molecular weight of about 20,000 Daltons. In some embodiments, the PEG group is a branched methoxy-PEG group with an average molecular weight of about 30,000 Daltons. In some embodiments, the PEG moiety is a branched methoxy-PEG moiety with an average molecular weight of about 50,000 daltons. In some embodiments, the PEG moiety is a branched methoxy-PEG moiety with an average molecular weight of about 60,000 daltons. In some embodiments, the PEG moiety contained in the IL-2 conjugates disclosed herein is a branched methoxy-PEG moiety with a particular molecular weight of ±10%, or 15%, or 20%, or 25%. For example, included within the scope of the present invention are IL-2 conjugates comprising a branched methoxy-PEG moiety having a molecular weight of 30,000 Da ±3,000 Da, or 30,000 Da ±4,500 Da, or 30,000 Da ±6,000 Da.

[00274] In some embodiments, exemplary water-soluble polymers include, but are not limited to, linear or branched discrete PEG (dPEG) from Quanta Biodesign, Ltd, linear, branched, or bifurcated PEG from Nektar Therapeutics, and Y-derivatized PEG from JenKem Technology.

[00275] In some embodiments, a cytokine polypeptide (e.g., interleukin, IFN, or TNF) described herein is conjugated to a water-soluble polymer selected from poly(alkylene glycols), such as polyethylene glycol ("PEG"), poly(propylene glycol) ("PPG"), a copolymer of ethylene glycol and propylene glycol, and the like, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), polymethylacrylamide (PDAAm), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polyphosphazene, polyoxazolines ("POZ"), poly(N-acryloylmorpholine), and combinations thereof. In some cases, the cytokine polypeptide is conjugated to PEG (e.g., pegylated). In some cases, the cytokine polypeptide is conjugated to PPG. In some cases, the cytokine polypeptide is conjugated to POZ. In some cases, the cytokine polypeptide is conjugated to PVP.

[00276] In some embodiments, the IL-2 polypeptide described herein is conjugated to a water-soluble polymer selected from poly(alkylene glycols) such as polyethylene glycol ("PEG"), poly(propylene glycol) ("PPG"), copolymers of ethylene glycol and propylene glycol, and the like, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), polymethylacrylamide (PDAAm), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polyphosphazene, polyoxazolines ("POZ"), poly(N-acryloylmorpholine), and combinations thereof. In some cases, the IL-2 polypeptide is conjugated to PEG (e.g., pegylated). In some cases, the IL-2 polypeptide is conjugated to PPG. In some cases, the IL-2 polypeptide is conjugated to POZ. In some cases, the IL-2 polypeptide is conjugated to PVP.

[00277] In some cases, the water-soluble polymer comprises polyglycerol (PG). In some cases, the polyglycerol is hyperbranched PG (HPG) (e.g., as described in Imran, et al. "Influence of architecture of high molecular weight linear and branched polyglycerols on their biocompatibility and biodistribution," Biomaterials 33 :9135-9147 (2012)). In other cases, the polyglycerol is linear PG (LPG). In additional cases, the polyglycerol is a semi-functional PG, a linear-block-hyperbranched PG (e.g., as described in Wurm et. Al., "Squaric acid mediated synthesis and biological activity of a library of linear and hyperbranched poly(glycerol)-protein conjugates," Biomacromolecules 13 :1161–1171 (2012)) or a PG with functional side chains (e.g., as described in Li, et. al., "Synthesis of linear polyether polyol derivatives as new materials for bioconjugation," Bioconjugate Chem . 20 :780–789 (2009).

[00278] In some cases, a cytokine polypeptide (e.g., interleukin, IFN, or TNF) described herein is conjugated to a PG, such as HPG, LPG, semi-functional PG, linear-block-hyperbranched PG, or PG with functional side chains. In some cases, the cytokine is an IL-2 polypeptide. In some cases, the IL-2 polypeptide is conjugated to a PG, semi-functional PG, linear-block-hyperbranched PG.

[00279] In some embodiments, the water-soluble polymer is a degradable synthetic alternative to PEG. Exemplary degradable synthetic alternatives to PEG include, but are not limited to, poly[oligo(ethylene glycol)methyl methacrylate] (POEGMA); backbone-modified PEG derivatives prepared by polymerizing telechelated or double-end-functionalized PEG-based macromonomers; PEG derivatives comprising comonomers containing a degradable bond such as [(ethylene oxide)-(methylene ethylene oxide)]copolymer[P(EO- co -MEO)], cyclic ketene acetals such as 5,6-benzo-2-methylene-1,3-dioxepane (BMDO), 2-methylene-1,3-dioxopane (MDO), and 2-methylene-4-phenyl-1,3-dioxolane (MPDL) copolymerized with OEGMA; or poly(ethylene oxide) grafted with poly(ε-caprolactone) (PCL-g-PEO).

[00280] In some cases, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide described herein is conjugated to a degradable synthetic alternative PEG, such as, for example, POEGM; backbone-modified PEG derivatives prepared by polymerization of telechelated or double-terminated PEG-based macromonomers; P(EO- co -MEO); cyclic ketene acetals such as BMDO, MDO, and MPDL copolymerized with OEGMA; or PCL-g-PEO. In some cases, the cytokine is an IL-2 polypeptide. In some cases, the IL-2 polypeptide is conjugated to a degradable synthetic alternative PEG, such as, for example, POEGM; backbone-modified PEG derivatives prepared by polymerization of telechelated or double-terminated PEG-based macromonomers; P(EO- co -MEO); cyclic ketene acetals such as BMDO, MDO and MPDL copolymerized with OEGMA; or PCL-g-PEO.

[00281] In some embodiments, the water-soluble polymer comprises poly(zwitterions). Exemplary poly(zwitterions) include, but are not limited to, poly(sulfobetaine methacrylate) (PSBMA), poly(carboxybetaine methacrylate) (PCBMA), and poly(2-methacryloyloxyethylphosphorylcholine) (PMPC). In some cases, a cytokine polypeptide (e.g., interleukin, IFN, or TNF) described herein is conjugated to a poly(zwitterion), such as PSBMA, PCBMA, or PMPC. In some cases, the cytokine is an IL-2 polypeptide. In some cases, the IL-2 polypeptide is conjugated to a poly(zwitterion), such as PSBMA, PCBMA, or PMPC.

[00282] In some embodiments, the water-soluble polymer comprises a polycarbonate. Exemplary polycarbonates include, but are not limited to, pentafluorophenyl 5-methyl-2-oxo-1,3-dioxane-5-carboxylate (MTC-OC ₆ F ₅ ). In some cases, a cytokine polypeptide (e.g., interleukin, IFN, or TNF) described herein is conjugated to a polycarbonate, such as MTC-OC ₆ F ₅ . In some cases, the cytokine is an IL-2 polypeptide. In some cases, the IL-2 polypeptide is conjugated to a polycarbonate, such as MTC-OC ₆ F ₅ .

[00283] In some embodiments, the water-soluble polymer comprises a polymer hybrid, such as, for example, a polycarbonate/PEG polymer hybrid, a peptide/protein polymer conjugate, or a hydroxyl-containing and/or zwitterionic derivatized polymer (e.g., a hydroxyl-containing and/or zwitterionic derivatized PEG polymer). In some cases, the cytokine (e.g., interleukin, IFN, or TNF) polypeptide described herein is conjugated to a polymer hybrid, such as a polycarbonate/PEG polymer hybrid, a peptide/protein-polymer conjugate, or a hydroxyl-containing and/or zwitterionic derivatized polymer (e.g., a hydroxyl-containing and/or zwitterionic derivatized PEG polymer). In some cases, the cytokine is an IL-2 polypeptide. In some cases, the IL-2 polypeptide is conjugated to a polymer hybrid, such as a polycarbonate/PEG polymer hybrid, a peptide/protein-polymer conjugate, or a hydroxyl-containing and/or zwitterionic derivatized polymer (e.g., a hydroxyl-containing and/or zwitterionic derivatized PEG polymer).

[00284] In some cases, the water-soluble polymer comprises a polysaccharide. Exemplary polysaccharides include, but are not limited to, dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some cases, a cytokine polypeptide (e.g., interleukin, IFN, or TNF) is conjugated to a polysaccharide. In some cases, an IL-2 polypeptide is conjugated to dextran. In some cases, an IL-2 polypeptide is conjugated to PSA. In some cases, an IL-2 polypeptide is conjugated to HA. In some cases, an IL-2 polypeptide is conjugated to amylose. In some cases, an IL-2 polypeptide is conjugated to heparin. In some cases, an IL-2 polypeptide is conjugated to HS. In some cases, the IL-2 polypeptide is conjugated to dextrin. In some cases, the IL-2 polypeptide is conjugated to HES.

[00285] In some cases, the water-soluble polymer includes a glycan. Exemplary classes of glycans include N-linked glycans, O-linked glycans, glycolipids, O-GlcNAc, and glycosaminoglycans. In some cases, a cytokine polypeptide (e.g., interleukin, IFN, or TNF) is conjugated to a glycan. In some cases, an IL-2 polypeptide is conjugated toN-linked glycans. In some cases, the IL-2 polypeptide is conjugated toO-linked glycans. In some cases, the IL-2 polypeptide is conjugated to glycolipids. In some cases, the IL-2 polypeptide is conjugated to O-GlcNAc. In some cases, the IL-2 polypeptide is conjugated to glycosaminoglycans.

[00286] In some embodiments, the water-soluble polymer comprises a polyoxazoline polymer. The polyoxazoline polymer is a linear synthetic polymer and, like PEG, has low polydispersity. In some cases, the polyoxazoline polymer is a polydisperse polyoxazoline polymer having an average molecular weight. In some cases, the average molecular weight of the polyoxazoline polymer is, for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, 100,000, 200,000, 300,000, 400,000, or 500,000 Da. In some cases, the polyoxazoline polymer comprises poly(2-methyl-2-oxazoline) (PMOZ), poly(2-ethyl-2-oxazoline) (PEOZ), or poly(2-propyl-2-oxazoline) (PPOZ). In some cases, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to the polyoxazoline polymer. In some cases, an IL-2 polypeptide is conjugated to a polyoxazoline polymer. In some cases, the IL-2 polypeptide is conjugated to PMOZ. In some cases, the IL-2 polypeptide is conjugated to PEOZ. In some cases, the IL-2 polypeptide is conjugated to PPOZ.

[00287] In some cases, the water-soluble polymer comprises a polyacrylic acid-based polymer. In some cases, a cytokine polypeptide (e.g., interleukin, IFN, or TNF) is conjugated to a polyacrylic acid-based polymer. In some cases, an IL-2 polypeptide is conjugated to a polyacrylic acid-based polymer.

[00288] In some cases, the water-soluble polymer comprises a polyamine. The polyamine is an organic polymer containing two or more primary amine groups. In some embodiments, the polyamine comprises a branched polyamine, a linear polyamine, or a cyclic polyamine. In some cases, the polyamine is a low molecular weight linear polyamine. Exemplary polyamines include putrescine, cadaverine, spermidine, spermine, ethylenediamine, 1,3-diaminopropane, hexamethylenediamine, tetraethylmethylenediamine, and piperazine. In some cases, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to the polyamine. In some cases, an IL-2 polypeptide is conjugated to the polyamine. In some cases, the IL-2 polypeptide is conjugated to putrescine, cadaverine, spermidine, spermine, ethylenediamine, 1,3-diaminopropane, hexamethylenediamine, tetraethylmethylenediamine, or piperazine.

[00289] In some cases, the water-soluble polymer is described in U.S. Patent Nos. 7,744,861, 8,273,833, and 7,803,777. In some cases, the cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to a linker described in U.S. Patent Nos. 7,744,861, 8,273,833, or 7,803,777. In some cases, the IL-2 polypeptide is conjugated to a linker described in U.S. Patent Nos. 7,744,861, 8,273,833, or 7,803,777.

Lipids

[00290] In some embodiments, a conjugate moiety described herein is a lipid. In some cases, the lipid is a fatty acid. In some cases, the fatty acid is a saturated fatty acid. In other cases, the fatty acid is an unsaturated fatty acid. Exemplary fatty acids include, but are not limited to, fatty acids having from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms. In some cases, the lipid binds to one or more serum proteins, thereby increasing serum stability and/or serum half-life.

[00291] In some embodiments, the lipid is conjugated to IL-2. In some cases, the lipid is a fatty acid, such as a saturated fatty acid or an unsaturated fatty acid. In some cases, the fatty acid comprises from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms. In some cases, a fatty acid contains approximately 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbon atoms in length. In some cases, the fatty acid includes caproic acid (hexanoic acid), enanthic acid (heptanoic acid), caprylic acid (octanoic acid), pelargonic acid (nonanoic acid), capric acid (decanoic acid), undecylic acid (undecanoic acid), lauric acid (dodecanoic acid), tridecylic acid (tridecanoic acid), myristic acid (tetradecanoic acid), pentadecylic acid (pentadecanoic acid), palmitic acid (hexadecanoic acid), margaric acid (heptadecanoic acid), stearic acid (octadecanoic acid), nonadecylic acid (nonadecanoic acid), arachidic acid (eicosanoic acid), heneicosylic acid (heneicosanoic acid), behenic acid (docosanoic acid), tricosylic acid (tricosanoic acid), lignoceric acid (tetracosanoic acid), pentacosylic acid (pentacosanoic acid) or cerotic acid (hexacosanoic acid).

[00292] In some embodiments, the IL-2 lipid conjugate enhances serum stability and/or serum half-life.

Squirrels

[00293] In some embodiments, the conjugate moiety described herein is a protein or a binding fragment thereof. Exemplary proteins include albumin, transferrin, or transthyretin. In some cases, the protein or binding fragment thereof comprises an antibody or binding fragments thereof. In some cases, the cytokine conjugate comprises a protein or a binding fragment thereof. In some cases, the IL-2 conjugate comprising the protein or binding fragment thereof has an increased serum half-life and/or increased stability. In some cases, the IL-2 conjugate comprising the protein or binding fragment thereof has a reduced interaction of IL-2 with one or more IL-2R subunits. In additional cases, the protein or binding fragment thereof blocks the interaction of IL-2 with one or more IL-2R subunits.

[00294] In some embodiments, the conjugating moiety is albumin. Albumin is a family of water-soluble globular proteins. It is typically found in blood plasma, accounting for approximately 55-60% of all blood plasma proteins. Human serum albumin (HSA) is a 585 amino acid polypeptide in which the tertiary structure is divided into three domains: domain I (amino acid residues 1-195), domain II (amino acid residues 196-383), and domain III (amino acid residues 384-585). Each domain further comprises a binding site that is capable of reversibly or irreversibly interacting with endogenous ligands such as long- and medium-chain fatty acids, bilirubin, or hemin, or exogenous compounds such as heterocyclic or aromatic compounds.

[00295] In some cases, a cytokine polypeptide (e.g., interleukin, IFN, or TNF) is conjugated to albumin. In some cases, the cytokine polypeptide is conjugated to human serum albumin (HSA). In additional cases, the cytokine polypeptide is conjugated to a functional fragment of albumin.

[00296] In some cases, the IL-2 polypeptide is conjugated to albumin. In some cases, the IL-2 polypeptide is conjugated to human serum albumin (HSA). In additional cases, the IL-2 polypeptide is conjugated to a functional fragment of albumin.

[00297] In some embodiments, the conjugating moiety is transferrin. Transferrin is a 679 amino acid polypeptide that is approximately 80 kDa in size and contains two Fe ³⁺ binding sites, one in the N-terminal domain and one in the C-terminal domain. In some cases, human transferrin has a half-life of approximately 7-12 days.

[00298] In some cases, a cytokine polypeptide (e.g., interleukin, IFN, or TNF) is conjugated to transferrin. In some cases, the cytokine polypeptide is conjugated to human transferrin. In additional cases, the cytokine polypeptide is conjugated to a functional fragment of transferrin.

[00299] In some cases, the IL-2 polypeptide is conjugated to transferrin. In some cases, the IL-2 polypeptide is conjugated to human transferrin. In additional cases, the IL-2 polypeptide is conjugated to a functional fragment of transferrin.

[00300] In some embodiments, the conjugating moiety is transthyretin (TTR). Transthyretin is a transport protein located in serum and cerebrospinal fluid that transports the thyroid hormone thyroxine ( _T4 ) and retinol-binding protein bound to retinol.

[00301] In some cases, a cytokine polypeptide (e.g., interleukin, IFN, or TNF) is conjugated to transthyretin (via one of its ends or via an internal hinge region). In some cases, the cytokine polypeptide is conjugated to a functional fragment of transthyretin.

[00302] In some cases, the IL-2 polypeptide is conjugated to transthyretin (via one of its ends or via an internal hinge region). In some cases, the IL-2 polypeptide is conjugated to a functional fragment of transthyretin.

[00303] In some embodiments, the conjugating moiety is an antibody or binding fragments thereof. In some cases, the antibody or binding fragments thereof comprises a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab ₂ , F(ab)' ₃ fragments, a single-chain variable fragment (scFv), a bis-scFv, (scFv) ₂ , a diabody, a minibody, a nanobody, a triantibody, a tetraantibody, a human antibody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camel antibody or binding fragment thereof, a bispecific antibody or binding fragment thereof, or a chemically modified derivative thereof.

[00304] In some cases, the conjugation moiety comprises an scFv, a bis-scFv, (scFv) ₂ , a dsFv, or an sdAb. In some cases, the conjugation moiety comprises an scFv. In some cases, the conjugation moiety comprises a bis-scFv. In some cases, the conjugation moiety comprises (scFv) ₂ . In some cases, the conjugation moiety comprises a dsFv. In some cases, the conjugation moiety comprises an sdAb.

[00305] In some cases, the conjugating fragment comprises an Fc portion of an antibody, such as IgG, IgA, IgM, IgE, or IgD. In some cases, the fragment comprises an Fc portion of an IgG (e.g., IgG ₁ , IgG ₃ , or IgG ₄ ).

[00306] In some cases, a cytokine polypeptide (e.g., interleukin, IFN, or TNF) is conjugated to an antibody or binding fragments thereof. In some cases, the cytokine polypeptide is conjugated to a humanized antibody or a binding fragment thereof, a murine antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, a monoclonal antibody or a binding fragment thereof, a monovalent Fab', a bivalent Fab ₂ , F(ab)' ₃ fragments, a single-chain variable fragment (scFv), a bis-scFv, (scFv) ₂ , a diabody, a minibody, a nanobody, a tribody, a tetrabody, a humanibody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camel antibody or a binding fragment thereof, a bispecific antibody or a binding fragment thereof, or a chemically modified derivative thereof. In additional cases, the cytokine polypeptide is conjugated to the Fc portion of an antibody. In additional cases, the cytokine polypeptide is conjugated to the Fc portion of an IgG (e.g., IgG ₁ , IgG ₃ , or IgG ₄ ).

[00307] In some cases, the IL-2 polypeptide is conjugated to an antibody or binding fragments thereof. In some cases, the IL-2 polypeptide is conjugated to a humanized antibody or a binding fragment thereof, a murine antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, a monoclonal antibody or a binding fragment thereof, a monovalent Fab', a bivalent Fab ₂ , F(ab)' ₃ fragments, a single-chain variable fragment (scFv), a bis-scFv, (scFv) ₂ , a diabody, a minibody, a nanobody, a tribody, a tetrabody, a human antibody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camel antibody or a binding fragment thereof, a bispecific antibody or a binding fragment thereof, or a chemically modified derivative thereof. In additional cases, the IL-2 polypeptide is conjugated to the Fc portion of an antibody. In additional cases, the IL-2 polypeptide is conjugated to the Fc portion of an IgG (e.g., IgG ₁ , IgG ₃ , or IgG ₄ ).

[00308] In some embodiments, the IL-2 polypeptide is conjugated to a water-soluble polymer (e.g., PEG) and an antibody or binding fragment thereof. In some cases, the antibody or binding fragments thereof comprises a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab ₂ , F(ab)' ₃ fragments, a single-chain variable fragment (scFv), a bis-scFv, (scFv) ₂ , a diabody, a minibody, a nanobody, a triantibody, a tetraantibody, a human antibody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camel antibody or binding fragment thereof, a bispecific antibody or binding fragment thereof, or a chemically modified derivative thereof. In some cases, the antibody or binding fragments thereof comprises an scFv, bis-scFv, (scFv) ₂ , dsFv, or sdAb. In some cases, the antibody or binding fragments thereof comprises an scFv. In some cases, the antibody or binding fragment thereof directs the IL-2 conjugate to a target cell of interest and the water-soluble polymer enhances its stability and/or serum half-life.

[00309] In some cases, one or more conjugates of an IL-2 polypeptide and a water-soluble polymer (e.g., PEG) are further linked to an antibody or binding fragments thereof. In some cases, the ratio of the IL-2 conjugate to the antibody is about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, or 12:1. In some cases, the ratio of the IL-2 conjugate to the antibody is about 1:1. In other cases, the ratio of the IL-2 conjugate to the antibody is about 2:1, 3:1, or 4:1. In additional cases, the ratio of the IL-2 conjugate to the antibody is about 6:1 or greater.

[00310] In some embodiments, one or more conjugates of an IL-2 polypeptide and a water-soluble polymer (e.g., PEG) are directly linked to the antibody or binding fragments thereof. In other cases, the IL-2 conjugate is indirectly linked to the antibody or binding fragments thereof via a linker. Exemplary linkers include homobifunctional linkers, heterobifunctional linkers, maleimide-based linkers, traceless linkers, self-cleaving linkers, spacers, and the like.

[00311] In some embodiments, the antibody or binding fragments thereof are directly or indirectly linked to a portion of the IL-2 polypeptide in a conjugate of the IL-2 polypeptide and a water-soluble polymer (e.g., PEG). In such cases, the conjugation site of the antibody to the IL-2 polypeptide is located in a site that will not interfere with binding of the IL-2 polypeptide to IL-2Rβγ. In additional cases, the conjugation site of the antibody to the IL-2 polypeptide is located in a region that partially blocks binding of the IL-2 polypeptide to IL-2Rβγ. In additional cases, the conjugation site of the antibody to the IL-2 polypeptide is located in a site that will interfere or further interfere with binding of the IL-2 polypeptide to IL-2Rα. In other embodiments, the antibody or binding fragments thereof are directly or indirectly linked to a water-soluble polymer moiety in a conjugate of an IL-2 polypeptide and a water-soluble polymer (e.g., PEG).

Peptides

[00312] In some embodiments, the conjugation moiety described herein is a peptide. In some cases, the peptide is an unstructured peptide. In some cases, a cytokine polypeptide (e.g., an interleukin, IFN, or TNF) is conjugated to the peptide. In some cases, the IL-2 conjugate comprising the peptide has an increased serum half-life and/or increased stability. In some cases, the IL-2 conjugate comprising the peptide has a reduced interaction of IL-2 with one or more IL-2R subunits. In additional cases, the peptide blocks the interaction of IL-2 with one or more IL-2R subunits.

[00313] In some cases, the conjugating moiety is an XTEN™ peptide (Amunix Operating Inc.) and the modification is referred to as XTENylation. XTENylation is the genetic fusion of a nucleic acid encoding a polypeptide of interest to a nucleic acid encoding an XTEN™ peptide (Amunix Operating Inc.), a long, unstructured, hydrophilic peptide containing varying percentages of six amino acids: Ala, Glu, Gly, Ser, and Thr. In some cases, the XTEN™ peptide is selected based on properties such as expression, genetic stability, solubility, resistance to aggregation, increased half-life, increased potency, and/or increased in vitro activity in combination with the polypeptide of interest. In some cases, a cytokine polypeptide (e.g., interleukin, IFN, or TNF) is conjugated to the XTEN peptide. In some cases, the IL-2 polypeptide is conjugated to the XTEN peptide.

[00314] In some cases, the conjugating moiety is a glycine-rich amino acid homopolymer (HAP), and the modification is referred to as HAPylation. HAPylation is the genetic fusion of a nucleic acid encoding a polypeptide of interest to a nucleic acid encoding a glycine-rich amino acid homopolymer (HAP). In some cases, the HAP polymer comprises a repeat motif of (Gly ₄ Ser) _n (SEQ ID NO: 85) and is sometimes about 50, 100, 150, 200, 250, 300, or more in length. In some cases, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to HAP. In some cases, an IL-2 polypeptide is conjugated to HAP.

[00315] In some embodiments, the conjugating moiety is a PAS polypeptide, and the modification is referred to as PASylation. PASylation is the genetic fusion of a nucleic acid encoding a polypeptide of interest to a nucleic acid encoding a PAS polypeptide. A PAS polypeptide is a hydrophilic, uncharged polypeptide consisting of Pro, Ala, and Ser residues. In some cases, the PAS polypeptide is at least about 100, 200, 300, 400, 500, or 600 amino acids in length. In some cases, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to a PAS polypeptide. In some cases, an IL-2 polypeptide is conjugated to a PAS polypeptide.

[00316] In some embodiments, the conjugating moiety is an elastin-like polypeptide (ELP), and the modification is referred to as ELPylation. ELPylation is a genetic fusion of a nucleic acid encoding a polypeptide of interest to a nucleic acid encoding an elastin-like polypeptide (ELP). ELP comprises a VPGxG repeat motif (SEQ ID NO: 86), wherein x is any amino acid except proline. In some cases, a cytokine polypeptide (e.g., interleukin, IFN, or TNF) is conjugated to ELP. In some cases, an IL-2 polypeptide is conjugated to ELP.

[00317] In some embodiments, the conjugating moiety is a CTP peptide. The CTP peptide provides a 31 amino acid residue peptide, namely FQSSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ (SEQ ID NO: 87), wherein S* denotes O-glycosylation sites (OPKO). In some cases, the CTP peptide is genetically fused to a cytokine polypeptide (e.g., an IL-2 polypeptide). In some cases, a cytokine polypeptide (e.g., an IL-2 polypeptide) is conjugated to the CTP peptide.

[00318] In some embodiments, a cytokine (e.g., an IL-2 polypeptide) is modified by glutamylation. Glutamylation (or polyglutamylation) is a reversible post-translational modification of glutamate in which the γ-carboxyl group of glutamate forms a peptide-like bond with the amino group of free glutamate, in which the α-carboxyl group is converted to a polyglutamate chain.

[00319] In some embodiments, a cytokine (e.g., an IL-2 polypeptide) is modified with a gelatin-like protein (GLK) polymer. In some cases, the GLK polymer comprises multiple Gly-Xaa-Yaa repeats, wherein Xaa and Yaa predominantly comprise proline and 4-hydroxyproline, respectively. In some cases, the GLK polymer further comprises Pro, Gly, Glu, Qln, Asn, Ser, and Lys amino acid residues. In some cases, the GLK polymer is about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150 residues, or more in length.

Additional conjugating fragments

[00320] In some cases, the conjugation moiety comprises an extracellular biomarker. In some cases, the extracellular biomarker is a tumor antigen. In some cases, an exemplary extracellular biomarker includes CD19, PSMA, B7-H3, B7-H6, CD70, CEA, CSPG4, EGFRvIII, EphA3, EpCAM, EGFR, ErbB2 (HER2), FAP, FRα, GD2, GD3, Lewis-Y, mesothelin, Muc1, Muc16, ROR1, TAG72, VEGFR2, CD11, Gr-1, CD204, CD16, CD49b, CD3, CD4, CD8, and B220. In some cases, the conjugation moiety is linked or conjugated to a cytokine (e.g., IL-2). In some cases, the conjugating moiety is genetically fused, for example at the N-terminus or C-terminus, to a cytokine (e.g. IL-2).

[00321] In some cases, the conjugate moiety comprises a molecule with a post-translational modification. In some cases, examples of a post-translational modification include myristoylation, palmitoylation, isoprenylation (or prenylation) (e.g., farnesylation or geranylgeranylation), glypylation, acylation (e.g., O-acylation, N-acylation, S-acylation), alkylation (e.g., with additional alkyl groups such as methyl or ethyl groups), amidation, glycosylation, hydroxylation, iodination, nucleotide addition, oxidation, phosphorylation, succinylation, sulfation, glycosylation, carbamylation, glutamylation, or deamidation. In some cases, the cytokine (e.g., IL-2) is modified by post-translational modification such as myristoylation, palmitoylation, isoprenylation (or prenylation) (e.g., farnesylation or geranylgeranylation), glypylation, acylation (e.g., O-acylation, N-acylation, S-acylation), alkylation (e.g., with additional alkyl groups such as methyl or ethyl groups), amidation, glycosylation, hydroxylation, iodination, nucleotide addition, oxidation, phosphorylation, succinylation, sulfation, glycosylation, carbamylation, glutamylation, or deamidation.

Conjugation

Linkers

[00322] In some embodiments, useful functional reactive groups for conjugating or linking a conjugate moiety to a cytokine polypeptide (e.g., an IL-2 polypeptide) described herein include, for example, zero-order or higher order linkers. In some cases, a non-naturally occurring amino acid incorporated into an interleukin described herein comprises a functional reactive group. In some cases, a linker comprises a functional reactive group that reacts with a non-naturally occurring amino acid incorporated into an interleukin described herein. In some cases, a conjugate moiety comprises a functional reactive group that reacts with a non-naturally occurring amino acid incorporated into an interleukin described herein. In some cases, a conjugate moiety comprises a functional reactive group that reacts with a linker (optionally pre-attached to a cytokine peptide) described herein. In some embodiments, the linker comprises a reactive group that reacts with a natural amino acid in a cytokine peptide described herein. In some cases, higher order linkers include bifunctional linkers, such as homobifunctional linkers or heterobifunctional linkers. Illustrative homobifunctional linkers include, but are not limited to, Lomant's reagent, which is dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propionate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl 3,3'-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), such as, for example, 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenylsulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N, N'-ethylene-bis(iodoacetamide) or N, N'-hexamethylene-bis(iodoacetamide).

[00323] In some embodiments, the bifunctional linker comprises a heterobifunctional linker. An illustrative heterobifunctional linker includes, but is not limited to, amino-reactive and sulfhydryl cross-linkers such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (LC-sPDP), water-soluble long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl 6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-sMPT), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBs), N-succinimidyl (4-iodacteyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodacteyl)aminobenzoate (sulfo-sIAB), succinimidyl 4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-sMPB), ester N-(γ-maleimidobutyryloxy)succinimide (GMBs), N-(γ-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs), succinimidyl-6-((iodaacetyl)amino)hexanoate (sIAX), succinimidyl-6-[6-((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl-6-(((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive cross-linkers such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxylhydrazide-8 ( _M2C2H ), 3-(2- _{pyridyldithio} )propionylhydrazide (PDPH), amine-reactive and photoreactive cross-linkers such as N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p-azidosalicylamido)ethyl 1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl 4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl 4-azidobenzoate (sulfo-HsAB), N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOs), sulfosuccinimidyl 2-(m-azido-o-nitrobenzamido)-ethyl 1,3'-dithiopropionate (sAND), N-succinimidyl 4-(4-azidophenyl)-1,3'-dithiopropionate (sADP), N-sulfosuccinimidyl (4-azidophenyl)-1,3'-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(p-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl 1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (ρNPDP), p-nitrophenyl 2-diazo-3,3,3-trifluoropropionate (PNP-DTP), sulfhydryl-reactive and photoreactive crosslinkers such as 1-(p-azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p-azidosalicylamido)butyl]-3′-(2′-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimide, carbonyl-reactive and photoreactive crosslinkers such as p-azidobenzoyl hydrazide (ABH), carboxylate-reactive and photoreactive crosslinkers such as 4-(p-azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive crosslinkers such as p-azidophenylglyoxal (APG).

[00324] In some cases, the reactive functional group provides a nucleophilic group that reacts with an electrophilic group present on the binding moiety (e.g., on the conjugate moiety or on IL-2). Exemplary electrophilic groups include carbonyl groups, such as an aldehyde, ketone, carbonyl group of a carboxylic acid, ester, amide, enone, acyl halide, or acid anhydride. In some embodiments, the reactive functional group is an aldehyde group. Exemplary nucleophilic groups include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In some embodiments, the unnatural amino acid incorporated into an interleukin described herein comprises an electrophilic group.

[00325] In some embodiments, the linker is a cleavable linker. In some embodiments, the cleavable linker is a dipeptide linker. In some embodiments, the dipeptide linker is valine-citrulline (Val-Cit), phenylalanine-lysine (Phe-Lys), valine-alanine (Val-Ala), and valine-lysine (Val-Lys). In some embodiments, the dipeptide linker is valine-citrulline.

[00326] In some embodiments, the linker is a peptide linker comprising, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids. In some cases, the peptide linker comprises no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, 50 or less amino acids. In additional cases, the peptide linker comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.

[00327] In some embodiments, the linker comprises a self-cleaving linker moiety. In some embodiments, the self-cleaving linker moiety comprises p -aminobenzyl alcohol (PAB), p -aminobenzyloxycarbonyl (PABC), or derivatives or analogs thereof. In some embodiments, the linker comprises a dipeptide linker moiety and a self-cleaving linker moiety. In some embodiments, the self-cleaving linker moiety is as described in U.S. Patent No. 9,089,614 and WIPO Application No. WO2015038426.

[00328] In some embodiments, the cleavable linker is a glucuronide. In some embodiments, the cleavable linker is an acid-cleavable linker. In some embodiments, the acid-cleavable linker is hydrazine. In some embodiments, the cleavable linker is a reducible linker.

[00329] In some embodiments, the linker comprises a maleimide group. In some cases, the maleimide group is also referred to as a maleimide spacer. In some cases, the maleimide group further comprises caproic acid, forming maleimidocaproyl (mc). In some cases, the linker comprises maleimidocaproyl (mc). In some cases, the linker is maleimidocaproyl (mc). In other cases, the maleimide group includes a maleimidomethyl group, such as succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC) or sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), described above.

[00330] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some cases, the self-stabilizing maleimide uses diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide to provide intramolecular catalysis of the hydrolysis of the thiosuccinimide ring, thereby eliminating the maleimide in a reverse Michael elimination reaction. In some cases, the self-stabilizing maleimide is the maleimide group described in Lyon, et al., "Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates," Nat. Biotechnol . 32 (10): 1059-1062 (2014). In some cases, the linker comprises a self-stabilizing maleimide. In some cases, the linker is a self-stabilizing maleimide.

Conjugation chemistry

[00331] Various conjugation reactions are used to conjugate linkers, conjugation moieties, and unnatural amino acids incorporated into the cytokine peptides described herein. Such conjugation reactions are often compatible with aqueous conditions, such as "biorthogonal" reactions. In some embodiments, the conjugation reactions are mediated by chemical reagents, such as catalysts, light, or reactive chemical groups found on linkers, conjugation moieties, or unnatural amino acids. In some embodiments, the conjugation reactions are mediated by enzymes. In some embodiments, a conjugation reaction used herein is described in Gong, Y., Pan, L. Tett. Lett. 2015, 56, 2123. In some embodiments, a conjugation reaction used herein is described in Chen, X.; Wu. Y-W. Org. Biomol. Chem. 2016, 14, 5417.

[00332] In some embodiments described herein, the conjugation reaction comprises reacting a ketone or aldehyde with a nucleophile. In some embodiments, the conjugation reaction comprises reacting a ketone with an aminooxy group to form an oxime. In some embodiments, the conjugation reaction comprises reacting a ketone with an aryl or heteroarylamino group to form an imine. In some embodiments, the conjugation reaction comprises reacting an aldehyde with an aryl or heteroarylamino group to form an imine. In some embodiments, the conjugation reaction described herein results in the production of a cytokine peptide comprising a linker or conjugation moiety attached via an oxime. In some embodiments, the conjugation reaction comprises a Pictet-Spengler reaction of an aldehyde or ketone with a tryptamine nucleophile. In some embodiments, the conjugation reaction comprises a Pictet-Spengler reaction with hydrazine. In some embodiments, the conjugation reaction comprises Pictet-Spengler ligation.

[00333] In some embodiments described herein, the conjugation reaction described herein comprises a reaction of an azide and a phosphine (Staudinger ligation). In some embodiments, the phosphine is an arylphosphine. In some embodiments, the arylphosphine comprises an orthoester group. In some embodiments, the phosphine has a methyl 2-(diphenylphosphanyl)benzoate structure. In some embodiments, the conjugation reaction described herein results in a cytokine peptide comprising a linker or conjugation moiety attached via an arylamide. In some embodiments, the conjugation reaction described herein results in a cytokine peptide comprising a linker or conjugation moiety attached via an amide.

[00334] In some embodiments described herein, a conjugation reaction described herein comprises a 1,3-dipolar cycloaddition reaction. In some embodiments, a 1,3-dipolar cycloaddition reaction comprises a click reaction of an azide and a phosphine. In some embodiments, the conjugation reaction is copper catalyzed. In some embodiments, a conjugation reaction described herein results in a cytokine peptide comprising a linker or conjugation moiety attached via a triazole. In some embodiments, a conjugation reaction described herein comprises a reaction of an azide with a strained olefin. In some embodiments, a conjugation reaction described herein comprises a reaction of an azide with a strained alkyne. In some embodiments, a conjugation reaction described herein comprises reacting an azide with a cycloalkyne, such as OCT, DIFO, DIFBO, DIBO, BARAC, TMTH, or another strained cycloalkyne, the structures of which are shown in Gong, Y., Pan, L. Tett. Lett. 2015, 56, 2123. In some embodiments, the 1,3-dipolar cycloaddition reaction is catalyzed by light ("photoclick"). In some embodiments, a conjugation reaction described herein comprises reacting a terminal allyl group with a tetrazole under light. In some embodiments, a conjugation reaction described herein comprises reacting a terminal alkynyl group with a tetrazole under light. In some embodiments, a conjugation reaction described herein comprises reacting an O-allyl amino acid with a tetrazine under light. In some embodiments, the conjugation reaction described herein comprises reacting O-allyl tyrosine with tetrazine in the presence of light.

[00335] In some embodiments described herein, the conjugation reaction described herein comprises

, wherein X is a position in an IL-2 conjugate comprising a non-natural amino acid, such as any of SEQ ID NOs: 5, 6, 7, 8, 9, 30, 31, 32, 33, and 34. In some embodiments, the conjugation moiety comprises a water-soluble polymer. In some embodiments, the reactive group comprises an alkyne or an azide. In some embodiments described herein, the conjugation reaction described herein comprises

, wherein X is a position in an IL-2 conjugate comprising a non-natural amino acid, such as in any of SEQ ID NOs: 5, 6, 7, 8, 9, 30, 31, 32, 33, and 34. In some embodiments described herein, the conjugation reaction described herein comprises

, wherein X is a position in an IL-2 conjugate comprising a non-natural amino acid, such as in any of SEQ ID NOs: 5, 6, 7, 8, 9, 30, 31, 32, 33, and 34. In some embodiments described herein, the conjugation reaction described herein comprises

, wherein X is a position in an IL-2 conjugate comprising a non-natural amino acid, such as in any of SEQ ID NOs: 5, 6, 7, 8, 9, 30, 31, 32, 33, and 34. In some embodiments described herein, the conjugation reaction described herein comprises

, wherein X is a position in an IL-2 conjugate comprising a non-natural amino acid such as any of SEQ ID NOs: 5, 6, 7, 8, 9, 30, 31, 32, 33, and 34. In some embodiments, the conjugation reaction described herein results in the IL-2 variant of Table 20 .

[00336] In some embodiments described herein, the conjugation reaction described herein involves a cycloaddition reaction between an azide moiety, such as one contained in a protein containing an amino acid residue derived from N6 -((2-azidoethoxy)-carbonyl)-L-lysine (AzK), and a strained cycloalkyne, such as one derived from DBCO, which is a chemical moiety containing a dibenzocyclooctyne group. PEG groups containing a DBCO moiety are commercially available or can be prepared by methods known to those skilled in the art.

[00337] Using conjugation reactions, such as the click reaction described herein, one can create a single regioisomer or a mixture of regioisomers. In some cases, the ratio of regioisomers is about 1:1. In some cases, the ratio of regioisomers is about 2:1. In some cases, the ratio of regioisomers is about 1.5:1. In some cases, the ratio of regioisomers is about 1.2:1. In some cases, the ratio of regioisomers is about 1.1:1. In some cases, the ratio of regioisomers is greater than 1:1.

[00338] This document describes IL-2 conjugates having the structure of formula (I),

formula (I),

Where

Z is CH ₂ and Y is ; or

Y is CH ₂ , and Z is ;

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa and 50 kDa; and

X is an amino acid position of recombinant human IL-2, wherein the amino acid position is among the positions in SEQ ID NO: 1; or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments of the IL-2 conjugate of Formula (I), Z is CH ₂ , and Y is . In some embodiments of the IL-2 conjugate of Formula (I), Y is CH ₂ and Z is . In some embodiments of the IL-2 conjugate of Formula (I), the PEG moiety has an average molecular weight selected from 5 kDa, 10 kDa, and 30 kDa. In some embodiments of the IL-2 conjugate of Formula (I), the PEG moiety has an average molecular weight of 5 kDa. In some embodiments of the IL-2 conjugate of Formula (I), the PEG moiety has an average molecular weight of 10 kDa. In some embodiments of the IL-2 conjugate of Formula (I), the PEG moiety has an average molecular weight of 20 kDa. In some embodiments of the IL-2 conjugate of Formula (I), the PEG moiety has an average molecular weight of 30 kDa. In some embodiments of the IL-2 conjugate of Formula (I), the PEG moiety has an average molecular weight of 40 kDa. In some embodiments of the IL-2 conjugate of Formula (I), the PEG moiety has an average molecular weight of 50 kDa. In some embodiments of the IL-2 conjugate of Formula (I), X is K35. In some embodiments of the IL-2 conjugate of Formula (I), X is F42. In some embodiments of the IL-2 conjugate of Formula (I), X is K43. In some embodiments of the IL-2 conjugate of Formula (I), X is E62. In some embodiments of the IL-2 conjugate of Formula (I), X is P65. In some embodiments of the IL-2 conjugate of Formula (I), X is R38. In some embodiments of the IL-2 conjugate of Formula (I), X is T41. In some embodiments of the IL-2 conjugate of Formula (I), X is E68. In some embodiments of the IL-2 conjugate of Formula (I), X is Y45. In some embodiments of the IL-2 conjugate of Formula (I), X is V69. In some embodiments of the IL-2 conjugate of Formula (I), X is selected from K35, F42, K43, E62, P65, R38, T41, E68, Y45 and V69. In some embodiments of the IL-2 conjugate of Formula (I), X is selected from F42, K43, E62 and P65. In some embodiments, the IL-2 conjugate of Formula (I) comprises a sequence of any of SEQ ID NOs: 5-84. In some embodiments, the IL-2 conjugate of Formula (I) comprises SEQ ID NOs: 15-29. In some embodiments, the IL-2 conjugate of Formula (I) comprises SEQ ID NOs: 40-54. In some embodiments, the IL-2 conjugate of Formula (I) comprises SEQ ID NOs: 55-69. In some embodiments, the IL-2 conjugate of Formula (I) comprises SEQ ID NOs: 70-84. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 3. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 4. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 5. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 6. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 7. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 8. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 9. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 10. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 11. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 12. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 13. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 14. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 15. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 16. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 17. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 18. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 19. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 20. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 21. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 22. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 23. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 24. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 25. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 26. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 27. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 28. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 29. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 30. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 31. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 32. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 33. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 34. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 35. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 36. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 37. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 38. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 39. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 40. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 41. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 42. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 43. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 44. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 45. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 46. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 47. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 48. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 49. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 50. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 51. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 52. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 53. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 54. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 55. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 56. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 57. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 58. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 59. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 60. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 61. In some embodiments, the conjugate IL-2 of Formula (I) comprises the sequence of SEQ ID NO: 62. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 63. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 64. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 65. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 66. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 67. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 68. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 69. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 70. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 71. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 72. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 73. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 74. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 75. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 76. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 77. In some embodiments, the IL-2 conjugate of Formula (I) comprises the sequence of SEQ ID NO: 78. In some embodiments the IL-2 conjugate of formula (I) comprises the sequence of SEQ ID NO: 79. In some embodiments, the IL-2 conjugate of formula (I) comprises the sequence of SEQ ID NO: 80. In some embodiments, the IL-2 conjugate of formula (I) comprises the sequence of SEQ ID NO: 81. In some embodiments, the IL-2 conjugate of formula (I) comprises the sequence of SEQ ID NO: 82. In some embodiments, the IL-2 conjugate of formula (I) comprises the sequence of SEQ ID NO: 83. In some embodiments, the IL-2 conjugate of formula (I) comprises the sequence of SEQ ID NO: 84.

[00339] This document describes IL-2 conjugates having the structure of formula (II),

formula (II),

where W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa and 30 kDa; and

X represents the position of the amino acid characterized by the structure

recombinant human IL-2 selected from F42, K43, E62 and P65, wherein the amino acid position corresponds to the positions in SEQ ID NO: 1.

[00340] This document describes IL-2 conjugates having the structure of formula (III),

formula (III),

where W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa and 30 kDa; and

X represents the position of the amino acid characterized by the structure

recombinant human IL-2 selected from F42, K43, E62 and P65, wherein the amino acid corresponds to the positions in SEQ ID NO: 1.

[00341] In some embodiments of the IL-2 conjugate of formula (II) or formula (III), X is position F42 of recombinant human IL-2. In some embodiments of the IL-2 conjugate of formula (II) or formula (III),

the PEG moiety has an average molecular weight of 5 kDa, and X is position F42 of recombinant human IL-2. In some embodiments of the IL-2 conjugate of Formula (II) or Formula (III), X is position K43 of recombinant human IL-2. In some embodiments of the IL-2 conjugate of Formula (II) or Formula (III), the PEG moiety has an average molecular weight of 5 kDa, and X is position K43 of recombinant human IL-2. In some embodiments of the IL-2 conjugate of Formula (II) or Formula (III), X is position E62 of recombinant human IL-2. In some embodiments of the IL-2 conjugate of Formula (II) or Formula (III), the PEG moiety has an average molecular weight of 5 kDa, and X is position E62 of recombinant human IL-2. In some embodiments of the IL-2 conjugate of Formula (II) or Formula (III), X is position P65 of recombinant human IL-2. In some embodiments of the IL-2 conjugate of Formula (II) or Formula (III), the PEG group has an average molecular weight of 5 kDa and X is position P65 of recombinant human IL-2. In some embodiments of the IL-2 conjugate of Formula (II) or Formula (III), X is position F42 of recombinant human IL-2 and W is a PEG group having an average molecular weight of 10 kDa. In some embodiments of the IL-2 conjugate of Formula (II) or Formula (III), X is position K43 of recombinant human IL-2 and W is a PEG group having an average molecular weight of 10 kDa. In some embodiments of the IL-2 conjugate of Formula (II) or Formula (III), X is position E62 of recombinant human IL-2 and W is a PEG moiety having an average molecular weight of 10 kDa. In some embodiments of the IL-2 conjugate of Formula (II) or Formula (III), position P65 of recombinant human IL-2 and W is a PEG moiety having an average molecular weight of 10 kDa. In some embodiments of the IL-2 conjugate of Formula (II) or Formula (III), position F42 of recombinant human IL-2 and W is a PEG moiety having an average molecular weight of 30 kDa. In some embodiments of the IL-2 conjugate of Formula (II) or Formula (III), position K43 of recombinant human IL-2 and W is a PEG moiety having an average molecular weight of 30 kDa. In some embodiments of the IL-2 conjugate of formula (II) or formula (III), position E62 of the recombinant human IL-2 and W is a PEG moiety having an average molecular weight of 30 kDa. In some embodiments of the IL-2 conjugate of formula (II) or formula (III), position P65 of the recombinant human IL-2 and W is a PEG moiety having an average molecular weight of 30 kDa. In some embodiments, the IL-2 conjugate of formula (II) or formula (III) comprises the sequence of any of SEQ NOs: 3-29 and 70-84. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 3. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 4. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 5. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 6. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 7. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 8. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 9. In some embodiments, the IL-2 conjugate of formula (II) or formula (III) comprises the sequence of SEQ ID NO: 10. In some embodiments, the IL-2 conjugate of formula (II) or formula (III) comprises the sequence of SEQ ID NO: 11. In some embodiments, the IL-2 conjugate of formula (II) or formula (III) comprises the sequence of SEQ ID NO: 12. In some embodiments, the IL-2 conjugate of formula (II) or formula (III) comprises the sequence of SEQ ID NO: 13. In some embodiments, the IL-2 conjugate of formula (II) or formula (III) comprises the sequence of SEQ ID NO: 14. In some embodiments, the IL-2 conjugate of formula (II) or formula (III) comprises the sequence of SEQ ID NO: 15. In some embodiments, the IL-2 conjugate of formula (II) or formula (III) comprises the sequence of SEQ ID NO: 16. In some embodiments, the IL-2 conjugate of formula (II) or formula (III) comprises the sequence of SEQ ID NO: 17. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 18. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 19. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 20. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 21. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 22. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 23. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 24. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 25. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 26. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 27. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 28. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 29. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 70. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 71. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 72. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 73. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 74. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 75. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 76. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 77. In some embodiments, the IL-2 conjugate of Formula (II) or Formula (III) comprises the sequence of SEQ ID NO: 78. In some embodiments, the IL-2 conjugate of formula (II) or formula (III) comprises the sequence of SEQ ID NO: 79. In some embodiments, the IL-2 conjugate of formula (II) or formula (III) comprises the sequence of SEQ ID NO: 80. In some embodiments, the IL-2 conjugate of formula (II) or formula (III) comprises the sequence of SEQ ID NO: 81. In some embodiments, the IL-2 conjugate of formula (II) or formula (III) comprises the sequence of SEQ ID NO: 82. In some embodiments, the IL-2 conjugate of formula (II) or formula (III) comprises the sequence of SEQ ID NO: 83. In some embodiments, the IL-2 conjugate of formula (II) or formula (III) comprises the sequence of SEQ ID NO: 84.

[00342] Described herein are pharmaceutical compositions of Formula (I), Formula (II), or Formula (III). In some embodiments, the pharmaceutical compositions of Formula (I), Formula (II), or Formula (III) comprise a sequence comprising any of SEQ ID NOs: 3-29 and 70-84. In some embodiments, the pharmaceutical compositions of Formula (I), Formula (II), or Formula (III) comprise a sequence comprising any of SEQ ID NO: 3. In some embodiments, the pharmaceutical compositions of Formula (I), Formula (II), or Formula (III) comprise a sequence comprising any of SEQ ID NO: 4.

[00343] This document describes IL-2 conjugates having the structure of formula (IV),

formula (IV),

where W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa and 30 kDa; and

X represents the position of the amino acid characterized by the structure

recombinant human IL-2 selected from F42, K43, E62 and P65, wherein the amino acid position corresponds to the positions in SEQ ID NO: 1.

[00344] This document describes IL-2 conjugates having the structure of formula (V),

formula (V),

where W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa and 30 kDa; and

X represents the position of the amino acid characterized by the structure

recombinant human IL-2 selected from F42, K43, E62 and P65, wherein the amino acid corresponds to the positions in SEQ ID NO: 1.

[00345] In some embodiments of the IL-2 conjugate of formula (IV) or formula (V), X is position F42 of recombinant human IL-2. In some embodiments of the IL-2 conjugate of formula (IV) or formula (V),

the PEG moiety has an average molecular weight of 5 kDa, and X is position F42 of recombinant human IL-2. In some embodiments of the IL-2 conjugate of Formula (IV) or Formula (V), X is position K43 of recombinant human IL-2. In some embodiments of the IL-2 conjugate of Formula (IV) or Formula (V), the PEG moiety has an average molecular weight of 5 kDa, and X is position K43 of recombinant human IL-2. In some embodiments of the IL-2 conjugate of Formula (IV) or Formula (V), X is position E62 of recombinant human IL-2. In some embodiments of the IL-2 conjugate of Formula (IV) or Formula (V), the PEG moiety has an average molecular weight of 5 kDa, and X is position E62 of recombinant human IL-2. In some embodiments of the IL-2 conjugate of Formula (IV) or Formula (V), X is position P65 of recombinant human IL-2. In some embodiments of the IL-2 conjugate of Formula (IV) or Formula (V), the PEG group has an average molecular weight of 5 kDa and X is position P65 of recombinant human IL-2. In some embodiments of the IL-2 conjugate of Formula (IV) or Formula (V), X is position F42 of recombinant human IL-2 and W is a PEG group having an average molecular weight of 10 kDa. In some embodiments of the IL-2 conjugate of Formula (IV) or Formula (V), X is position K43 of recombinant human IL-2 and W is a PEG group having an average molecular weight of 10 kDa. In some embodiments of the IL-2 conjugate of Formula (IV) or Formula (V), X is position E62 of recombinant human IL-2 and W is a PEG moiety having an average molecular weight of 10 kDa. In some embodiments of the IL-2 conjugate of Formula (IV) or Formula (V), position P65 of recombinant human IL-2 and W is a PEG moiety having an average molecular weight of 10 kDa. In some embodiments of the IL-2 conjugate of Formula (IV) or Formula (V), position F42 of recombinant human IL-2 and W is a PEG moiety having an average molecular weight of 30 kDa. In some embodiments of the IL-2 conjugate of Formula (IV) or Formula (V), position K43 of recombinant human IL-2 and W is a PEG moiety having an average molecular weight of 30 kDa. In some embodiments of the IL-2 conjugate of formula (IV) or formula (V), position E62 of the recombinant human IL-2 and W is a PEG moiety having an average molecular weight of 30 kDa. In some embodiments of the IL-2 conjugate of formula (IV) or formula (V), position P65 of the recombinant human IL-2 and W is a PEG moiety having an average molecular weight of 30 kDa. In some embodiments, the IL-2 conjugate of formula (IV) or formula (V) comprises the sequence of any one of SEQ ID NOs: 3, 4, 40-69. In some embodiments, the IL-2 conjugate of Formula (IV) or Formula (V) comprises the sequence of SEQ ID NO: 3. In some embodiments, the IL-2 conjugate of Formula (IV) or Formula (V) comprises the sequence of SEQ ID NO: 4. In some embodiments, the IL-2 conjugate of Formula (IV) or Formula (V) comprises the sequence of SEQ ID NO: 40. In some embodiments, the IL-2 conjugate of Formula (IV) or Formula (V) comprises the sequence of SEQ ID NO: 41. In some embodiments, the IL-2 conjugate of Formula (IV) or Formula (V) comprises the sequence of SEQ ID NO: 42. In some embodiments, the IL-2 conjugate of Formula (IV) or Formula (V) comprises the sequence of SEQ ID NO: 43. In some embodiments, the IL-2 conjugate of Formula (IV) or Formula (V) comprises the sequence of SEQ ID NO: 44. In some embodiments the IL-2 conjugate of formula (IV) or formula (V) comprises the sequence of SEQ ID NO: 45. In some embodiments, the IL-2 conjugate of formula (IV) or formula (V) comprises the sequence of SEQ ID NO: 46. In some embodiments, the IL-2 conjugate of formula (IV) or formula (V) comprises the sequence of SEQ ID NO: 47. In some embodiments, the IL-2 conjugate of formula (IV) or formula (V) comprises the sequence of SEQ ID NO: 48. In some embodiments, the IL-2 conjugate of formula (IV) or formula (V) comprises the sequence of SEQ ID NO: 49. In some embodiments, the IL-2 conjugate of formula (IV) or formula (V) comprises the sequence of SEQ ID NO: 50. In some embodiments, the IL-2 conjugate of formula (IV) or formula (V) comprises the sequence of SEQ ID NO: 51. In some embodiments, the IL-2 conjugate of formula (IV) or formula (V) comprises the sequence of SEQ ID NO: 52. In some embodiments, the IL-2 conjugate of formula (IV) or formula (V) comprises the sequence of SEQ ID NO: 53. In some embodiments, the IL-2 conjugate of formula (IV) or formula (V) comprises the sequence of SEQ ID NO: 54. In some embodiments, the IL-2 conjugate of formula (IV) or formula (V) comprises the sequence of SEQ ID NO: 55. In some embodiments, the IL-2 conjugate of formula (IV) or formula (V) comprises the sequence of SEQ ID NO: 56. In some embodiments, the IL-2 conjugate of formula (IV) or formula (V) comprises the sequence of SEQ ID NO: 57. In some embodiments, the IL-2 conjugate of formula (IV) or formula (V) comprises the sequence of SEQ ID NO: 58. In some embodiments, the IL-2 conjugate of formula (IV) or formula (V) comprises the sequence of SEQ ID NO: 59. In some embodiments, the IL-2 conjugate of Formula (IV) or Formula (V) comprises the sequence of SEQ ID NO: 60. In some embodiments, the IL-2 conjugate of Formula (IV) or Formula (V) comprises the sequence of SEQ ID NO: 61. In some embodiments, the IL-2 conjugate of Formula (IV) or Formula (V) comprises the sequence of SEQ ID NO: 62. In some embodiments, the IL-2 conjugate of Formula (IV) or Formula (V) comprises the sequence of SEQ ID NO: 63. In some embodiments, the IL-2 conjugate of Formula (IV) or Formula (V) comprises the sequence of SEQ ID NO: 64. In some embodiments, the IL-2 conjugate of Formula (IV) or Formula (V) comprises the sequence of SEQ ID NO: 65. In some embodiments, the IL-2 conjugate of Formula (IV) or Formula (V) comprises the sequence of SEQ ID NO: 66. In some embodiments, the IL-2 conjugate of formula (IV) or formula (V) comprises the sequence of SEQ ID NO: 67. In some embodiments, the IL-2 conjugate of formula (IV) or formula (V) comprises the sequence of SEQ ID NO: 68. In some embodiments, the IL-2 conjugate of formula (IV) or formula (V) comprises the sequence of SEQ ID NO: 69.

[00346] Described herein are pharmaceutical compositions of Formula (I), Formula (IV), or Formula (V). In some embodiments, the pharmaceutical compositions of Formula (I), Formula (IV), or Formula (V) comprise a sequence comprising any of SEQ ID NOs: 3, 4, and 40-69. In some embodiments, the pharmaceutical compositions of Formula (I), Formula (IV), or Formula (V) comprise a sequence comprising SEQ ID NO: 3.

[00347] In some embodiments described herein, a conjugation reaction described herein comprises an inverse electron demand cycloaddition reaction involving a diene and a dienophile. In some embodiments, the diene comprises a tetrazine. In some embodiments, the dienophile comprises an alkene. In some embodiments, the dienophile comprises an alkyne. In some embodiments, the alkyne is a strained alkyne. In some embodiments, the alkene is a strained diene. In some embodiments, the alkyne is trans-cyclooctyne. In some embodiments, the alkyne is cyclooctene. In some embodiments, the alkene is cyclopropene. In some embodiments, the alkene is fluorocyclopropene. In some embodiments, a conjugation reaction described herein results in the formation of a cytokine peptide attached to a linker or conjugate moiety via a 6-membered ring heterocycle containing two nitrogen atoms in the ring.

[00348] In some embodiments described herein, a conjugation reaction described herein comprises an olefin metathesis reaction. In some embodiments, a conjugation reaction described herein comprises reacting an alkene and an alkyne with a ruthenium catalyst. In some embodiments, a conjugation reaction described herein comprises reacting two alkenes with a ruthenium catalyst. In some embodiments, a conjugation reaction described herein comprises reacting two alkynes with a ruthenium catalyst. In some embodiments, a conjugation reaction described herein comprises reacting an alkene or alkyne with a ruthenium catalyst and an amino acid containing an allyl group. In some embodiments, a conjugation reaction described herein comprises reacting an alkene or alkyne with a ruthenium catalyst and an amino acid containing an allyl sulfide or selenide. In some embodiments, the ruthenium catalyst is a Hoveyda-Grubbs 2-type catalyst. generations. In some embodiments, the olefin metathesis reaction comprises reacting one or more strained alkenes or alkynes.

[00349] In some embodiments described herein, the conjugation reaction described herein comprises a cross-coupling reaction. In some embodiments, the cross-coupling reactions comprise transition metal catalysts, such as iridium, gold, ruthenium, rhodium, palladium, nickel, platinum, or another transition metal catalyst, and one or more ligands. In some embodiments, the transition metal catalysts are water-soluble. In some embodiments described herein, the conjugation reaction described herein comprises a Suzuki-Miyaura cross-coupling reaction. In some embodiments described herein, the conjugation reaction described herein comprises the reaction of an aryl halide (or triflate or tosylate), an aryl or alkenyl boronic acid, and a palladium catalyst. In some embodiments described herein, the conjugation reaction described herein comprises a Sonogashira cross-coupling reaction. In some embodiments described herein, the conjugation reaction described herein comprises a reaction of an aryl halide (or triflate or tosylate), an alkyne, and a palladium catalyst. In some embodiments, the cross-coupling reactions result in the attachment of a linker or conjugation moiety to a cytokine peptide via a carbon-carbon bond.

[00350] In some embodiments described herein, a conjugation reaction described herein comprises a deprotection reaction or "release" of a reactive group prior to conjugation. In some embodiments, a conjugation reaction described herein comprises release of a reactive group using light, followed by a conjugation reaction. In some embodiments, the reactive group is protected by an aralkyl moiety containing one or more nitro groups. In some embodiments, release of the reactive group results in the formation of a free amine, sulfide, or other reactive group. In some embodiments, a conjugation reaction described herein comprises release of a reactive group using a transition metal catalyst, followed by a conjugation reaction. In some embodiments, the transition metal catalyst comprises palladium and one or more ligands. In some embodiments, the reactive group is protected by an allyl moiety. In some embodiments, the reactive group is protected by an allyl carbamate. In some embodiments, the reactive group is protected by a propargyl moiety. In some embodiments, the reactive group is protected by a propargyl carbamate. In some embodiments, the reactive group is protected by a dienophile, wherein exposure to a diene (such as tetrazine) results in deprotection of the reactive group.

[00351] In some embodiments described herein, the conjugation reaction described herein comprises a ligand-directed reaction, wherein a ligand (optionally) attached to a reactive group) provides a conjugation site between the reactive group and the cytokine peptide. In some embodiments, the ligand is cleaved during or after the reaction of the cytokine peptide with the reactive group. In some embodiments, the cytokine peptide conjugation site is a naturally occurring amino acid. In some embodiments, the cytokine peptide conjugation site is lysine, cysteine, or serine. In some embodiments, the cytokine peptide conjugation site is a non-naturally occurring amino acid as described herein. In some embodiments, the reactive group comprises a leaving group, such as an electron-poor aryl or heteroaryl group. In some embodiments, the reactive group provides a leaving group, such as an electron-poor alkyl group, that is displaced by the cytokine peptide. In some embodiments, the conjugation reaction described herein comprises a reaction of a radical scavenger with a chemical radical. In some embodiments, the conjugation reaction described herein comprises an addition reaction of an oxidizing radical. In some embodiments, the radical scavenger is an arylamine. In some embodiments, the chemical radical is a tyrosyl radical. In some embodiments, the chemical radicals are generated by a ruthenium catalyst (such as [Ru(bpy) ₃ ]) and light.

[00352] Enzymatic reactions are optionally used for the conjugation reactions described herein. Examples of enzymatic conjugations include SortA-mediated conjugation, TG-mediated conjugation, or FGE-mediated conjugation. In some embodiments, a conjugation reaction described herein comprises ligating a native protein (NPL) at a terminal 1-amino-2-thio group to a thioester to form an amide bond.

[00353] Described herein are various conjugation reactions for reacting a linker or conjugation moiety with a cytokine peptide, wherein the reaction occurs with a naturally occurring ("canonical") amino acid in the cytokine peptide. In some embodiments, the naturally occurring amino acid is found at a conjugation position that is in the wild-type sequence, or, alternatively, the position has been mutated. In some embodiments, the conjugation reaction involves the formation of a disulfide bond at a cysteine residue. In some embodiments, the conjugation reaction involves a Michael 1,4-addition reaction of cysteine or lysine. In some embodiments, the conjugation reaction involves the ligation of a cyanobenzothiazole to a cysteine. In some embodiments, the conjugation reaction involves cross-linking with an acetone moiety, such as 1,3-dichloro-2-propionone. In some embodiments, the conjugation reaction comprises a 1,4-Michael addition to a dehydroalanine formed by the reaction of cysteine with O-mesitylenesulfonylhydroxylamine. In some embodiments, the conjugation reaction comprises the reaction of tyrosine with a triazolidinedione (TAD) or a TAD derivative. In some embodiments, the conjugation reaction comprises the reaction of tryptophan with a rhodium carbenoid.

Methods of application

Proliferative diseases or conditions

[00354] In some embodiments, described herein is a method of treating a proliferative disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a cytokine conjugate (e.g., an IL-2 conjugate) described herein. In some cases, the cytokine conjugate comprises SEQ ID NOs: 5-84. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 15-29. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 40-54. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 55-69. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 70-84. In some cases, the IL-2 conjugate comprises an isolated and purified IL-2 polypeptide and a conjugation moiety, wherein the IL-2 conjugate has a reduced affinity for the IL-2 receptor α subunit (IL-2Rα) compared to a wild-type IL-2 polypeptide. In some cases, the IL-2 conjugate comprises an isolated and purified IL-2 polypeptide and a conjugation moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105, and Y107, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some cases, the IL-2 conjugate preferentially interacts with IL-2Rβ and IL-2Rβγ subunits to form an IL-2/IL-2Rβγ complex. In some cases, the IL-2/IL-2Rβγ complex stimulates and/or enhances the expansion of CD4+ helper cells, CD8+ effector naive and memory T cells, NK cells, and/or NKT cells. In additional cases, the expansion of Teff cells shifts the Teff:Treg ratio toward the Teff population. In some embodiments, an IL-2 conjugate comprising a mutation at residue F42, wherein the residue corresponds to position 42 of SEQ ID NO: 1, comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has a minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has a minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of a wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of a modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of a wild-type IL-2 polypeptide and IL-2Rα.

[00355] In some embodiments, described herein is a method of treating a proliferative disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a cytokine conjugate (e.g., an IL-2 conjugate) described in Table 20. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 1-84. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 15-29. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 40-54. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 55-69. In some embodiments, the IL-2 conjugate comprises SEQ ID NOs: 70-84. In some embodiments, the IL-2 conjugate comprises a structure of Formula (I). In some embodiments, the IL-2 conjugate comprises a structure of Formula (II). In some embodiments, the IL-2 conjugate comprises a structure of formula (III). In some embodiments, the IL-2 conjugate comprises a structure of formula (IV). In some embodiments, the IL-2 conjugate comprises a structure of formula (V). In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 1. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 2. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 3. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 4. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 5. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 6. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 7. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 8. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 9. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 10. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 11. In some embodiments, the conjugate IL-2 comprises SEQ ID NO: 12. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 13. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 14. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 15. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 16. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 17. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 18. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 19. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 20. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 21. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 22. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 23. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 24. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 25. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 26. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 27. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 28. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 24. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 25. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 26. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 27. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 28. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: NO: 29. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 30. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 31. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 32. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 33. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 34. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 35. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 36. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 37. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 38. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 39. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 40. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 41. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 42. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 43. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 44. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 45. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 46. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 47. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 48. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 49. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 50. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 51. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 52. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 53. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 54. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 55. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 56. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 57. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 58. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 59. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 60. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 61. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 62. In some embodiments the IL-2 conjugate comprises SEQ ID NO: 63. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 64. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 65. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 66. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 67. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 68. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 69. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 70. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 71. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 72. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 73. In some embodiments, the conjugate IL-2 comprises SEQ ID NO: 74. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 75. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 76. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 77. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 78. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 79. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 80. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 81. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 82. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 83. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 84.

[00356] In some embodiments, the proliferative disease or condition is cancer. In some cases, the cancer is a solid tumor. Exemplary solid tumors include, but are not limited to, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer. In some cases, the solid tumor is metastatic cancer. In some cases, the solid tumor is recurrent or refractory cancer. In some cases, the solid tumor is castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, or DNA damage response (DDR)-deficient metastatic castration-resistant prostate cancer.

[00357] In some cases, a cytokine conjugate (e.g., an interleukin, IFN, or TNF) described herein is administered to a subject in need thereof to treat a solid tumor. In such cases, the subject has bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer. In some cases, the solid tumor is metastatic cancer. In some cases, the solid tumor is relapsed or refractory cancer. In some cases, the solid tumor is castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, or DNA damage response (DDR)-deficient metastatic castration-resistant prostate cancer.

[00358] In some cases, the IL-2 conjugate described herein is administered to a subject in need thereof to treat a solid tumor. In such cases, the subject has bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer. In some cases, the IL-2 conjugate is administered to the subject to treat bladder cancer. In some cases, the IL-2 conjugate is administered to the subject to treat breast cancer. In some cases, the IL-2 conjugate is administered to the subject to treat colorectal cancer. In some cases, the IL-2 conjugate is administered to the subject to treat esophageal cancer. In some cases, the IL-2 conjugate is administered to the subject to treat head and neck cancer. In some cases, the IL-2 conjugate is administered to the subject to treat kidney cancer. In some cases, the IL-2 conjugate is administered to the subject to treat lung cancer. In some cases, the IL-2 conjugate is administered to the subject to treat melanoma. In some cases, the IL-2 conjugate is administered to the subject to treat ovarian cancer. In some cases, the IL-2 conjugate is administered to the subject to treat pancreatic cancer. In some cases, the IL-2 conjugate is administered to the subject to treat prostate cancer. In some cases, the IL-2 conjugate is administered to the subject to treat castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, or DNA damage response (DDR)-deficient metastatic castration-resistant prostate cancer.

[00359] In some embodiments, the IL-2 conjugate is administered to a subject to treat metastatic cancer. In some cases, the metastatic cancer comprises metastatic bladder cancer, metastatic bone cancer, metastatic brain cancer, metastatic breast cancer, metastatic colorectal cancer, metastatic esophageal cancer, metastatic eye cancer, metastatic head and neck cancer, metastatic kidney cancer, metastatic lung cancer, metastatic melanoma, metastatic ovarian cancer, metastatic pancreatic cancer, or metastatic prostate cancer. In some cases, the IL-2 conjugate is administered to a subject to treat metastatic bladder cancer, metastatic bone cancer, metastatic brain cancer, metastatic breast cancer, metastatic colorectal cancer, metastatic esophageal cancer, metastatic eye cancer, metastatic head and neck cancer, metastatic renal cell cancer, metastatic lung cancer, metastatic melanoma, metastatic ovarian cancer, metastatic pancreatic cancer, or metastatic prostate cancer. In some cases, the IL-2 conjugate is administered to a subject to treat castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, or DNA damage response (DDR)-deficient metastatic castration-resistant prostate cancer.

[00360] In some cases, the IL-2 conjugate is administered to a subject to treat a relapsed or refractory cancer. In some cases, the relapsed or refractory cancer includes relapsed or refractory bladder cancer, relapsed or refractory bone cancer, relapsed or refractory brain cancer, relapsed or refractory breast cancer, relapsed or refractory colorectal cancer, relapsed or refractory esophageal cancer, relapsed or refractory eye cancer, relapsed or refractory head and neck cancer, relapsed or refractory kidney cancer, relapsed or refractory lung cancer, relapsed or refractory melanoma, relapsed or refractory ovarian cancer, relapsed or refractory pancreatic cancer, or relapsed or refractory prostate cancer. In some cases, the IL-2 conjugate is administered to a subject for the treatment of recurrent or refractory bladder cancer, recurrent or refractory bone cancer, recurrent or refractory brain cancer, recurrent or refractory breast cancer, recurrent or refractory colorectal cancer, recurrent or refractory esophageal cancer, recurrent or refractory eye cancer, recurrent or refractory head and neck cancer, recurrent or refractory kidney cancer, recurrent or refractory lung cancer, recurrent or refractory melanoma, recurrent or refractory ovarian cancer, recurrent or refractory pancreatic cancer, or recurrent or refractory prostate cancer.

[00361] In some embodiments, the cancer is a previously untreated cancer. In such cases, the previously untreated cancer is a cancer that has not previously been treated with a therapy. In some cases, the previously untreated cancer is a solid tumor, such as bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer. In some embodiments, described herein is a method of treating a previously untreated solid tumor in a subject in need thereof, comprising administering to the subject a cytokine conjugate (e.g., an IL-2 conjugate) described herein.

[00362] In some embodiments, the cancer is a hematologic malignancy. In some cases, the hematologic malignancy includes leukemia, lymphoma, or myeloma. In some cases, the hematologic malignancy is a T-cell malignancy. In other cases, the hematologic malignancy is a B-cell malignancy. Illustrative hematologic malignancies include, but are not limited to, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, high-grade non-Burkitt's B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-cell lymphoblastic lymphoma, prolymphocytic B-cell leukemia, lymphoplasmacytic lymphoma, marginal zone cell lymphoma spleen, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

[00363] In some cases, the hematologic malignancy is a metastatic cancer. In some cases, the metastatic cancer is a metastatic T-cell malignancy or a metastatic B-cell malignancy.

[00364] In some cases, the hematologic malignancy is a relapsed or refractory cancer. In some cases, the relapsed or refractory cancer is a relapsed or refractory T-cell malignancy or a relapsed or refractory B-cell malignancy.

[00365] In some cases, a cytokine (e.g., an interleukin, IFN, or TNF) described herein is administered to a subject in need thereof to treat a hematological malignancy. In some cases, the subject has a T-cell malignancy. In some cases, the subject has a B-cell malignancy. In some cases, the subject has chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, high-grade non-Burkitt's B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-cell lymphoblastic lymphoma, prolymphocytic B-cell leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

[00366] In some cases, an IL-2 conjugate described herein is administered to a subject in need thereof to treat a hematological malignancy. In some cases, the subject has a T-cell malignancy. In some cases, the subject has a B-cell malignancy. In some cases, the subject has chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, high-grade non-Burkitt's B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-cell lymphoblastic lymphoma, prolymphocytic B-cell leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. In some cases, the IL-2 conjugate is administered to a subject to treat CLL. In some cases, the IL-2 conjugate is administered to a subject to treat SLL. In some cases, the IL-2 conjugate is administered to a subject to treat FL. In some cases, the IL-2 conjugate is administered to a subject to treat DLBCL. In some cases, the IL-2 conjugate is administered to a subject to treat MCL. In some cases, the IL-2 conjugate is administered to a subject to treat Waldenstrom's macroglobulinemia. In some cases, the IL-2 conjugate is administered to a subject to treat multiple myeloma. In some cases, the IL-2 conjugate is administered to a subject to treat Burkitt's lymphoma.

[00367] In some cases, the IL-2 conjugate is administered to a subject to treat a metastatic hematological malignancy. In some cases, the IL-2 conjugate is administered to a subject to treat a metastatic T-cell malignancy. In some cases, the IL-2 conjugate is administered to a subject to treat a metastatic B-cell malignancy. In some cases, the IL-2 conjugate is administered to a subject for the treatment of metastatic chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, high-grade non-Burkitt's B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-cell lymphoblastic lymphoma, prolymphocytic B-cell leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myelomas, plasmacytomas, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or metastatic lymphomatoid granulomatosis.

[00368] In some cases, the IL-2 conjugate is administered to a subject to treat a relapsed or refractory hematological malignancy. In some cases, the IL-2 conjugate is administered to a subject to treat a relapsed or refractory T-cell malignancy. In some cases, the IL-2 conjugate is administered to a subject to treat a relapsed or refractory B-cell malignancy. In some cases, the IL-2 conjugate is administered to a subject for the treatment of relapsed or refractory chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, high-grade B-cell non-Burkitt's lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-cell lymphoblastic lymphoma, prolymphocytic B-cell leukemia, lymphoplasmacytic lymphoma, splenic marginal zone cell lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

Complementary Therapies

[00369] In some embodiments, the subject is further administered an additional therapeutic agent. In some cases, the additional therapeutic agent is administered concurrently with the cytokine conjugate (e.g., IL-2 conjugate). In other cases, the additional therapeutic agent and the IL-2 conjugate are administered sequentially, such as the cytokine conjugate (e.g., IL-2 conjugate) is administered before the additional therapeutic agent or the cytokine conjugate (e.g., IL-2 conjugate) is administered after the additional therapeutic agent is administered.

[00370] In some cases, the additional therapeutic agent includes a chemotherapeutic agent, an immunotherapeutic agent, a targeted therapy agent, radiation therapy, or a combination thereof. Illustrative additional therapeutic agents include, but are not limited to, alkylating agents such as altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxalaplatin, temozolomide, or thiotepa; antimetabolites such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, or pemetrexed; anthracyclines such as daunorubicin, doxorubicin, epirubicin, or idarubicin; topoisomerase I inhibitors such as topotecan or irinotecan (CPT-11); topoisomerase II inhibitors such as etoposide (VP-16), teniposide, or mitoxantrone; mitotic inhibitors such as docetaxel, estramustine, ixabepilone, paclitaxel, vinblastine, vincristine, or vinorelbine; or corticosteroids such as prednisone, methylprednisolone, or dexamethasone.

[00371] In some cases, the additional therapeutic agent includes first-line therapy. As used herein, the term "first-line therapy" includes the primary treatment of a subject with cancer. In some cases, the cancer is a primary cancer. In other cases, the cancer is a metastatic or recurrent cancer. In some cases, the first-line therapy includes chemotherapy. In other cases, the first-line treatment includes radiation therapy. One of skill in the art will readily appreciate that different first-line treatments may be appropriate for different types of cancer.

[00372] In some cases, the cytokine conjugate (e.g., IL-2 conjugate) is administered with an additional therapeutic agent selected from an alkylating agent such as altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxalaplatin, temozolomide, or thiotepa; an antimetabolite such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, or pemetrexed; an anthracycline such as daunorubicin, doxorubicin, epirubicin, or idarubicin; a topoisomerase I inhibitor such as topotecan or irinotecan (CPT-11); a topoisomerase II inhibitor such as etoposide (VP-16), teniposide, or mitoxantrone; a mitotic inhibitor such as docetaxel, estramustine, ixabepilone, paclitaxel, vinblastine, vincristine, or vinorelbine; or a corticosteroid such as prednisone, methylprednisolone, or dexamethasone.

[00373] In some cases, the cytokine conjugate (e.g., IL-2 conjugate) described herein is administered with an inhibitor of the enzyme poly-ADP-ribose polymerase (PARP). Illustrative PARP inhibitors include, but are not limited to, olaparib (AZD-2281, Lynparza®, from Astra Zeneca), rucaparib (PF-01367338, Rubraca®, from Clovis Oncology), niraparib (MK-4827, Zejula®, from Tesaro), talazoparib (BMN-673, from BioMarin Pharmaceutical Inc.), veliparib (ABT-888, from AbbVie), CK-102 (formerly CEP 9722, from Teva Pharmaceutical Industries Ltd.), E7016 (from Eisai), iniparib (BSI 201, from Sanofi), and pamiparib (BGB-290, from BeiGene). In some cases, a cytokine conjugate (eg, IL-2 conjugate) is administered in combination with a PARP inhibitor such as olaparib, rucaparib, niraparib, talazoparib, veliparib, CK-102, E7016, iniparib, or pamiparib.

[00374] In some cases, a cytokine conjugate (e.g., an IL-2 conjugate) described herein is administered with an immune checkpoint inhibitor. Exemplary immune checkpoint inhibitors include

[00375] PD-L1 inhibitors such as MPDL3280A (RG7446) from Genentech, murine anti-PD-L1 antibody clone 10F.9G2 (catalog number BE0101) from BioXcell, monoclonal anti-PD-L1 antibody MDX-1105 (BMS-936559) and BMS-935559 from Bristol-Myer's Squibb, MSB0010718C, murine anti-PD-L1 antibody clone 29E.2A3, MEDI4736 from AstraZeneca, atezolizumab (also known as Tecentriq®), bavelizumab (also known as Imfinzi®), and avelumab (also known as Bavencio®);

[00376] PD-L2 inhibitors such as AMP-224 (amplimmune) from GlaxoSmithKline and rHIgM12B7;

[00377] PD-1 inhibitors such as the murine PD-1 antibody, clone J43, (catalog number BE0033-2) from BioXcell, the murine anti-PD-1 antibody, clone RMP1-14, (catalog number BE0146) from BioXcell, the murine anti-PD-1 antibody, clone EH12, the murine anti-PD-1 antibody, MK-3475 (Keytruda, pembrolizumab, lambrolizumab) from Merck, the anti-PD-1 antibody from AnaptysBio known as ANB011, the MDX-1 106 antibody (ONO-4538), the human IgG4-based monoclonal antibody nivolumab (Opdivo®, BMS-936558, MDX1106) from Bristol-Myers Squibb, AMP-514 and AMP-224 from AstraZeneca, cemiplimab from Regeneron and pidilizumab (CT-011) from CureTech Ltd;

[00378] CTLA-4 inhibitors such as the anti-CTLA-4 antibody ipilimumab (also known as Yervoy®, MDX-010, BMS-734016, and MDX-101) from Bristol Myers Squibb, the anti-CTLA4 antibody clone 9H10 from Millipore, tremelimumab (CP-675,206, ticilimumab) from Pfizer, and the anti-CTLA4 antibody clone BNI3 from Abcam;

[00379] LAG3 inhibitors such as anti-Lag-3 antibody, clone eBioC9B7W, (C9B7W) from eBioscience, anti-Lag3 antibody LS-B2237 from LifeSpan Biosciences, IMP321 (ImmuFact) from Immutep, anti-Lag3 antibody BMS-986016, and chimeric anti-LAG-3 antibody A9H12;

[00380] B7-H3 inhibitors such as MGA271;

[00381] KIR inhibitors such as lirilumab (IPH2101);

[00382] CD137 inhibitors such as urelumab (BMS-663513, Bristol-Myers Squibb), PF-05082566 (k4-1BB, PF-2566, Pfizer), or XmAb-5592 (Xencor);

[00383] PS inhibitors such as bavituximab;

[00384] as well as inhibitors, such as an antibody or fragments thereof (e.g., a monoclonal antibody, human, humanized, or chimeric antibody), RNAi molecules, or small molecules to TIM3, CD52, CD30, CD20, CD33, CD27, OX40, GITR, ICOS, BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2, or SLAM.

[00385] In some cases, a cytokine conjugate (eg, an IL-2 conjugate) is administered in combination with pembrolizumab, nivolumab, tremelimumab, or ipilimumab.

[00386] In some cases, a cytokine conjugate (e.g., an IL-2 conjugate) described herein is administered with an antibody such as alemtuzumab, trastuzumab, ibritumomab tiuxetan, brentuximab vedotin, ado-trastuzumab emtansine, or blinatumomab.

[00387] In some cases, a cytokine conjugate (e.g., an IL-2 conjugate) is administered with an additional therapeutic agent selected from a receptor agonist. In some cases, the receptor agonist comprises a Toll-like receptor (TLR) ligand. In some cases, the TLR ligand comprises TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9. In some cases, the TLR ligand comprises a synthetic ligand, such as, for example, Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, poly I:C, poly A:U, AGP, MPL A, RC-529, MDF2β, CFA, or flagellin. In some cases, the cytokine conjugate (e.g., IL-2 conjugate) is administered with one or more TLR agonists selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In some cases, the cytokine conjugate (e.g., IL-2 conjugate) is administered with one or more TLR agonists selected from Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, poly I:C, poly A:U, AGP, MPL A, RC-529, MDF2β, CFA, and flagellin.

[00388] In some embodiments, a cytokine conjugate (e.g., an IL-2 conjugate) is used in combination with adoptive T cell transfer (ACT) therapy. In one embodiment, ACT comprises identifying autologous T lymphocytes in a subject, e.g., with anti-tumor activity, expanding the autologous T lymphocytes in vitro , and then reinfusing the expanded T lymphocytes into the subject. In another embodiment, ACT comprises using allogeneic T lymphocytes, e.g., with anti-tumor activity, expanding the T lymphocytes in vitro, and then infusing the expanded allogeneic T lymphocytes into a subject in need thereof. In some cases, a cytokine conjugate (e.g., an IL-2 conjugate) described herein is used in combination with autologous T lymphocytes as part of ACT therapy. In other cases, a cytokine conjugate (e.g., an IL-2 conjugate) described herein is used in combination with allogeneic T lymphocytes as part of ACT therapy. In some cases, a cytokine conjugate (e.g., an IL-2 conjugate) is administered concurrently with ACT therapy to a subject in need thereof. In other cases, a cytokine conjugate (e.g., an IL-2 conjugate) is administered sequentially with ACT therapy to a subject in need thereof.

[00389] In some embodiments, a cytokine conjugate (e.g., an IL-2 conjugate) is used to ex vivo activate and/or expand autologous and/or allogeneic T cells upon transfer. In such cases, the cytokine conjugate (e.g., an IL-2 conjugate) is used to activate and/or expand a sample comprising autologous and/or allogeneic T cells, and the cytokine conjugate (e.g., an IL-2 conjugate) is optionally removed from the sample prior to administration of the sample to a subject in need thereof.

[00390] In some embodiments, a cytokine conjugate (e.g., an IL-2 conjugate) is administered with a vaccine. In some cases, a cytokine conjugate (e.g., an IL-2 conjugate) is used in combination with an oncolytic virus. In such cases, the cytokine conjugate (e.g., an IL-2 conjugate) acts as a stimulatory agent to modulate the immune response. In some cases, the cytokine conjugate (e.g., an IL-2 conjugate) is used with an oncolytic virus as part of an adjuvant therapy. Exemplary oncolytic viruses include T-Vec (Amgen), G47Δ (Todo et al.), JX-594 (Sillajen), CG0070 (Cold Genesys), and reolysin (Oncolytics Biotech). In some cases, a cytokine conjugate (eg, IL-2 conjugate) is used in combination with an oncolytic virus such as T-Vec, G47Δ, JX-594, CG0070, or reolysin.

[00391] In some embodiments, a cytokine conjugate (e.g., an IL-2 conjugate) is administered in combination with radiation therapy.

[00392] In some embodiments, a cytokine conjugate (e.g., an IL-2 conjugate) is administered in combination with surgery.

Pathogenic infections

[00393] In some embodiments, described herein is a method of treating a pathogenic infection in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of a cytokine conjugate (e.g., an IL-2 conjugate) described herein. In some cases, the IL-2 conjugate comprises an isolated and purified IL-2 polypeptide and a conjugate moiety, wherein the IL-2 conjugate has a reduced affinity for the IL-2 receptor α subunit (IL-2Rα) compared to a wild-type IL-2 polypeptide. In some cases, the IL-2 conjugate comprises an isolated and purified IL-2 polypeptide and a conjugation moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105, and Y107, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some cases, the IL-2 conjugate preferentially interacts with an IL-2Rβ subunit and IL-2Rβγ to form an IL-2/IL-2Rβγ complex that stimulates and/or enhances the expansion of CD4+ helper cells, CD8+ effector naive cells, and memory cells, NK cells, and/or NKT cells. In further instances, the IL-2 conjugate facilitates recognition of a pathogenic site by CD8+ T cells. In some embodiments, an IL-2 conjugate comprising a mutation at residue F42, wherein the residue corresponds to position 42 of SEQ ID NO: 1, comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has a minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00394] In some embodiments, the pathogenic infection is a viral infection in which, following treatment with an antiviral therapy, a viral reservoir (e.g., resting CD4+ T cells) persists in the treated host. In such cases, a cytokine conjugate (e.g., an IL-2 conjugate) described herein induces recognition of the viral reservoir by CD8+ T cells (or cytotoxic T cells). In some cases, the cytokine conjugate (e.g., an IL-2 conjugate) is used as a monotherapy to redirect CD8+ T cells to infected resting cells for the purpose of eliminating them. In some cases, the cytokine conjugate (e.g., an IL-2 conjugate) is used in combination with an additional therapy to redirect CD8+ T cells to infected resting cells for the purpose of eliminating them. Illustrative adjunctive therapy includes antiviral drugs such as acyclovir, brivudine, docosanol, famciclovir, foscarnet, idoxuridine, penciclovir, trifluridine, valacyclovir, and pritelivir.

[00395] In some embodiments, the virus is a DNA virus or an RNA virus. DNA viruses include single-stranded (ss) DNA viruses, double-stranded (ds) DNA viruses, or DNA viruses that contain regions of ssDNA and dsDNA. RNA viruses include single-stranded (ss) RNA viruses, double-stranded (ds) RNA viruses. In some cases, ssRNA viruses are further divided into positive-sense RNA viruses or negative-sense RNA viruses.

[00396] Exemplary dsDNA viruses include those from the families Myoviridae, Podoviridae, Siphoviridae, Alloherpesviridae, Herpesviridae, Malacoherpesviridae, Lipothrixviridae, Rudiviridae, Adenoviridae, Ampullaviridae, Ascoviridae, Asfaviridae, Baculoviridae, Bicaudaviridae, , Corticoviridae, Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae , Iridoviridae, Marseilleviridae, Mimiviridae, Nimaviridae, Pandoraviridae, Papillomaviridae, Phycodnaviridae, Plasmaviridae, Polydnaviruses, Polyomaviridae, Poxviridae, Sphaerolipoviridae, and Tectiviridae.

[00397] Illustrative ssDNA viruses include viruses from the families Anelloviridae, Bacillariodnaviridae, Bidnaviridae, Circoviridae, Geminiviridae, Inoviridae, Microviridae, Nanoviridae, Parvoviridae, and Spiraviridae.

[00398] Exemplary DNA viruses that contain regions of ssDNA and dsDNA include viruses from the Pleolipoviruses group. In some cases, Pleolipoviruses include Haloarcula hispanica pleomorphic virus 1 , Halogeometricum pleomorphic virus 1 , Halorubrum pleomorphic virus 1 , Halorubrum pleomorphic virus 2 , Halorubrum pleomorphic virus 3 , and Halorubrum pleomorphic virus 6 .

[00399] Illustrative dsDNA viruses include those of the families Birnaviridae, Chrysoviridae, Cystoviridae, Endornaviridae, Hypoviridae, Megavirnaviridae, Partitiviridae, Picobirnaviridae, Reoviridae, Rotavirus, and Totiviridae.

[00400] Exemplary positive-sense ssRNA viruses include those from the families Alphaflexiviridae, Alphatetraviridae, Alvernaviridae, Arteriviridae, Astroviridae, Barnaviridae, Betaflexiviridae, Bromoviridae, Caliciviridae, Carmotetraviridae, Closteroviridae, Coronaviridae, Dicistroviridae, Flaviviridae, iviridae, Iflaviridae, Leviviridae, Luteoviridae, Marnaviridae, Mesoniviridae, Narnaviridae, Nodaviridae, Permutotetraviridae, Picornaviridae, Potyviridae, Roniviridae, Retroviridae, Secoviridae, Togaviridae, Tombusviridae, Tymoviridae, and Virgaviridae.

[00401] Illustrative negative-sense sRNA viruses include viruses from the families Arenaviridae, Bornaviridae, Bunyaviridae, Filoviridae, Nyamiviridae, Ophioviridae, Orthomyxoviridae, Paramyxoviridae, and Rhabdoviridae.

[00402] In some embodiments, the pathogenic infection is caused by Abelson leukemia virus, Abelson murine leukemia virus, Abelson virus, acute laryngotracheobronchitis virus, Adelaide River virus, adeno-associated virus group, adenovirus, African horse disease virus, African swine fever virus, HIV virus, Aleutian mink parvovirus, alpharetrovirus, alphavirus, ALV-related virus, Amapari virus, aphthovirus, aquareovirus, arbovirus, arbovirus C, arbovirus group A, arbovirus group B, arenavirus group, Argentine hemorrhagic fever virus, Argentine hemorrhagic fever virus, arterivirus, astrovirus, Atheline herpesvirus group, Aujeszky's disease virus, aura virus, Ausduc disease, Australian bat lyssavirus, aviadenovirus, avian erythroblastosis virus, avian infectious bronchitis virus, avian leukemia virus, avian leukosis virus, avian lymphomatosis virus, avian myeloblastosis virus, avian paramyxovirus, avian pneumopague virus, avian reticuloendotheliosis virus, avian sarcoma virus, avian retrovirus type C group, avigepadnavirus, avipoxvirus, B virus, B19 virus, Babanki virus, baboon herpesvirus, baculovirus, Barmah Forest virus, Bebaru virus, Berrimah virus, betaretrovirus, birnavirus, Bitner virus, BK virus, Black Creek Canal virus, bluetongue virus, Bolivian hemorrhagic fever virus, Woma disease virus, border disease virus sheep, bornavirus, bovine herpesvirus 1, bovine herpesvirus 2, bovine coronavirus, bovine ephemeral fever virus, bovine immunodeficiency virus, bovine leukemia virus, bovine leukosis virus, bovine mamillitis virus, bovine papillomavirus, bovine papular stomatitis virus, bovine parvovirus, bovine syncytial virus, bovine oncovirus type C, bovine diarrhea virus, Baggy Creek virus, rod-shaped virus group, Bunyamwera supergroup of viruses, bunyavirus, Burkitt lymphoma virus, Bwamba fever virus, CA virus, calicivirus, California encephalitis virus, camelpox virus, smallpox virus canary, canine herpes virus, canine coronavirus, canine distemper virus, canine herpesvirus, small canine virus, canine parvovirus, Cano Delgatido virus, caprine arthritis virus, caprine encephalitis virus, caprine herpesvirus, capripoxvirus, cardiovirus, pig herpesvirus 1, Cercopithecidae herpesvirus 1, monkey herpesvirus 1, monkey herpesvirus 2, Chandipura virus, Changuinola virus, channel catfish virus, Charleville virus, varicella-zoster virus, chimpanzee herpesvirus, chub reovirus, chum salmon virus, Cocal virus, coho reovirus, coital exanthema virus, Colorado tick fever virus, Coltivirus, Columbia SK virus, Virus, Acute respiratory disease, Contagious ecthyma virus, Contagious pustular dermatitis virus, Coronavirus, Bat virus, Coronavirus, Cowpox virus, Cowpox virus, Coxsackie virus, CPV (Cytoplasmic polyhedrosis virus), Cricket paralysis virus, Crimean-Congo hemorrhagic fever virus, Pseudocroup virus, Cryptovirus, Cypovirus, Cytomegalovirus, Cytomegalovirus group, Cytoplasmic polyhedrosis virus, Deer papillomavirus, Deltaretrovirus, Dengue virus, Densovirus, Dependovirus, Dhori virus, Diploma virus, Drosophila C virus, Duck hepatitis B virus, Duck hepatitis 1 virus, Hepatitis virus 2 ducks, duovirus, Duvenhage virus, deformed wing virus DWV, eastern equine encephalomyelitis virus, eastern equine encephalomyelitis virus, EB virus, Ebola virus, Ebola virus-like virus, ECHO group virus, ECHO virus, ECHO virus 10, ECHO virus 28, ECHO virus 9, ectromelia virus, EEE virus, EIA virus, EIA virus, encephalitis virus, encephalomyocarditis group virus, encephalomyocarditis virus, enterovirus, enzyme-elevating virus, enzyme-elevating virus (LDH), epidemic hemorrhagic fever virus, epizootic hemorrhagic disease virus, Epstein-Barr virus, equine herpes virus 1, virus equine herpesvirus 4, equine herpesvirus 2, mare abortion virus, equine arteritis virus, equine encephalosis virus, equine infectious anemia virus, equine morbillivirus, equine rhinopneumonia virus, equine rhinovirus, Eubenangu virus, elk papillomavirus, European swine fever virus, Everglades virus, Eyach virus, feline herpesvirus 1, feline calicivirus, feline fibrosarcoma virus, feline herpesvirus, feline immunodeficiency virus, feline infectious peritonitis virus, feline leukemia/sarcoma virus, feline leukemia virus, feline panleukopenia virus, feline parvovirus, feline sarcoma virus, feline syncytial virus, filovirus, Flanders virus, flavivirus, foot-and-mouth disease virus, Fort Morgan virus, Four Corners hantavirus, fowl adenovirus 1, fowl pox virus, Friend virus, gammaretrovirus, hepatitis GB virus, GB virus, German measles virus, Getah virus, gibbon leukemia virus, glandular fever virus, goat pox virus, golden blue virus, Gonomet virus, goose parvovirus, granulosis virus, Gross virus, ground squirrel hepatitis B virus, arbovirus group A, Guanarito virus, guinea pig cytomegalovirus, guinea pig type C virus, Hantaan virus, hantavirus, molluscum reovirus, rabbit fibroma virus, HCMV (human cytomegalovirus), hemadsorption virus 2, Japanese hemagglutinating virus, hemorrhagic fever virus, virus hendra, heniparaviruses, hepadnavirus, hepatitis A virus, hepatitis B group of viruses, hepatitis C virus, hepatitis D virus, hepatitis delta virus, hepatitis E virus, hepatitis F virus, hepatitis G virus, hepatitis virus other than A and B, hepatitis virus, hepatitis virus (non-human), hepatitis reovirus 3, hepatovirus, hepatitis B virus of the heron, herpes B virus, herpes simplex virus, herpes simplex virus 1, herpes simplex virus 2, herpesvirus, herpesvirus 7, herpesvirus ateles, human herpesvirus, herpesvirus infection, herpes squirrel virus, Herpesvirus suis, Herpesvirus varicellae, virus Highlands G, Hiram rhabdovirus, swine cholera virus, human adenovirus 2, human alphaherpesvirus 1, human alphaherpesvirus 2, human alphaherpesvirus 3, human lymphotropic virus B, human betaherpesvirus 5, human coronavirus, human cytomegalovirus group, human spumavirus, human gammaherpesvirus 4, human gammaherpesvirus 6, human hepatitis A virus, human herpesvirus group 1, human herpesvirus group 2, human herpesvirus group 3, human herpesvirus group 4, human herpesvirus 6, human herpesvirus 8, human immunodeficiency virus, human immunodeficiency virus 1, human immunodeficiency virus 2, human papillomavirus, human T-cell leukemia virus, human T-cell human leukemia virus I, human T-cell leukemia virus II, human T-cell leukemia virus III, human T-cell lymphoma virus I, human T-cell lymphoma virus II, human T-cell lymphotropic virus type 1, human T-cell lymphotropic virus type 2, human T-cell lymphotropic virus I, human T-cell lymphotropic virus II, human T-cell lymphotropic virus III, ichnovirus, infantile gastroenteritis virus, infectious bovine rhinotracheitis virus, infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, influenza A virus, influenza B virus, influenza C virus, influenza D virus, influenza virus pr8, insect rainbow virus, insect virus, iridovirus, Japanese virus B, Japanese encephalitis virus, JC virus, Junin fever virus, Kaposi's sarcoma-associated herpesvirus, Kemerovo virus, Kilham rat virus, Klamath virus, Kolongo virus, Korean hemorrhagic fever virus, Kumba virus, Kyasanur forest disease virus, Kizilagac virus, La Crosse virus, lactate dehydrogenase-increasing virus, lactate dehydrogenase virus, Lagos Bath virus, Langur virus, rabbit parvovirus, Lassa fever virus, Lassa virus, latent rat virus, LCM virus, Leakey virus, lentivirus, leporipoxvirus, leukemia virus, leukovirus, nodular dermatitis virus, lymphadenopathy-associated virus, lymphocryptovirus, lymphocytic choriomeningitis virus, lymphoproliferative group of viruses diseases, Machupo virus, bulbar paralysis virus, mammalian oncovirus type B group, mammalian retrovirus type B, mammalian retrovirus type C, mammalian retrovirus type D, mammary tumor virus, Mapuera virus, Marburg disease virus, Marburg disease-like virus, Mazen-Pfeiser monkey virus, Mastadenovirus, Mayago virus, ME virus, measles virus, Menangle virus, Mengo virus, Mengovirus, Middelburg virus, false cowpox virus, mink enteritis virus, small mouse virus, MLV-related virus, MM virus, Mokola virus, Molluscipoxvirus, Molluscum contagiosum virus, Monkey B virus, Monkeypox virus, Mononegavirales, Morbillivirus, Mount Elgon bat virus, murine cytomegalovirus, murine encephalomyelitis virus, murine hepatitis virus, murine virus K, murine leukemia virus, murine mammary tumor virus, murine small virus, murine pneumonia virus, murine poliovirus, murine polyomavirus, murine sarcoma virus, mousepox virus, Mozambique virus, Mukambo virus, mucosal disease virus, mumps virus, murine betaherpevirus 1, murine cytomegalovirus 2, murine cytomegalovirus group, murine encephalomyelitis virus, murine hepatitis virus, murine leukemia virus, murine nodule virus, murine polyomavirus, murine sarcoma virus, Muromegalovirus, Murray Valley encephalitis virus, myxoma virus, myxovirus, multiforme myxovirus, mumps myxovirus, Nairobian sheep disease virus, nairovirus, Nanimavirus, Nariva virus, Ndumo virus, Neethling virus, Nelson Bay virus, neurotropic virus, New World arenavirus, neonatal pneumonitis virus, Newcastle disease virus, Nipah virus, non-cytopathogenic virus, Norwalk virus, nuclear polyhedrosis virus (NPV), nipple neck virus, o'Nyong-Nyong virus, Ockelbo virus, oncogenic virus, oncogenic virus-like particle, oncornavirus, orbivirus, Orf virus, Oropouche virus, orthohepadnavirus, orthomyxovirus, orthopoxvirus, orthoreovirus, Orungo virus, ovine papillomavirus, bluetongue virus, herpesvirus tribandi durukuli, Palyam virus, papillomavirus, Papillomavirus sylvilagi, papovavirus, parainfluenza virus, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, parainfluenza virus type 4, paramyxovirus, parapoxvirus, paravaccinia virus, parvovirus, parvovirus B19, parvovirus group, pestivirus, phlebovirus, seal distemper virus, picodnavirus, picornavirus, swine cytomegalovirus and pigeonpox virus, Piri virus, Pixuna virus, mouse pneumonia virus, pneumovirus, poliovirus, poliovirus, polydnavirus, polyhedral virus, polyoma virus, polyomavirus, bovine polyomavirus, monkey polyomavirus, human polyomavirus 2, macaque polyomavirus 1, murine polyomavirus 1, murine polyomavirus 2, baboon polyomavirus 1, baboon polyomavirus 2, rabbit polyomavirus, orangutan herpesvirus 1, porcine epidemic diarrhea virus, porcine hemagglutinating encephalomyelitis virus, porcine parvovirus, porcine transmissible gastroenteritis virus, porcine type C virus, poxvirus, Poxvirus, Poxvirus variolae, Prospect Hill virus, provirus, pseudocowpox virus, pseudorabies virus, psittacinepox virus, quailpox virus, rabbit fibroma virus, rabbit kidney vacuolating virus, rabbit papillomavirus, virus rabies, raccoon parvovirus, raccoon pox virus, Ranikhet virus, rat cytomegalovirus, rat parvovirus, rat virus, Rauscher virus, recombinant vaccinia virus, recombinant virus, reovirus, reovirus 1, reovirus 2, reovirus 3, reptile type C virus, respiratory infection virus, respiratory syncytial virus, respiratory virus, reticuloendotheliosis virus, Rhabdovirus, Rhabdovirus carpia, Rhadinovirus, Rhinovirus, Rhizidiovirus, Rift Valley fever virus, Riley virus, distemper virus, RNA oncogenic virus, Ross River virus, rotavirus, measles virus, Rous sarcoma virus, rubella virus, measles virus, rubivirus, Russian autumn encephalitis, monkey virus SA 11, SA2 virus, Sabia virus, Sagiyama virus, Saimirine herpesvirus 1, salivary gland virus, phlebotomine fever group of viruses, Sanjimba virus, SARS virus, SDAV (sialodacryoadenitis virus), sealpox virus, Semliki Forest virus, Seoul virus, sheeppox virus, Shope fibroma virus, Shope papillomavirus, foamy monkey virus, monkey hepatitis A virus, simian-human immunodeficiency virus, simian immunodeficiency virus, simian parainfluenza virus, simian T-cell lymphotropic virus, monkey virus, simian virus 40, Simplexvirus, Sin Nombre virus, Sindbis virus, smallpox virus, South American hemorrhagic fever viruses, sparrowpox virus, Spumavirus, Squirrel fibroma virus, Squirrel monkey retrovirus, SSV 1 group of viruses, STLV (Simonee T-cell lymphotropic virus) type I, STLV (Simonee T-cell lymphotropic virus) type II, STLV (Simonee T-cell lymphotropic virus) type III, Papular stomatitis virus, Submaxillary virus, Pseudorabies virus 1, Pseudorabies virus 2, Suipoxvirus, Swamp fever virus, Swinepox virus, Murine leukemia virus, TAC virus, Tacaribe complex virus, Tacaribe virus, Tanapox virus, Taterapox virus, Tench reovirus, Theiler's encephalomyelitis virus, Theiler virus, Togoto virus, Thottapalayam virus, Tick-borne encephalitis virus, Tioman, Togavirus, Torovirus, Tumor Virus, Tupaia Virus, Turkey Rhinotracheitis Virus, Turkey Pox Virus, Retrovirus Type C, Oncovirus Type D, Retrovirus Group D, Ulcer Rhabdovirus, Una Virus, Uukuniemi Virus Group, Vaccinia Virus, Vacuolating Virus, Varicella Zoster Virus, Varicellovirus, Varicola Virus, Smallpox Virus, Smallpox Virus, Wasingishu Disease Virus, VEE Virus, Venezuelan Equine Encephalitis Virus, Venezuelan Equine Encephalomyelitis Virus, Venezuelan Hemorrhagic Fever Virus, Vesicular Stomatitis Virus, Vesiculovirus, Vilyuy Virus, Viper Retrovirus, Hemorrhagic septicemia, Visna-Maedi virus, Visna virus, volepox virus, VSV (vesicular stomatitis virus), Wallal virus, Warrego virus, pipilloma virus, WEE virus, West Nile virus, Western equine encephalitis virus, Western equine encephalomyelitis virus, Whataroa virus, winter vomiting virus, woodchuck hepatitis B virus, woolly monkey sarcoma virus, wound tumor virus, WRSV virus, Yaba monkey tumor virus, Yaba virus, Yatapoxvirus, yellow fever virus and Yug Bogdanovac virus.

[00403] In some embodiments, the pathogenic infection is caused by a retrovirus. Exemplary retroviruses include, but are not limited to, human immunodeficiency virus (HIV), human T-cell leukemia viruses (HTLV), Moloney murine leukemia virus (MuLV), mouse mammary tumor virus (MMTV), avian leukemia and sarcoma viruses, or Mazen-Pfizer simian virus.

[00404] In some embodiments, a cytokine conjugate (e.g., an IL-2 conjugate) described herein is administered to a subject with a retroviral infection or in latency to reduce and/or eliminate infected cells that are in a dormant state. In some cases, the retrovirus includes human immunodeficiency virus (HIV), human T-cell leukemia viruses (HTLV), Moloney murine leukemia virus (MuLV), murine mammary tumor virus (MMTV), avian leukemia and sarcoma viruses, or Mazen-Pfizer simian virus. In some cases, the cytokine conjugate redirects CD8+ T cells to recognize and eliminate infected cells that are in a dormant state.

[00405] In some cases, the cytokine conjugate is an IL-2 conjugate. In some cases, the IL-2 conjugate is administered to a subject with a retroviral infection or in a latency period to reduce the number and/or eliminate infected cells that are in a dormant state. In some cases, the retrovirus includes human immunodeficiency virus (HIV), human T-cell leukemia viruses (HTLV), Moloney murine leukemia virus (MuLV), murine mammary tumor virus (MMTV), avian leukemia and sarcoma viruses, or Mazen-Pfizer monkey virus. In some cases, the IL-2 conjugate redirects CD8+ T cells to recognize and eliminate infected cells that are in a dormant state. In additional cases, the IL-2 conjugate is administered to the subject in combination with antiretroviral therapy.

In some embodiments, the retrovirus is HIV. In some cases, a cytokine conjugate (e.g., an IL-2 conjugate) described herein is administered to a subject who has acquired immunodeficiency syndrome (AIDS) or is latently infected to reduce and/or eliminate HIV-infected cells (e.g., CD4+ T cells) that are in a resting state. In some cases, the cytokine conjugate is an IL-2 conjugate. In some cases, the IL-2 conjugate is administered to the subject in combination with antiretroviral therapy. Exemplary antiretroviral therapy for HIV includes: (a) nucleoside reverse transcriptase inhibitors (NRTIs) such as abacavir, emtricitabine, lamivudine, tenofovir disoproxil fumarate, and zidovudine; (b) non-nucleoside reverse transcriptase inhibitors (NNRTIs), such as efavirenz, etravirine, nevirapine, or rilpivirine; (c) protease inhibitors (PIs), such as atazanavir, darunavir, fosamprenavir, ritonavir, saquinavir, and tipranavir; (d) fusion inhibitors, such as enfuvirtide; (e) CCR5 agonists, such as maraviroc; (f) integrase inhibitors, such as dolutegravir and raltegravir; (g) post-attachment inhibitors, such as ibalizumab; (h) pharmacokinetic enhancers, such as cobicistat; and (i) cocktails, such as abacavir and limivudine; abacavir, dolutegravir, and limivudine; abacavir, limivudine, and zidovudine; Atazanavir and cobicistat; Bictegravir, emtricitabine and tenofovir alafenamide; Darunavir and cobicistat; Dolutegravir and rilpivirine; Efavirenz, emtricitabine and tenofovir disoproxil fumarate; Efavirenz, limivudine and tenofovir disoproxil fumarate; Efavirenz, limivudine and tenofovir disoproxil fumarate; Elvitegravir, cobicistat, emtricitabine and tenofovir alafenamide fumarate; Elvitegravir, cobicistat, emtricitabine and tenofovir disoproxil fumarate; Emtricitabine, rilpivirine and tenofovir alafenamide; emtricitabine, rilpivirine, and tenofovir disoproxil fumarate; emtricitabine and tenofovir alafenamide; emtricitabine and tenofovir disoproxil fumarate; limivudine and tenofovir disoproxil fumarate; limivudine and zidovudine; and lopinavir and ritonavir.

[00406] In some cases, the IL-2 conjugate is administered to a subject in combination with an antiviral therapy such as nucleoside reverse transcriptase inhibitors (NRTIs) such as abacavir, emtricitabine, lamivudine, tenofovir disoproxil fumarate, and zidovudine; non-nucleoside reverse transcriptase inhibitors (NNRTIs) such as efavirenz, etravirine, nevirapine, or rilpivirine; protease inhibitors (PIs) such as atazanavir, darunavir, fosamprenavir, ritonavir, saquinavir, and tipranavir; fusion inhibitors such as enfuvirtide; CCR5 agonists such as maraviroc; integrase inhibitors such as dolutegravir and raltegravir; post-attachment inhibitors such as ibalizumab; Pharmacokinetic enhancers such as cobicistat; or cocktails such as abacavir and limivudine; abacavir, dolutegravir and limivudine; abacavir, limivudine and zidovudine; atazanavir and cobicistat; bictegravir, emtricitabine and tenofovir alafenamide; darunavir and cobicistat; dolutegravir and rilpivirine; efavirenz, emtricitabine and tenofovir disoproxil fumarate; efavirenz, limivudine and tenofovir disoproxil fumarate; efavirenz, limivudine and tenofovir disoproxil fumarate; elvitegravir, cobicistat, emtricitabine and tenofovir alafenamide fumarate; elvitegravir, cobicistat, emtricitabine, and tenofovir disoproxil fumarate; emtricitabine, rilpivirine, and tenofovir alafenamide; emtricitabine, rilpivirine, and tenofovir disoproxil fumarate; emtricitabine and tenofovir alafenamide; emtricitabine and tenofovir disoproxil fumarate; limivudine and tenofovir disoproxil fumarate; limivudine and zidovudine; and lopinavir and ritonavir.

[00407] In some embodiments, the virus is a hepatitis virus, such as hepatitis A, B, C, D, or E. In some cases, a cytokine conjugate (e.g., an IL-2 conjugate) described herein is administered to a subject with infectious hepatitis or in a latent state to reduce the number and/or eliminate infected cells that are in a resting state. In some cases, the cytokine conjugate redirects CD8+ T cells to recognize and eliminate infected cells that are in a resting state.

[00408] In some cases, the cytokine conjugate is an IL-2 conjugate. In some cases, the IL-2 conjugate is administered to a subject with or in latency with hepatitis to reduce and/or eliminate infected cells that are in a resting state. In some cases, the IL-2 conjugate redirects CD8+ T cells to recognize and eliminate infected cells that are in a resting state. In some cases, the IL-2 conjugate is administered to the subject in combination with an antiviral therapy. Exemplary antiviral therapy for hepatitis includes ribavirin; NS3/4A protease inhibitors such as paritaprevir, simeprevir, and grazoprevir; NS5A protease inhibitors such as ledipasvir, ombitasvir, elbasvir, and daclatasvir; NS5B nucleotide/nucleoside and non-nucleoside polymerase inhibitors such as sofosbuvir and dasabavir; and combinations such as ledipasvir-sofosbuvir, dasabavir-ombitasvir-paritaprevir-ritonavir; elbasvir-grazoprevir, ombitasvir-paritaprevir-ritonavir, sofosbuvir-velpatasvir, sofosbuvir-velpatasvir-voxilaprevir and glecaprevir-pibrentasvir; and interferons such as peg-interferon alfa-2a, peg-interferon alfa-2b and interferon alfa-2b. In some cases, the IL-2 e conjugate is administered to a subject in combination with an antiviral therapy such as ribavirin; NS3/4A protease inhibitors such as paritaprevir, simeprevir and grazoprevir; NS5A protease inhibitors such as ledipasvir, ombitasvir, elbasvir and daclatasvir; NS5B nucleotide/nucleoside and non-nucleoside polymerase inhibitors such as sofosbuvir and dasabavir; and combinations such as ledipasvir-sofosbuvir, dasabavir-ombitasvir-paritaprevir-ritonavir; elbasvir-grazoprevir, ombitasvir-paritaprevir-ritonavir, sofosbuvir-velpatasvir, sofosbuvir-velpatasvir-voxilaprevir and glecaprevir-pibrentasvir; and interferons such as peg-interferon alfa-2a, peg-interferon alfa-2b and interferon alfa-2b.

Autoimmune disease or disorder

[00409] In some embodiments, described herein is also a method of treating an autoimmune disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a cytokine conjugate (e.g., an IL-2 conjugate) described herein. In some cases, the IL-2 conjugate comprises an isolated and purified IL-2 polypeptide and a conjugate moiety, wherein the IL-2 conjugate has reduced affinity for an IL-2 receptor β subunit (IL-2Rβ), an IL-2 receptor γ subunit (IL-2Rγ), or a combination thereof, compared to a wild-type IL-2 polypeptide. In some cases, the IL-2 conjugate comprises an isolated and purified IL-2 polypeptide and a conjugation moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid residue selected from P2, T3, S4, S5, S6, T7, K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, R81, P82, R83, D84, S87, N88, N89, V91, I92, L94, E95, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, T113, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some cases, the amino acid residue is selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, I92, L94, E95, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133. In some cases, the amino acid residue is selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95, and Q126. In some cases, the IL-2 conjugate interacts with the IL-2Rαβγ complex, but with reduced affinity for the IL-2Rβ and IL-2Rγ subunits, or it will reduce the recruitment of the IL-2R γ subunit to the IL-2/IL-2Rβ complex. In some cases, the modified IL-2 polypeptide retains binding affinity for IL-2Rα compared to the wild-type IL-2 polypeptide. In such cases, the IL-2/IL-2Rαβγ complex stimulates or enhances the expansion of CD4+ Tregs. In additional cases, the modified IL-2 polypeptide provides an increase in the dose required to activate Teff and/or NK cells via the IL-2Rβγ complex, thereby expanding the dose range for Tregs activation via the IL-2Rαβγ complex (or expanding the therapeutic window of IL-2 for Tregs activation via the IL-2Rαβγ complex).

[00410] In some cases, the autoimmune disease or disorder includes alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes, juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, multiple sclerosis, pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis, vitiligo, or Wegener's granulomatosis.

[00411] In some cases, a cytokine conjugate (e.g., interleukin, IFN, or TNF) is administered to a subject who has alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes, juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, multiple sclerosis, pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis, vitiligo, or Wegener's granulomatosis.

[00412] In some cases, the IL-2 conjugate is administered to a subject having alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes, juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, multiple sclerosis, pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis, vitiligo, or Wegener's granulomatosis. In some cases, the IL-2 conjugate is administered to a subject having type 1 diabetes. In some cases, the IL-2 conjugate is administered to a subject who has Graves' disease. In some cases, the IL-2 conjugate is administered to a subject who has multiple sclerosis. In some cases, the IL-2 conjugate is administered to a subject who has psoriasis. In some cases, the IL-2 conjugate is administered to a subject who has rheumatoid arthritis. In some cases, the IL-2 conjugate is administered to a subject who has Sjogren's syndrome. In some cases, the IL-2 conjugate is administered to a subject who has systemic lupus erythematosus. In some cases, the IL-2 conjugate is administered to a subject who has uveitis. In some cases, the IL-2 conjugate is administered to a subject who has Wegener's granulomatosis.

[00413] In some cases, a cytokine conjugate (e.g., an IL-2 conjugate) is administered to a subject to treat graft-versus-host disease (GVHD).

[00414] In some embodiments, the subject is further administered an additional therapeutic agent. In some cases, the additional therapeutic agent is administered concurrently with the cytokine conjugate (e.g., IL-2 conjugate). In other cases, the additional therapeutic agent and the cytokine conjugate (e.g., IL-2 conjugate) are administered sequentially, such as the cytokine conjugate (e.g., IL-2 conjugate) is administered before the additional therapeutic agent or the cytokine conjugate (e.g., IL-2 conjugate) is administered after the additional therapeutic agent is administered.

[00415] Illustrative additional therapeutic agents for treating an autoimmune disease or disorder include, but are not limited to, corticosteroids such as prednisone, budesonide, or prednisolone; calcineurin inhibitors such as cyclosporine or tacrolimus; mTOR inhibitors such as sirolimus or everolimus; IMDH inhibitors such as azathioprine, leflunomide, or mycophenolate; biologics such as abacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, or vedolizumab; and monoclonal antibodies such as basiliximab, daclizumab, or muromonab.

[00416] In some cases, the cytokine conjugate (e.g., IL-2 conjugate) is administered with an additional therapeutic agent selected from a corticosteroid such as prednisone, budesonide, or prednisolone; a calcineurin inhibitor such as cyclosporine or tacrolimus; an mTOR inhibitor such as sirolimus or everolimus; an IMDH inhibitor such as azathioprine, leflunomide, or mycophenolate; a biologic such as abacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, or vedolizumab; as well as a monoclonal antibody such as basiliximab, daclizumab, or muromonab.

Development of tools for adoptive cell therapy

[00417] Disclosed herein in some embodiments are methods for producing an adoptive cell therapy composition useful for treating a disease or condition described herein (e.g., a proliferative disease or condition, a pathogenic infection, and/or an autoimmune disease or condition) in a subject in need thereof, comprising a) providing immune cells obtainable from a subject in need thereof; b) engineering the immune cells to express a modified IL-2 polypeptide, IL-2 conjugate, IL-2Rβγ binding protein, or immune cell activator, wherein the immune cell comprises a CD4+ helper cell, a CD8+ effector naive and memory cell, a CD8+ cytotoxic T cell, a suppressor T cell, a natural killer (NK) cell, or a natural killer T (NKT) cell. In some embodiments, the immune cell is engineered to additionally express a chimeric antigen receptor (CAR). In some embodiments, step (b) of constructing comprises contacting immune cells obtained from a subject with a vector (e.g., polynucleotide sequence) encoding a modified IL-2 polypeptide, an IL-2 conjugate, an IL-2Rβγ binding protein, or an immune cell activator. In some cases, the vector comprises the articles of manufacture disclosed herein. In some cases, methods for producing an adoptive cell therapy agent are performed using the kits disclosed herein. In some embodiments, a subject is treated with an adoptive cell therapy agent by administering a therapeutically effective amount of the adoptive cell therapy agent. In some cases, the subject is diagnosed with a disease or condition. In some cases, the adoptive cell therapy agent is effective for treating the disease or condition in the subject. In some embodiments, the disease or condition comprises a proliferative disease (e.g., cancer). In some embodiments, the disease or condition comprises a pathogenic infection. In some cases, the disease or condition comprises an autoimmune disease, is a cancer, such as those described herein.

[00418] Disclosed herein in some embodiments are methods for producing an adoptive cell therapy composition useful for treating a disease or condition described herein (e.g., proliferative disease or condition, pathogenic infection, and/or autoimmune disease or condition) in a subject in need thereof, comprising a) providing immune cells obtainable from a subject in need thereof; b) contacting the immune cells with a modified IL-2 polypeptide, IL-2 conjugate, IL-2Rβγ binding protein, or immune cell activator, wherein the immune cell comprises a CD4+ helper cell, a CD8+ effector naive and memory cell, a CD8+ cytotoxic T cell, a suppressor T cell, a natural killer (NK) cell, or a natural killer T (NKT) cell. In some embodiments, the immune cell is engineered to additionally express a chimeric antigen receptor (CAR). In some cases, the modified IL-2 polypeptide, IL-2 conjugate, IL-2Rβγ binding protein, or immune cell activator comprises the articles disclosed herein. In some cases, methods for producing an agent for adoptive cell therapy of cells are performed using the kits disclosed herein. In some embodiments, a subject is treated with an agent for adoptive cell therapy by administering a therapeutically effective amount of the agent for adoptive cell therapy. In some cases, the subject is diagnosed with a disease or pathological condition. In some cases, the agent for adoptive cell therapy is effective for treating the disease or pathological condition in the subject. In some embodiments, the disease or pathological condition comprises a proliferative disease (e.g., cancer). In some embodiments, the disease or pathological condition comprises a pathogenic infection. In some cases, the disease or pathological condition comprises an autoimmune disease, is a cancer, such as described herein.

[00419] In some embodiments, the modified IL-2 polypeptide or IL-2 conjugate comprises a mutation at residue F42, corresponding to position 42 of SEQ ID NO: 1, and comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da.

[00420] In some embodiments, the modified IL-2 polypeptide or IL-2 conjugate comprises a mutation at residue P65 corresponding to position 65 of SEQ ID NO: 1 and comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da.

[00421] In some embodiments, the modified IL-2 polypeptide or IL-2 conjugate comprises a mutation at residue E62, corresponding to position 62 of SEQ ID NO: 1, and comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da.

[00422] In some cases, the molecular weight of PEG is effective to improve the manufacturing process of the IL-2 polypeptide or IL-2 conjugate as a reagent for adoptive cell therapy agents. In some embodiments, the molecular weight of PEG improves the solubility of the IL-2 polypeptide or IL-2 conjugate. In some cases, the molecular weight of PEG improves the purification process in the manufacture of an adoptive cell therapy agent. In some cases, the molecular weight of PEG improves the stability of the IL-2 polypeptide or IL-2 conjugate.

[00423] Disclosed herein in some embodiments are methods of treating an autoimmune disease or disorder in a subject in need thereof that comprise administering to the subject an adoptive cell therapy agent described herein. In some cases, the adoptive cell therapy agent is developed using the methods described herein. In some cases, the adoptive cell therapy agent is administered to the subject in addition to a cytokine conjugate (e.g., an IL-2 conjugate) described herein. In some cases, the cytokine conjugate is administered prior to the adoptive cell therapy agent. In some cases, the cytokine conjugate is administered subsequent to the adoptive cell therapy agent. In some cases, the adoptive cell therapy agent is effective in promoting expansion of an immune cell population in the subject (e.g., populations of CD4+ helper cells, CD8+ effector naive and memory cells, NK cells and/or NKT cells, populations of Treg cells).

[00424] Disclosed herein in some embodiments are methods of treating a pathogenic infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an adoptive cell therapy agent described herein. In some cases, the adoptive cell therapy agent is developed using the methods described herein. In some cases, the adoptive cell therapy agent is administered to the subject in addition to a cytokine conjugate (e.g., an IL-2 conjugate) described herein. In some cases, the cytokine conjugate is administered prior to the adoptive cell therapy agent. In some cases, the cytokine conjugate is administered subsequent to the adoptive cell therapy agent. In some cases, the adoptive cell therapy agent is effective in promoting expansion of an immune cell population in the subject (e.g., populations of CD4+ helper cells, CD8+ effector naive and memory cells, NK cells and/or NKT cells, populations of Treg cells).

[00425] Disclosed herein in some embodiments are methods for treating a proliferative disease or disorder (e.g., cancer) in a subject in need thereof, which comprises administering to the subject an adoptive cell therapy agent described herein. In some cases, the adoptive cell therapy agent is developed using the methods described herein. In some cases, the adoptive cell therapy agent is administered to the subject in addition to the cytokine conjugate (e.g., the IL-2 conjugate) described herein. In some cases, the cytokine conjugate is administered before the adoptive cell therapy agent. In some cases, the cytokine conjugate is administered after the adoptive cell therapy agent. In some cases, the adoptive cell therapy agent is effective in promoting expansion of an immune cell population in the subject (e.g., populations of CD4+ helper cells, CD8+ effector naive and memory cells, NK cells and/or NKT cells, populations of Treg cells).

Methods of reproduction of cell populations

[00426] In some embodiments, methods for expanding populations of lymphocytes, such as populations of CD4+ helper cells, CD8+ effector naive and memory cells, NK cells and/or NKT cells, or methods for expanding a population of Treg cells, are further described herein. In some cases, the method comprises contacting a cell with a cytokine conjugate described herein and interacting the cytokine with a cytokine receptor to form a complex, wherein the complex stimulates expansion of a distinct population of lymphocytes.

[00427] In some cases, a method of expanding a population of CD4+ helper cells, CD8+ effector naive and memory cells, natural killer (NK) cells, or natural killer T (NKT) cells comprises contacting the population of cells with an isolated and modified IL-2 polypeptide described above for a time sufficient to induce formation of a complex with IL-2Rβ, thereby stimulating expansion of the population of Teff and/or NK cells. In some cases, a method of expanding populations of CD4+ helper cells, CD8+ effector naive and memory cells, NK cells, and/or NKT cells comprises (a) contacting the population of cells with an IL-2 conjugate described herein; and (b) allowing IL-2 to interact with IL-2Rβ and IL-2Rγ subunits to form an IL-2/IL-2Rβγ complex; wherein the IL-2 conjugate has reduced affinity for the IL-2Rα subunit, and wherein the IL-2/IL-2Rβγ complex stimulates the expansion of CD4+ helper cells, CD8+ effector naive and memory cells, NK cells and/or NKT cells. As described above, the IL-2 conjugate comprises an isolated and purified IL-2 polypeptide and a conjugation moiety that binds to the isolated and purified IL-2 polypeptide at an amino acid position selected from K35, T37, R38, T41, F42, K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105, and Y107, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some cases, the amino acid position is selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some cases, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In some cases, the amino acid position is selected from T37, R38, T41, F42, F44, Y45, E61, E62, E68, P65, V69, L72, and Y107. In some cases, the amino acid position is selected from T37, T41, F42, F44, Y45, P65, V69, L72, and Y107. In some cases, the amino acid position is selected from R38 and K64. In some cases, the amino acid position is selected from E61, E62, and E68. In some cases, the amino acid position is E62.

[00428] In some cases, the IL-2 conjugate provides for expansion of CD4+ regulatory T cells (Treg) by less than 20%, 15%, 10%, 5%, or 1% of the cell population. In some cases, the IL-2 conjugate does not provide for expansion of CD4+ Treg cells in the cell population. In some cases, the ratio of Teff cells to Treg cells in the cell population after incubation with the isolated and modified IL-2 polypeptide is at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, or 100:1. In some cases, the ratio of Teff cells to Treg cells in the cell population after incubation with the isolated and modified IL-2 polypeptide is approximately 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, or 100:1.

[00429] In some cases, the time sufficient to induce complex formation with IL-2Rβ is at least 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days. In some cases, the time sufficient to induce complex formation with IL-2Rβ is approximately 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 8 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.

[00430] In some cases, the method is an in vivo method.

[00431] In some cases, the method is an in vitro method.

[00432] In some cases, the method is an ex vivo method.

Obtaining cytokine polypeptide

[00433] In some cases, the cytokine polypeptides (e.g., interleukin, IFN, or TNF) described herein, comprising either a mutation of a natural amino acid or a mutation of a non-natural amino acid, are produced recombinantly or are chemically synthesized. In some cases, the cytokine polypeptides (e.g., IL-2) described herein are produced recombinantly, such as in either a host cell system or a cell-free system.

[00434] In some cases, the cytokine polypeptides (e.g., IL-2) are produced recombinantly using a host cell system. In some cases, the host cell is a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell, or a plant cell), a prokaryotic cell (e.g., a gram-positive bacterium or a gram-negative bacterium). In some cases, the eukaryotic host cell is a mammalian host cell. In some cases, the mammalian host cell is an established cell line or a cell line that contains the genetic material of interest integrated into its own genome and has the ability to express the product of the genetic material after many generations of cell division. In other cases, the mammalian host cell is a transient cell line or a cell line that does not contain the genetic material of interest integrated into its own genome and does not have the ability to express the product of the genetic material after many generations of cell division.

[00435] Exemplary mammalian host cells include the 293T cell line, the 293A cell line, the 293FT cell line, the 293F cells, the 293 H cells, the A549 cells, the MDCK cells, the CHO DG44 cells, the CHO-S cells, the CHO-K1 cells, the Expi293F™ cells, the Flp-In™ T-REx™ 293 cell line, the Flp-In™-293 cell line, the Flp-In™-3T3 cell line, the Flp-In™-BHK cell line, the Flp-In™-CHO cell line, the Flp-In™-CV-1 cell line, the Flp-In™-Jurkat cell line, the FreeStyle™ 293-F cells, the FreeStyle™ CHO-S cells, the GripTite™ 293 MSR, GS-CHO cell line, HepaRG™ cells, T-REx™ Jurkat cell line, Per.C6 cells, T-REx™-293 cell line, T-REx™-CHO cell line, and T-REx™-HeLa cell line.

[00436] In some embodiments, the eukaryotic host cell is an insect host cell. Exemplary insect host cells include Drosophila S2 cells, Sf9 cells, Sf21 cells, High Five™ cells, and expresSF+® cells.

[00437] In some embodiments, the eukaryotic host cell is a yeast host cell. Exemplary yeast host cells include Pichia pastoris yeast strains such as GS115, KM71H, SMD1168, SMD1168H, and X-33, and a Saccharomyces cerevisiae yeast strain such as INVSc1.

[00438] In some embodiments, the eukaryotic host cell is a plant host cell. In some cases, the plant cell comprises an algal cell. Exemplary plant cell lines include strains from Chlamydomonas reinhardtii 137c or Synechococcus elongatus PPC 7942.

[00439] In some embodiments, the host cell is a prokaryotic host cell. Exemplary prokaryotic host cells include BL21, Mach1™, DH10B™, TOP10, DH5α, DH10Bac™, OmniMax™, MegaX™, DH12S™, INV110, TOP10F', INVαF, TOP10/P3, ccdB Survival, PIR1, PIR2, Stbl2™, Stbl3™, or Stbl4™.

[00440] In some cases, suitable polynucleic acid molecules or vectors for producing the IL-2 polypeptide described herein include any suitable vectors derived from either a eukaryotic or prokaryotic source. Exemplary polynucleic acid molecules or vectors include vectors from bacteria (e.g., E. coli ), insects, yeast (e.g., Pichia pastoris ), algae, or a mammalian source. Bacterial vectors include, for example, pACYC177, pASK75, the pBAD series of vectors, the pBADM series of vectors, the pET series of vectors, the pETM series of vectors, the pGEX series of vectors, pHAT, pHAT2, pMal-c2, pMal-p2, the pQE series of vectors, pRSET A, pRSET B, pRSET C, the pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC or pTAC-MAT-2.

[00441] Insect vectors include, for example, pFastBac1, pFastBac DUAL, pFastBac ET, pFastBac HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac, M30c, pVL1392, pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, FLAG vectors such as pPolh-FLAG1 or pPolh-MAT 2, or MAT vectors such as pPolh-MAT1 or pPolh-MAT2.

[00442] Yeast vectors include, for example, the Gateway® vector pDEST™ 14, Gateway® vector pDEST™ 15, Gateway® vector pDEST™ 17, Gateway® vector pDEST™ 24, Gateway® vector pYES-DEST52, pBAD-DEST49 Gateway® Delivery Vector, pAO815 VectorPichia, vector pFLD1Pichi pastoris, pGAPZA vector, B & CPichia pastoris, pPIC3.5K vectorPichia, pPIC6 vector A, B & CPichia, pPIC9K vectorPichia, pTEF1/Zeo, yeast vector pYES2, yeast vector pYES2/CT, yeast vector pYES2/NT A, B & C, or yeast vector pYES3/CT.

[00443] Algal vectors include, for example, the pChlamy-4 vector or the MCS vector.

[00444] Mammalian vectors include, for example, transient expression vectors or stable expression vectors. Exemplary mammalian transient expression vectors include p3xFLAG-CMV 8, pFLAG-Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a, b,c, pFLAG-CMV 5.1, pFLAG-CMV 5a, b,c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. Exemplary mammalian stable expression vectors include pFLAG-CMV 3, p3xFLAG-CMV 9, p3xFLAG-CMV 13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10, p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1 or pBICEP-CMV 2.

[00445] In some cases, a cell-free system is used to produce a cytokine polypeptide (e.g., IL-2) described herein. In some cases, the cell-free system comprises a mixture of cytoplasmic and/or nuclear components from a cell and is suitable for in vitro nucleic acid synthesis. In some cases, the cell-free system uses prokaryotic cell components. In other cases, the cell-free system uses eukaryotic cell components. The nucleic acid synthesis is achieved in a cell-free system, such as a Drosophila cell, a Xenopus egg, an archaea, or a HeLa cell. Exemplary cell-free systems include the E. coli S30 extract system, the E. coli T7 S30 system, or the PURExpress®, XpressCF, and XpressCF+ system.

[00446] Cell-free translation systems contain various components, such as plasmids, mRNA, DNA, tRNA, synthetases, release factors, ribosomes, chaperone proteins, translation initiation and elongation factors, natural and/or unnatural amino acids, and/or other components used to express the protein. Such components are optionally modified to improve yield, increase the rate of synthesis, improve the fidelity of the protein product, or incorporate unnatural amino acids. In some embodiments, the cytokines described herein are synthesized using the cell-free translation systems described in US 8,778,631; US 2017/0283469; US 2018/0051065; US 2014/0315245; or US 8,778,631. In some embodiments, the cell-free translation systems include modified release factors or even the removal of one or more release factors from the system. In some embodiments, the cell-free translation systems include a reduced concentration of protease. In some embodiments, the cell-free translation systems include modified tRNAs with reassigned codons used to encode unnatural amino acids. In some embodiments, the synthetases described herein for incorporating unnatural amino acids are used in the cell-free translation systems. In some embodiments, the tRNAs are preloaded with unnatural amino acids using enzymatic or chemical methods prior to addition to the cell-free translation system. In some embodiments, the components for the cell-free translation system are obtained from modified organisms, such as modified bacteria, yeast, or other organism.

[00447] In some embodiments, a cytokine polypeptide (e.g., IL-2) is produced in a circularly permutated form using either a host expression system or a cell-free system.

Obtaining a cytokine polypeptide containing an unnatural amino acid

[00448] The present invention may utilize an orthogonal or expanded genetic code in which one or more specific codons present in the nucleic acid sequence of a cytokine polypeptide (e.g., IL-2) are designed to encode an unnatural amino acid so that it can be genetically inserted into the cytokine (e.g., IL-2) using an orthogonal tRNA synthetase/tRNA pair. The orthogonal tRNA synthetase/tRNA pair is capable of loading a tRNA with an unnatural amino acid and is capable of inserting the unnatural amino acid into a polypeptide chain in response to a codon.

[00449] In some cases, the codon is an amber codon, an ochre codon, an opal codon, or a quadruplet codon. In some cases, the codon corresponds to an orthogonal tRNA that will be used to transfer the unnatural amino acid. In some cases, the codon is an amber codon. In other cases, the codon is an orthogonal codon.

[00450] In some cases, the codon is a quadruplet codon that can be decoded by the orthogonal ribosome ribo-Q1. In some cases, the quadruplet codon is as shown in Neumann, et al ., "Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome," Nature , 464 (7287): 441–444 (2010).

[00451] In some cases, a codon used in the present invention is a recoded codon, such as a synonymous codon or a rare codon that is replaced by an alternative codon. In some cases, the recoded codon is as described in Napolitano, et al., "Emergent rules for codon choice elucidated by editing rare argine codons in Escherichia coli ," PNAS , 113 (38): E5588-5597 (2016). In some cases, the recoded codon is as described in Ostrov et al ., "Design, synthesis, and testing toward a 57-codon genome," Science 353 (6301): 819-822 (2016).

[00452] In some cases, non-natural nucleic acids are used, which results in the incorporation of one or more non-natural amino acids into the cytokine (e.g., IL-2). Illustrative non-naturally occurring nucleic acids include, but are not limited to, uracil-5-yl, hypoxanthine-9-yl (I), 2-aminoadenine-9-yl, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl- and other alkyl derivatives of adenine and guanine, 2-propyl- and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halogen, in particular 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, as well as 3-deazaguanine and 3-deazaadenine. Certain unnatural nucleic acids such as 5-substituted pyrimidines, 6-azapyrimidines and N-2-substituted purines, N-6-substituted purines, O-6-substituted purines, 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, 5-methylcytosine, those that enhance the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, enlarged nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines and N-2-, N-6- and O-6-substituted purines including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-Methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl(-C≡C-CH₃)-uracil, 5-propynylcytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogen, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halogen, in particular 5-bromo, 5-trifluoromethyl, other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines, phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps, phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindolcytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one), those in which the purine or pyrimidine base is replaced by other heterocycles, 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil and 5-iodouracil, 2-aminoadenine, 6-thioguanine, 2-thiothymine, 4-thiothymine, 5-propynyluracil, 4-thiouracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine and those described in U.S. Patent Nos. 3,687,808; 4,845,205; 4,910,300; 4,948,882; 5093232; 5130302; 5134066; 5175273; 5367066; 5432272; 5457187; 5459255; 5484908; 5502177; 5525711; 5552540; 5587469; 5594121; 5596091; 5614617; 5645985; 5681941; 5750692; 5763588; 5830653 and 6005096; WO 99/62923; Kandimalla et al., (2001) Bioorg. Med. Chem. 9:807-813; The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, J.I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research and Applications, Crooke and Lebleu Eds., CRC Press, 1993, 273-288. Further base modifications can be found, for example, in U.S. Patent No. 3,687,808; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke and Lebleu ed., CRC Press, 1993.

[00453] Non-naturally occurring nucleic acids containing various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and the nucleic acids in some cases contain one or more heterocyclic bases other than the basic five base components of naturally occurring nucleic acids. For example, a heterocyclic base includes in some cases uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo[2,3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyrimidin-3-yl groups, wherein purines are attached to the sugar moiety of the nucleic acid through position 9, pyrimidines through position 1, pyrrolopyrimidines through position 7, and pyrazolopyrimidines through position 1.

[00454] In some embodiments, the nucleotide analogs are also modified in the phosphate moiety. Modified phosphate moieties include, but are not limited to, those with a modification in the linkage between two nucleotides and include, for example, phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkylphosphonates including 3'-alkylenephosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidate and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. It should be understood that these phosphate or modified phosphate linkages between two nucleotides are formed by a 3'-5' linkage or a 2'-5' linkage and the linkage is characterized by inverted polarity, such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms. Many U.S. patents describe how to make and use nucleotides containing modified phosphates and include, but are not limited to, 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5453496; 5455233; 5466677; 5476925; 5519126; 5536821; 5541306; 5550111; 5563253; 5571799; 5587361 and 5625050.

[00455] In some embodiments, non-naturally occurring nucleic acids include 2',3'-dideoxy-2',3'-didehydronucleosides (PCT/US2002/006460), 5'-substituted derivatives of DNA and RNA (PCT/US2011/033961; Saha et al., J. Org Chem., 1995, 60, 788-789; Wang et al., Bioorganic & Medicinal Chemistry Letters, 1999, 9, 885-890; and Mikhailov et al., Nucleosides & Nucleotides, 1991, 10(1-3), 339-343; Leonid et al., 1995, 14(3-5), 901-905; and Eppacher et al., Helvetica Chimica Acta, 2004, 87, 3004-3020; PCT/JP2000/004720; PCT/JP2003/002342; PCT/JP2004/013216; PCT/JP2005/020435; PCT/JP2006/315479; PCT/JP2006/324484; PCT/JP2009/056718; PCT/JP2010/067560), or 5'-substituted monomers prepared as monophosphate with modified bases (Wang et al., Nucleosides Nucleotides & Nucleic Acids, 2004, 23 (1 & 2), 317-337).

[00456] In some embodiments, the non-natural nucleic acids include modifications at the 5'-position and 2'-position of the sugar ring (PCT/US94/02993), such as 5'-CH2 _- substituted 2'-O-protected nucleosides (Wu et al., Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al., Bioconjugate Chem. 1999, 10, 921-924). In some cases, unnatural nucleic acids include amide-linked nucleoside dimers prepared for incorporation into oligonucleotides, wherein the 3'-linked nucleoside in the dimer (5' to 3') contains 2'- _OCH3 and 5'-(S) _-CH3 (Mesmaeker et al., Synlett, 1997, 1287-1290). Unnatural nucleic acids may contain 2'-substituted 5'- _CH2 (or O) modified nucleosides (PCT/US92/01020). Unnatural nucleic acids may include 5'-methylenephosphonate DNA and RNA monomers and dimers (Bohringer et al., Tet. Lett., 1993, 34, 2723-2726; Collingwood et al., Synlett, 1995, 7, 703-705; and Hutter et al., Helvetica Chimica Acta, 2002, 85, 2777-2806). Unnatural nucleic acids may include 5'-phosphonate monomers having a 2'-substitution (US2006/0074035) and other modified 5'-phosphonate monomers (WO1997/35869). Unnatural nucleic acids may include 5'-modified methylene phosphonate monomers (EP614907 and EP629633). Unnatural nucleic acids may include analogs of 5'- or 6'-phosphonate ribonucleosides containing a hydroxyl group at the 5'- and/or 6'-position (Chen et al., Phosphorus, Sulfur and Silicon, 2002, 777, 1783-1786; Jung et al., Bioorg. Med. Chem., 2000, 8, 2501-2509; Gallier et al., Eur. J. Org. Chem., 2007, 925-933; and Hampton et al., J. Med. Chem., 1976, 19(8), 1029-1033). Unnatural nucleic acids may include 5'-phosphonate deoxyribonucleoside monomers and dimers having a 5'-phosphate group (Nawrot et al., Oligonucleotides, 2006, 16(1), 68-82). Unnatural nucleic acids may include nucleosides having a 6'-phosphonate group in which the 5'- and/or 6'-position is unsubstituted or substituted with a thiot- tert -butyl group (SC( _CH3 ) ₃ ) (and its analogs); methyleneamino group (CH ₂ NH ₂ ) (and its analogues) or cyano group (CN) (and its analogues) (Fairhurst et al., Synlett, 2001, 4, 467-472; Kappler et al., J. Med. Chem., 1986, 29, 1030-1038; Kappler et al., J. Med. Chem., 1982, 25, 1179-1184; Vrudhula et al., J. Med. Chem., 1987, 30, 888-894; Hampton et al., J. Med. Chem., 1976, 19, 1371-1377; Geze et al., J. Am. Chem. Soc, 1983, 105(26), 7638-7640; and Hampton et al., J. Am. Chem. Soc, 1973, 95(13), 4404-4414).

[00457] In some embodiments, the non-natural nucleic acids also comprise modifications of the sugar moiety. In some cases, the nucleic acids comprise one or more nucleosides wherein the sugar group has been modified. Such sugar-modified nucleosides are capable of imparting increased stability to nucleases, increased binding affinity, or certain other beneficial biological properties. In certain embodiments, the nucleic acids comprise a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituent groups (including 5' and/or 2' substituent groups, bridging two ring atoms to form bicyclic nucleic acids (BNAs), replacing the oxygen atom of the ribosyl ring with S, N(R) or C(R ₁ )(R ₂ ) (R=H, C ₁ -C ₁₂ alkyl or a protecting group); and combinations thereof. Examples of chemically modified sugars can be found in WO2008/101157, US2005/0130923 and WO2007/134181.

[00458] In some cases, the modified nucleic acid comprises modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or a cyclopentyl group representing a sugar "analog". The sugar can be in the pyranosyl or furanosyl form. The sugar moiety can be a furanoside of ribose, deoxyribose, arabinose, or 2'-O-alkylribose, and the sugar can be attached to the corresponding heterocyclic bases in either the [alpha] or [beta] anomeric configuration. Sugar modifications include, but are not limited to, 2'-alkoxy RNA analogs, 2'-amino RNA analogs, 2'-fluoro DNA, and 2'-alkoxy or amino RNA/DNA chimeras. For example, a sugar modification can include 2'-O-methyluridine or 2'-O-methylcytidine. Sugar modifications include 2'-O-alkyl substituted deoxyribonucleosides and 2'-O-ethylene glycol-like ribonucleosides. It is known to prepare these sugars or sugar analogs and the corresponding "nucleosides" wherein such sugars or analogs are attached to a heterocyclic base (nucleic acid base). Sugar modifications can also be prepared and combined with other modifications.

[00459] Modifications of the sugar moiety include naturally occurring modifications of ribose and deoxyribose, as well as non-naturally occurring modifications. Sugar modifications include, but are not limited to, the following modifications at the 2'-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C ₁ -C ₁₀ alkyl or C ₂ -C ₁₀ alkenyl and alkynyl. 2'-Sugar modifications also include, but are not limited to, -O[(CH ₂ ) _n O] _m CH ₃ , -O(CH ₂ ) _n OCH ₃ , -O(CH ₂ ) _n NH ₂ , -O(CH ₂ ) _n CH ₃ , -O(CH ₂ ) _n ONH ₂ , and -O(CH ₂ ) _n ON[(CH ₂ )n CH ₃ )] ₂ , where n and m are from 1 to about 10.

[00460] Other modifications at the 2'-position include, but are not limited to, lower _C1 - _C10 alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, _SCH3 , OCN, Cl, Br, CN, _CF3 , _OCF3 , _SOCH3 , SO2CH3, _ONO2 , _NO2 , _N3 , _NH2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, _{polyalkylamino} , substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for _improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents with similar properties. Similar modifications can also be made at other positions in the sugar, particularly at the 3' position of the sugar in the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and at the 5' position of the 5' terminal nucleotide. Modified sugars also include sugars that contain modifications in the bridging oxygen of the ring such as _CH2 and S. Sugar analog nucleotides can also contain sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. There are numerous U.S. patents that teach the preparation of such modified sugar structures and that detail and describe a number of base modifications, such as U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5466786; 5514785; 5519134; 5567811; 5576427; 5591722; 5597909; 5610300; 5627053; 5639873; 5646265; 5658873; 5670633; 4845205; 5130302; 5134066; 5175273; 5367066; 5432272; 5,457,187; 5459255; 5484908; 5502177; 5525711; 5552540; 5587469; 5594121, 5596091; 5614617; 5681941 and 5700920, each of which is incorporated herein by reference in its entirety.

[00461] Examples of nucleic acids having modified sugar moieties include, but are not limited to, nucleic acids containing substituent groups that are 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH ₃ , and 2'-O(CH ₂ ) ₂ OCH ₃ . The substituent at the 2'-position may also be selected from allyl, amino, azido, thio, O-allyl, O-(C ₁ -C _1O alkyl), OCF ₃ , O(CH ₂ ) ₂ SCH ₃ , O(CH ₂ ) ₂ -ON(R _m )(R _n ) and O-CH ₂ -C(=O)-N(R _m )(R _n ), where each R _m and R _n is independently H or substituted or unsubstituted C ₁ -C ₁₀ alkyl.

[00462] In certain embodiments, the nucleic acids described herein include one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acid comprises a bridge between the 4' and 2' atoms of the ribosyl ring. In certain embodiments, the nucleic acids provided herein include one or more bicyclic nucleic acids, wherein the bridge comprises a 4'-2' bicyclic nucleic acid. Examples of such 4'-2' bicyclic nucleic acids include, but are not limited to, one of the formulas: 4'-(CH ₂ )-O-2'(LNA);4'-(CH ₂ )-S-2';4'-(CH ₂ ) ₂ -O-2'(ENA);4'-CH(CH ₃ )-O-2' and 4'-CH(CH ₂ OCH ₃ )-O-2' and their analogs (see U.S. Patent No. 7,399,845); 4'-C(CH ₃ )(CH ₃ )-O-2' and its analogs (see WO2009/006478, WO2008/150729, US2004/0171570, U.S. Patent No. 7,427,672, Chattopadhyaya et al., J. Org. Chem., 209, 74, 118-134 and WO2008/154401). See also, for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Oram et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Patents 4,849,513; 5,015,733; 5,118,800; 5,118,802; 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; 6,525,191; 6,670,461; and 7,399,845; International Publication Nos. WO2004/106356, WO1994/14226, WO2005/021570, WO2007/090071, and WO2007/134181; U.S. Patent Publication Nos. US2004/0171570, US2007/0287831, and US2008/0039618; U.S. Provisional Patent Application Nos. 60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and 61/099,844; and International Patent Application Nos. PCT/US2008/064591, PCT US2008/066154, PCT US2008/068922 and PCT/DK98/00393.

[00463] In certain embodiments, nucleic acids comprise linked nucleic acids. Nucleic acids can be linked together using any linkage between nucleic acids. Two main classes of nucleic acid linkage groups are defined by the presence or absence of a phosphorus atom. Exemplary phosphorus-containing linkages between nucleic acids include, but are not limited to, phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Exemplary non-phosphorus-containing nucleic acid linkage groups include, but are not limited to, methylenemethylimino (-CH₂-N(CH₃)-O-CH₂-), thiodiester (-O-C(O)-S-), thionocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)₂-O-) and N, N*-dimethylhydrazine (-CH₂-N(CH₃)-N(CH₃)). In certain embodiments, bonds between nucleic acids having a chiral atom can be obtained as a racemic mixture, as individual enantiomers, for example, alkyl phosphonates and phosphorothioates. Unnatural nucleic acids may contain one modification. Unnatural nucleic acids may contain several modifications in one of the fragments or between different fragments.

[00464] Modifications of the backbone phosphate in a nucleic acid include, but are not limited to, methylphosphonate, phosphorothioate, phosphoramidate (bridged or unbridged), phosphotriester, phosphorodithioate, phosphodithioate, and boranophosphate, and may be used in any combination. Other non-phosphate linkages may also be used.

[00465] In some embodiments, modifications to the backbone (e.g., methylphosphonate, phosphorothioate, phosphoramidate and phosphorodithioate internucleotide linkages) can impart immunomodulatory activity to the modified nucleic acid and/or increase its stabilityin vivo.

[00466] In some cases, a phosphorus derivative (or modified phosphate group) is attached to the sugar or sugar analog moiety and may be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, or the like. Exemplary polynucleotides containing modified phosphate linkages or non-phosphate linkages can be found in Peyrottes et al., 1996, Nucleic Acids Res. 24: 1841-1848; Chaturvedi et al., 1996, Nucleic Acids Res. 24:2318-2323; and Schultz et al., (1996) Nucleic Acids Res. 24:2966-2973; Matteucci, 1997, “Oligonucleotide Analogs: an Overview” in Oligonucleotides as Therapeutic Agents, (Chadwick and Cardew, ed.) John Wiley and Sons, New York, NY; Zon, 1993, "Oligonucleoside Phosphorothioates" in Protocols for Oligonucleotides and Analogs, Synthesis and Properties, Humana Press, pp. 165-190; Miller et al., 1971, JACS 93:6657-6665; Jager et al., 1988, Biochem. 27:7247-7246; Nelson et al., 1997, JOC 62:7278-7287; US Patent No. 5453496 and Micklefield, 2001, Curr. Med. Chem. 8: 1157-1179.

[00467] In some cases, the backbone modification involves replacing the phosphodiester linkage with an alternative moiety, such as an anionic, neutral, or cationic group. Examples of such modifications include an anionic internucleoside linkage; a phosphoramidate modification from N3' to P5'; boranophosphate DNA; prooligonucleotides; neutral internucleoside linkages such as methylphosphonates; amide-linked DNA; methylene (methylimino) linkages; formatetal and thioformacetal linkages; backbones containing sulfonyl groups; morpholine oligonucleotides; peptide nucleic acids (PNA), and positively charged deoxyribonucleic guanidine (DNG) oligonucleotides (Micklefield, 2001, Current Medicinal Chemistry 8: 1157-1179). The modified nucleic acid may comprise a chimeric or mixed backbone containing one or more modifications, such as, a combination of phosphate bonds, such as a combination of phosphodiester and phosphorothioate bonds.

[00468] Substituents for the phosphate include, for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include those having morpholine linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimine and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones and others having mixed parts of N, O, S and CH components₂. Many U.S. patents disclose how to make and use these types of phosphate substitutes and include, without limitation, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439. It should also be understood that when replacing a nucleotide, both the sugar and phosphate moieties of the nucleotide can be replaced, for example, by an amide-type (aminoethylglycine) linkage (PNA). U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262 describe how to make and use PNA molecules, each of which is incorporated herein by reference. See also Nielsen et al., Science, 1991, 254, 1497-1500. It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance, for example, cellular uptake. Conjugates may be chemically linked to a nucleotide or nucleotide analogs. Such conjugates include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioether, for example, hexyl-S-tritylthiol (Manoharan et al., Ann. KY. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, for example, dodecanediol or undecyl residues (Saison-Behmoaras et al., EM5OJ, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipid, for example, di-hexadecyl-rac-glycerol or triethylammonium 1-di-O-hexadecyl-rac-glycero-S-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973) or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochem. Biophys. Acta, 1995, 1264, 229-237) or an octadecylamine or hexylaminocarbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). Numerous U.S. patents describe the preparation of such conjugates and include, but are not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5414077; 5486603; 5512439; 5578718; 5608046; 4587044; 4605735; 4667025; 4762779; 4789737; 4824941; 4835263; 4876335; 4904582; 4958013; 5082830; 5112963; 5214136; 5082830; 5112963; 5214136; 5245022; 5254469; 5258506; 5262536; 5272250; 5292873; 5317098; 5371241, 5391723; 5416203, 5451463; 5510475; 5512667; 5514785; 5565552; 5567810; 5574142; 5585481; 5587371; 5595726; 5597696; 5599923; 5599928 and 5688941.

[00469] In some cases, the non-natural nucleic acids further form non-natural base pairs. Exemplary non-natural nucleotides capable of forming a non-natural DNA or RNA base pair (UBP) in vivo include, but are not limited to, TPT3, dTPT3, 5SICS, d5SICS, NaM, dNaM, CNMO, dCNMO, and combinations thereof.

[00470] In some cases, the non-natural nucleic acids further form non-natural base pairs. Exemplary non-natural nucleotides capable of forming a non-natural DNA or RNA base pair (UBP) in vivo include, but are not limited to, SICS, d5SICS, NAM, dNaM, and combinations thereof. Other examples of non-natural nucleotides capable of forming non-natural UBPs that can be used to make the IL-2 conjugates disclosed herein can be found in Dien et al., J Am Chem Soc., 2018, 140:16115-16123; Feldman et al., J Am Chem Soc, 2017, 139:11427-11433; Ledbetter et al., J Am Chem Soc., 2018, 140:758-765; Dhami et al., Nucleic Acids Res. 2014, 42:10235-10244; Malyshev et al., Nature, 2014, 509:385-388; Betz et al., J Am Chem Soc., 2013, 135:18637-18643; Lavergne et al., J Am Chem Soc. 2013, 135:5408-5419 and Malyshev et al. Proc Natl Acad Sci USA, 2012, 109:12005-12010. In some embodiments, the non-natural nucleotides include:

.

[00471] In some embodiments, non-natural nucleotides that can be used to produce the IL-2 conjugates disclosed herein can be derived from a compound of the formula,

, where R ₂ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano and azido; and

The wavy line indicates a bond with ribosyl or 2'-deoxyribosyl, where the 5'-hydroxy group of the ribosyl or 2'-deoxyribosyl moiety is in free form or optionally linked to a monophosphate, diphosphate or triphosphate group.

[00472] In some embodiments, non-natural nucleotides that can be used to produce the IL-2 conjugates disclosed herein can be derived from , , , , And In some embodiments, non-natural nucleotides that can be used to produce the IL-2 conjugates disclosed herein include , , , , And or their salts.

[00473] In some embodiments, the unnatural base pair generates an unnatural amino acid as described in Dumas et al., "Designing logical codon reassignment - Expanding the chemistry in biology," Chemical Science , 6 : 50-69 (2015).

[00474] The host cell into which the constructs or vectors disclosed herein are introduced is cultured or maintained in a suitable medium such that tRNA, tRNA synthetase, and the protein of interest are produced. The medium also contains unnatural amino acid(s) such that the unnatural amino acid(s) is(are) incorporated into the protein of interest. In some embodiments, a nucleoside triphosphate transporter (NTT) from a bacterium, plant, or algae is also present in the host cell. In some embodiments, the IL-2 conjugates disclosed herein are produced by using a host cell that expresses NTT. In some embodiments, the nucleotide nucleoside triphosphate transporter used in the host cell can be selected from TpNTT1, TpNTT2, TpNTT3, TpNTT4, TpNTT5, TpNTT6, TpNTT7, TpNTT8 (T. pseudonana), PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, PtNTT6 (P. tricornutum), GsNTT (Galdieria sulphuraria), AtNTT1, AtNTT2 (Arabidopsis thaliana), CtNTT1, CtNTT2 (Chlamydia trachomatis), PamNTT1, PamNTT2 (Protochlamydia amoebophila), CcNTT (Caedibacter caryophilus), RpNTT1 (Rickettsia prowazekii). In some embodiments, NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6. In some embodiments, NTT is PtNTT1. In some embodiments, NTT is PtNTT2. In some embodiments, NTT is PtNTT3. In some embodiments, NTT is PtNTT4. In some embodiments, NTT is PtNTT5. In some embodiments, NTT is PtNTT6. Other NTTs that may be used are disclosed in Zhang et al., Nature 2017, 551(7682): 644-647; Malyshev et al. Nature 2014 (509(7500), 385-388; and Zhang et al. Proc Natl Acad Sci USA, 2017, 114:1317-1322.

[00475] An orthogonal tRNA synthetase/tRNA pair loads a tRNA with an unnatural amino acid and incorporates the unnatural amino acid into a polypeptide chain in response to a codon. Exemplary aaRS-tRNA pairs include, but are not limited to, Methanococcus jannaschii aaRS/tRNA ( Mj-Tyr ) pairs, E. coli TyrRS (Ec- Tyr )/ B. stearothermophilus tRNA _CUA pairs, E. coli LeuRS ( Ec-Leu )/ B. stearothermophilus tRNA _CUA pairs, and pyrrolizyl-tRNA pairs. Other aaRS-tRNA pairs that can be used in accordance with the present invention include those derived from M. mazei described in Feldman et al., J Am Chem Soc., 2018 140:1447-1454 and Zhang et al. Proc Natl Acad Sci USA, 2017, 114:1317-1322.

[00476] In some embodiments, methods for producing IL-2 conjugates disclosed herein are provided in a cellular system that expresses NTT and a tRNA synthetase. In some embodiments described herein, NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6, and the tRNA synthetase is selected from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , and M. mazei . In some embodiments, NTT is PtNTT1 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei . In some embodiments, NTT is PtNTT2 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei . In some embodiments, NTT is PtNTT3 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei . In some embodiments, NTT is PtNTT3 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei . In some embodiments, NTT is PtNTT4 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei . In some embodiments, NTT is PtNTT5 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei . In some embodiments, NTT is PtNTT6 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei .

[00477] A cytokine polypeptide (e.g., IL-2) comprising unnatural amino acid(s) is produced by introducing into a host cell the nucleic acid constructs described herein comprising a tRNA and tRNA synthetase and comprising a nucleic acid sequence of interest with one or more orthogonal (stop) codons in frame. The host cell is exposed to a saline solution containing the unnatural amino acid(s), and the host cells are then maintained under conditions that allow expression of the coding sequence of the protein of interest. The unnatural amino acid(s) is(are) incorporated into the polypeptide chain in response to a codon. For example, one or more unnatural amino acids are incorporated into a cytokine polypeptide (e.g., IL-2). Alternatively, two or more unnatural amino acids may be inserted into a cytokine polypeptide (e.g., IL-2) at two or more sites on the protein.

[00478] In some embodiments, the IL-2 conjugates disclosed herein can be produced in a cell, such asE. coli, containing (a) a nucleoside triphosphate transporterPtNTT2 (including a truncated variant in which the first 65 amino acid residues of the full-length protein are deleted), (b) a plasmid containing a double-stranded oligonucleotide that encodes an IL-2 variant having the desired amino acid sequence and that contains a non-natural base pair comprising a first non-natural nucleotide and a second non-natural nucleotide to provide a codon at the desired position into which a non-natural amino acid will be inserted, such asN6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), (c) plasmid encoding tRNA, which is obtained fromM. mazeiand which contains a non-natural nucleotide to provide a recognizable anticodon (to the IL-2 variant codon) in place of its native sequence, and (d) a plasmid encoding the derivedM. barkeripyrrolysyl-tRNA synthetase (MbPylRS), which may be the same plasmid that encodes the tRNA or a different plasmid. Furthermore, in some embodiments, the cell is supplemented with deoxyribotriphosphates containing one or more unnatural bases. Furthermore, in some embodiments, the cell is supplemented with ribotriphosphates containing one or more unnatural bases. Furthermore, in some embodiments, the cells are supplemented with one or more unnatural amino acids, such asN6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, a double-stranded oligonucleotide that encodes the amino acid sequence of a desired IL-2 variant comprises an AXC codon, such as at position 35, 42, 43, 62, or 65 of the sequence that encodes the protein of SEQ ID NO: 4 (IL-2_C125S), or at position 34, 41, 42, 61, or 64 of the sequence that encodes the protein of SEQ ID NO: 3 (aldesleukin), wherein X is a non-natural nucleotide. In some embodiments, the cell further comprises a plasmid, which may be a protein expression plasmid or another plasmid, that encodes an orthogonal tRNA gene fromM. mazei, which comprises an AXC-corresponding GYT anticodon in place of its native sequence, wherein Y is a non-natural nucleotide that is complementary to and may be the same as or different from a non-natural nucleotide in the codon. In some embodiments, the non-natural nucleotide in the codon is different from the non-natural nucleotide and is complementary to the non-natural nucleotide in the anticodon. In some embodiments, the non-natural nucleotide in the codon is the same as the non-natural nucleotide in the anticodon. In some embodiments, the first and second non-natural nucleotides of a non-natural base pair in the double-stranded oligonucleotide can be derived from, And . In some embodiments, the first and second non-natural nucleotides of a non-natural base pair in a double-stranded oligonucleotide may be derived from And In some embodiments, the triphosphates of the first and second non-natural nucleotides comprise, And or salts thereof. In some embodiments, the triphosphates of the first and second non-natural nucleotides comprise And or salts thereof. In some embodiments, mRNA derived from a double-stranded oligonucleotide comprising a first non-natural nucleotide and a second non-natural nucleotide may comprise a codon comprising a non-natural nucleotide derived from, And . In some embodiments, the tRNA M. mazeimay contain an anticodon containing an unnatural nucleotide that recognizes a codon containing an unnatural nucleotide in mRNA. Anticodon in tRNAM. mazeimay contain an unnatural nucleotide derived from, And . In some embodiments, the mRNA comprises a non-natural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from. In some embodiments, the tRNA comprises a non-natural nucleotide derived from. In some embodiments, the tRNA comprises a non-natural nucleotide derived from. In some embodiments, the tRNA comprises a non-natural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from, and tRNA contains an unnatural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from, and tRNA contains an unnatural nucleotide derived from. The host cell is cultured in a medium containing appropriate nutrients and supplemented with (a) deoxyribonucleoside triphosphates containing one or more unnatural bases that are necessary for the replication of the plasmid(s) encoding the cytokine gene(s) containing the codon, (b) ribonucleoside triphosphates containing one or more unnatural bases necessary for the transcription of (i) mRNA corresponding to the cytokine coding sequence and containing a codon containing one or more unnatural bases, and (ii) tRNA containing an anticodon containing one or more unnatural bases, and (c) unnatural amino acid(s) to be inserted into the polypeptide sequence of the cytokine of interest. The host cells are then maintained under conditions that allow expression of the protein of interest.

[00479] In some cases, the codon containing the non-natural base and the anticodon containing the non-natural base may be selected from the following pairs, wherein each X and Y contains a base independently selected from the group consisting of , , , , , , , , , , , , And , where R ₂ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano and azido; and in each case the wavy line indicates a bond to ribosyl if X and Y comprise mRNA or tRNA, or to 2'-deoxyribosyl if X and Y comprise DNA.

Codon (mRNA) Anticodon (tRNA) UUX YAA or XAA UGX YCA or XCA CGX YCG or XCG AGX YCU or XCU GAX YUC or XUC CAX YUG or XUG GXU AYC CXU AYG GXG CYC AXG CYU GXC GYC AXC GYU GXA UYC CXC GYG UXC GYA AUX YAU or XAU CUX XAG or YAG GUX XAC or YAC UAX XUA or YUA GGX XCC or YCC

[00480] The resulting protein containing one or more unnatural amino acids, such as Azk, that is expressed can be purified by methods known to those skilled in the art and then reacted with an alkyne, such as DBCO, containing a PEG chain of the desired average molecular weight, as disclosed herein, under conditions known to those skilled in the art, to produce the IL-2 conjugates disclosed herein. Other methods are known to those skilled in the art, such as those disclosed in Zhang et al., Nature 2017, 551(7682): 644-647, WO 2015157555, WO 2015021432, WO 2016115168, WO 2017106767, WO 2017223528, WO 2019014262, WO 2019014267, WO 2019028419, and WO2019/028425.

[00481] Alternatively, a cytokine polypeptide (e.g., IL-2) comprising a non-natural amino acid(s) is produced by introducing into a host cell the nucleic acid constructs described herein comprising a tRNA and an aminoacyl-tRNA synthetase and comprising a nucleic acid sequence of interest with one or more orthogonal (stop) codons in frame. The host cell is cultured in a medium containing appropriate nutrients and supplemented with (a) deoxyribonucleoside triphosphates containing one or more unnatural bases required for replication of the plasmid(s) encoding the cytokine gene(s) containing the novel codon and anticodon, (b) ribonucleoside triphosphates required for transcription of mRNA corresponding to (i) the cytokine sequence containing the codon and (ii) the orthogonal tRNA containing the anticodon, and (c) unnatural amino acid(s). The host cells are then maintained under conditions that allow expression of the protein of interest. The unnatural amino acid(s) is(are) incorporated into the polypeptide chain in response to the unnatural codon. For example, one or more unnatural amino acids are incorporated into the cytokine polypeptide (e.g., IL-2). Alternatively, two or more unnatural amino acids may be inserted into a cytokine polypeptide (e.g., IL-2) at two or more sites on the protein.

[00482] Once a cytokine (e.g., IL-2) with an incorporated non-natural amino acid(s) has been produced in a host cell, it can be recovered from the host cell using a variety of techniques known in the art, including enzymatic, chemical, and/or osmotic lysis and physical disruption. The cytokine polypeptide (e.g., IL-2) can be purified using standard techniques known in the art, such as preparative ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, or any other suitable technique known to those skilled in the art.

[00483] Suitable host cells may include bacterial cells (e.g., E. coli, BL21(DE3)), but most suitable host cells are eukaryotic cells, such as insect cells (e.g., fruit flies such as Drosophila melanogaster), yeast cells, nematode cells (e.g., C. elegans), mice (e.g., Mus musculus), or mammalian cells (such as Chinese hamster ovary (CHO) cells, or COS cells, human 293T cells, HeLa cells, NIH 3T3 cells, and mouse erythroleukemia (MEL) cells), or human cells or other eukaryotic cells. Other suitable host cells are known to those skilled in the art. Accordingly, the host cell is a mammalian cell, such as a human cell, or an insect cell. In some embodiments, suitable host cells include E. coli .

[00484] Other suitable host cells that can be generally used in embodiments of the present invention include those mentioned in the Examples section. Vector DNA can be introduced into host cells using conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to cover a variety of well-known techniques for introducing a foreign nucleic acid molecule (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells are well known in the art.

[00485] When generating cell lines, it is generally preferred to generate stable cell lines. For example, it is known that in the case of stable transfection of mammalian cells, depending on the expression vector and transfection technique used, only a small proportion of the cells can integrate foreign DNA into their genome. To identify and select these integrants, a gene encoding a selectable marker (e.g., for antibiotic resistance) is typically introduced into the host cells along with the gene of interest. Preferred selectable markers include markers that confer resistance to drugs such as G418, hygromycin, or methotrexate. Nucleic acid molecules encoding a selectable marker can be introduced into the host cell in the same vector or can be introduced in a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection (e.g. cells that contain the inserted selectable marker gene will survive while other cells die).

[00486] In one embodiment, the constructs described herein are integrated into the genome of a host cell. The advantage of stable integration is that homogeneity is achieved between individual cells or clones. Another advantage is that selection of the best producers can be performed. Accordingly, stable cell lines must be created. In another embodiment, the constructs described herein are transfected into a host cell. The advantage of transfecting a host cell with the constructs is that protein yield can be maximized. In one aspect, a cell is described that contains a nucleic acid construct or vector described herein.

[00487] If multiple unnatural amino acids are to be incorporated into a cytokine polypeptide (e.g., IL-2), it will be appreciated that multiple codons will need to be incorporated into the coding nucleic acid sequence at the desired positions so that tRNA synthetase/tRNA pairs can control the incorporation of the unnatural amino acids in response to the codon(s). At least 1, 2, 3, 4, or more nucleic acids encoding a codon may be incorporated into the nucleic acid sequence of interest.

[00488] When it is desired to incorporate more than one type of unnatural amino acid into a single protein of interest, a second or additional orthogonal pair of tRNA-tRNA synthetase may be used to incorporate the second or additional unnatural amino acid; accordingly, said second or additional orthogonal pair of tRNA-tRNA synthetase recognizes a different codon in the nucleic acid encoding the protein of interest, so that two or more unnatural amino acids can be specifically incorporated into different defined sites in the protein in a single manufacturing step. Accordingly, in certain embodiments, two or more orthogonal pairs of tRNA-tRNA synthetase may be used.

[00489] Once a cytokine (e.g., IL-2) with an incorporated non-natural amino acid(s) has been produced in a host cell, it can be recovered from the host cell using a variety of techniques known in the art, including enzymatic, chemical, and/or osmotic lysis and physical disruption. The cytokine polypeptide (e.g., IL-2) can be purified using standard techniques known in the art, such as preparative chromatography, affinity purification, or any other suitable technique.

[00490] Suitable host cells may include bacterial cells (e.g., E. coli, BL21(DE3)), but particularly suitable host cells are eukaryotic cells, such as insect cells (e.g., fruit flies such as Drosophila melanogaster), yeast cells, nematode cells (e.g., C. elegans), mice (e.g., Mus musculus), or mammalian cells (such as Chinese hamster ovary (CHO) cells, or COS cells, human 293T cells, HeLa cells, NIH 3T3 cells, and mouse erythroleukemia (MEL) cells), or human cells or other eukaryotic cells. Other suitable host cells are known to those skilled in the art. Accordingly, the host cell is a mammalian cell, such as a human cell, or an insect cell.

[00491] Other suitable host cells that can be generally used in embodiments of the present invention include those mentioned in the Examples section. Vector DNA can be introduced into host cells using conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to cover a variety of well-known techniques for introducing a foreign nucleic acid molecule (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells are well known in the art.

[00492] When generating cell lines, it is generally preferred to generate stable cell lines. For example, it is known that in the case of stable transfection of mammalian cells, depending on the expression vector and transfection technique used, only a small proportion of the cells can integrate foreign DNA into their genome. To identify and select these integrants, a gene encoding a selectable marker (e.g., for antibiotic resistance) is typically introduced into the host cells along with the gene of interest. Preferred selectable markers include markers that confer resistance to drugs such as G418, hygromycin, or methotrexate. Nucleic acid molecules encoding a selectable marker can be introduced into the host cell in the same vector or can be introduced in a separate vector. Cells stably transfected with the introduced nucleic acid molecule can be identified by drug selection (e.g. cells that contain the inserted selectable marker gene will survive while other cells die).

[00493] In one embodiment, the constructs described herein are integrated into the genome of a host cell. The advantage of stable integration is that homogeneity is achieved between individual cells or clones. Another advantage is that selection of the best producers can be performed. Accordingly, stable cell lines must be created. In another embodiment, the constructs described herein are transfected into a host cell. The advantage of transfecting a host cell with the constructs is that protein yield can be maximized. In one aspect, a cell is described that contains a nucleic acid construct or vector described herein.

Pharmaceutical compositions and formulations

[00494] In some embodiments, the pharmaceutical composition and formulations described herein are administered to a subject via multiple routes of administration, including, but not limited to, parenteral, oral, buccal, rectal, sublingual, or transdermal routes of administration. In some cases, parenteral administration includes intravenous, subcutaneous, intramuscular, intracerebral, intranasal, intraarterial, intra-articular, intradermal, intravitreal, intraosseous infusion, intraperitoneal, or intrathecal administration. In some cases, the pharmaceutical composition is formulated for topical administration. In other cases, the pharmaceutical composition is formulated for systemic administration. In some embodiments, the pharmaceutical composition and formulations described herein are administered to a subject via intravenous, subcutaneous, intramuscular administration. In some embodiments, the pharmaceutical composition and formulations described herein are administered to a subject via intravenous administration. In some embodiments, the pharmaceutical composition and formulations described herein are administered to a subject via administration. In some embodiments, the pharmaceutical composition and formulations described herein are administered to a subject via intramuscular administration.

[00495] In some embodiments, pharmaceutical formulations include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast-melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsed release formulations, multiparticulate formulations (e.g., nanoparticulate formulations), and mixed immediate and controlled release formulations.

[00496] In some embodiments, the pharmaceutical formulations comprise a carrier or carrier materials selected based on compatibility with the composition disclosed herein and the release profile characteristics of the desired dosage form. Illustrative carrier materials include, for example, binders, suspending agents, disintegrating agents, fillers, surfactants, solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. Pharmaceutically compatible carrier materials include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars, sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and etc. See, for example, Remington: The Science and Practice of Pharmacy , Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995), Hoover, John E., Remington 's Pharmaceutical Sciences , Mack Publishing Co., Easton, Pennsylvania 1975, Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms , Marcel Decker, New York, NY, 1980, and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins1999).

[00497] In some cases, the pharmaceutical composition is formulated as an immunoliposome that comprises a plurality of IL-2 conjugates directly or indirectly bound to a lipid bilayer of the liposomes. Exemplary lipids include, but are not limited to, fatty acids; phospholipids; sterols such as cholesterols; sphingolipids such as sphingomyelin; glycosphingolipids such as gangliosides, globosides, and cerebrosides; surfactant amines such as stearyl, oleyl, and linoleylamines. In some cases, the lipid comprises a cationic lipid. In some cases, the lipid comprises a phospholipid. Illustrative phospholipids include, but are not limited to, phosphatidic acid ("PA"), phosphatidylcholine ("PC"), phosphatidylglycerol ("PG"), phosphatidylethanolamine ("PE"), phosphatidylinositol ("PI"), and phosphatidylserine ("PS"), sphingomyelin (including brain sphingomyelin), lecithin, lysolecithin, lysophosphatidylethanolamine, cerebrosides, diarachidoylphosphatidylcholine ("DAPC"), didecanoyl-L-alpha-phosphatidylcholine ("DDPC"), dielaidoylphosphatidylcholine ("DEPC"), dilauroylphosphatidylcholine ("DLPC"), dilinoleoylphosphatidylcholine, dimyristoylphosphatidylcholine ("DMPC"), dioleoylphosphatidylcholine ("DOPC"), dipalmitoylphosphatidylcholine ("DPPC"), Distearoylphosphatidylcholine ("DSPC"), 1-palmitoyl-2-oleoylphosphatidylcholine ("POPC"), diarachidoylphosphatidylglycerol ("DAPG"), didecanoyl-L-alpha-phosphatidylglycerol ("DDPG"), dielaidoylphosphatidylglycerol ("DEPG"), dilauroylphosphatidylglycerol ("DLPG"), dilinoleoylphosphatidylglycerol, dimyristoylphosphatidylglycerol ("DMPG"), dioleoylphosphatidylglycerol ("DOPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG"), 1-palmitoyl-2-oleoylphosphatidylglycerol ("POPG"), diarachidoylphosphatidylethanolamine ("DAPE"), didecanoyl-L-alpha-phosphatidylethanolamine ("DDPE"), dielaidoylphosphatidylethanolamine ("DEPE"), dilauroylphosphatidylethanolamine ("DLPE"), dilinoleoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine ("DMPE"), dioleoylphosphatidylethanolamine ("DOPE"), dipalmitoylphosphatidylethanolamine ("DPPE"), distearoylphosphatidylethanolamine ("DSPE"), 1-palmitoyl-2-oleoylphosphatidylethanolamine ("POPE"), diarachidoylphosphatidylinositol ("DAPI"), didecanoyl-L-alpha-phosphatidylinositol ("DDPI"), dielaidoylphosphatidylinositol ("DEPI"), dilauroylphosphatidylinositol ("DLPI"), dilinoleoylphosphatidylinositol, dimyristoylphosphatidylinositol ("DMPI"), dioleoylphosphatidylinositol ("DOPI"), Dipalmitoylphosphatidylinositol ("DPPI"), Distearoylphosphatidylinositol ("DSPI"), 1-Palmitoyl-2-oleoylphosphatidylinositol ("POPI"), Diarachidoylphosphatidylserine ("DAPS"), Didecanoyl-L-alpha-phosphatidylserine ("DDPS"), Dielaidoylphosphatidylserine ("DEPS"), Dilauroylphosphatidylserine ("DLPS"), Dilinoleoylphosphatidylserine, Dimyristoylphosphatidylserine ("DMPS"), Dioleoylphosphatidylserine ("DOPS"), Dipalmitoylphosphatidylserine ("DPPS"), Distearoylphosphatidylserine ("DSPS"), 1-Palmitoyl-2-oleoyl-phosphatidylserine ("POPS"), Diarachidoylsphingomyelin, Didecanoylsphingomyelin, dielaidoylsphingomyelin, dilauroylsphingomyelin, dilinoleoylsphingomyelin, dimyristoylsphingomyelin, sphingomyelin, dioleoylsphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin and 1-palmitoyl-2-oleoylsphingomyelin.

[00498] In some cases, the pharmaceutical compositions further comprise pH adjusting or buffering agents, which include acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids, bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane, and buffers such as citrate/dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount necessary to maintain the pH of the composition in an acceptable range.

[00499] In some cases, the pharmaceutical composition contains one or more salts in an amount necessary to bring the osmolality of the composition into an acceptable range. Such salts include salts containing sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions, suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[00500] In some embodiments, pharmaceutical formulations include, but are not limited to, sugars such as trehalose, sucrose, mannitol, maltose, glucose, or salts such as potassium phosphate, sodium citrate, ammonium sulfate, and/or other agents such as heparin, designed to enhance the solubility and stability of the polypeptides in vivo .

[00501] In some cases, pharmaceutical compositions further comprise a diluent, which is used to stabilize the compounds because they are capable of providing a more stable environment. Salts dissolved in buffer solutions (which are also capable of providing pH control or maintenance) are used as diluents in the art, including, but not limited to, phosphate-buffered sodium chloride solution. In some cases, diluents increase the volume of the composition to facilitate compression or to create sufficient volume for a homogeneous mixture for filling capsules. Such compounds may include, for example, lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Avicel®, dibasic calcium phosphate, dicalcium phosphate dihydrate, tricalcium phosphate, calcium phosphate, anhydrous lactose, spray-dried lactose, pregelatinized starch, compressible sugar such as Di-Pac® (Amstar), mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, confectioners' sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, kaolin, mannitol, sodium chloride, inositol, bentonite, and the like. In some embodiments, the conjugates The IL-2s disclosed herein can be used in pharmaceutical formulations comprising histidine, sorbitol, and polysorbate 80, or any combination that results in a stable formulation, and can be administered to subjects in need thereof. In one embodiment, the IL-2 conjugates disclosed herein can be presented as a finished drug product in a suitable container, such as a vial, as follows: IL-2 conjugate (about 2 mg to about 10 mg); L-histidine (about 0.5 mg to about 2 mg); L-histidine hydrochloride (about 1 mg to about 2 mg); sorbitol (about 20 mg to about 80 mg), and polysorbate 80 (about 0.1 mg to about 0.2 mg); with sufficient water for injection to provide a liquid formulation suitable for use in the disclosed methods.

[00502] In some cases, pharmaceutical formulations include disintegrating agents or disintegrants to facilitate distribution or disintegration of the substance. The term "disintegrating" includes both dissolution and dispersion of the dosage form upon contact with gastrointestinal juice. Examples of disintegrating agents include starch, e.g., natural starch such as corn starch or potato starch, pregelatinized starch such as National 1551 or Amijel®, or sodium starch glycolate such as Promogel® or Explotab®, cellulose such as a wood product, crystalline methylcellulose such as Avicel®, Avicel® PH101, Avicel® PH102, Avicel® PH105, Elcema® P100, Emcocel®, Vivacel®, Ming Tia® and Solka-Floc®, methylcellulose, croscarmellose or cross-linked cellulose such as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol®), cross-linked carboxymethylcellulose or cross-linked croscarmellose, cross-linked starch such as sodium starch glycolate, cross-linked polymer such as crospovidone, cross-linked polyvinylpyrrolidone, alginate such as alginic acid or an alginic acid salt such as sodium alginate, clay such as Veegum® HV (magnesium aluminum silicate), gum such as agar, guar gum, locust bean gum, karaya gum, pectin or gum tragacanth, sodium starch glycolate, bentonite, natural sponge, surfactant, resin such as cation exchange resin, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination with starch, etc.

[00503] In some cases, pharmaceutical formulations include excipients such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, etc.

[00504] Lubricants and glidants are also optionally included in the pharmaceutical compositions described herein to prevent, reduce, or suppress adhesion or friction of materials. Illustrative lubricants include, for example, stearic acid, calcium hydroxide, talc, sodium stearyl fumarate, a hydrocarbon such as mineral oil or hydrogenated vegetable oil such as hydrogenated soybean oil (Sterotex®), higher fatty acids and their alkali metal and alkaline earth metal salts such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, glycerin, talc, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g., PEG-4000) or methoxypolyethylene glycol such as Carbowax™, sodium oleate, sodium benzoate, glyceryl behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silicon dioxide such as Syloid™, Cab-O-Sil®, starch such as corn starch, silicone oil, surfactant, etc.

[00505] Plasticizers include compounds used to soften the microencapsulated material or film coatings to make them less brittle. Suitable plasticizers include, for example, polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350 and PEG 800, stearic acid, propylene glycol, oleic acid, triethylcellulose and triacetin. Plasticizers can also function as dispersants or wetting agents.

[00506] Solubilizers include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium doccusate, vitamin E TPGS, dimethylacetamide, N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrins, ethanol, n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600, glycofurol, transcutol, propylene glycol, dimethyl isosorbide, etc.

[00507] Stabilizers include such as any antioxidant agents, buffers, acids, preservatives, etc. Illustrative stabilizers include L-arginine hydrochloride, tromethamine, albumin (human), citric acid, benzyl alcohol, phenol, disodium hydrogen phosphate dihydrate, propylene glycol, meta-cresol or m-cresol, zinc acetate, polysorbate 20 or Tween® 20, or trometamol.

[00508] Suspending agents include compounds such as polyvinylpyrrolidone, for example polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25 or polyvinylpyrrolidone K30, vinylpyrrolidone-vinyl acetate copolymer (S630), polyethylene glycol, for example polyethylene glycol can have a molecular weight of from about 300 to about 6000, or from about 3350 to about 4000, or from about 7000 to about 5400, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, hydroxymethylcellulose acetate stearate, polysorbate 80, hydroxyethylcellulose, sodium alginate, gums such as, for example, gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulose compounds such as, for example, sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, polysorbate 80, sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone, etc.

[00509] Surfactants include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, ethylene oxide and propylene oxide copolymers such as Pluronic® (BASF), and the like. Additional surfactants include polyoxyethylene glycerides of fatty acids and vegetable oils, for example, polyoxyethylene (60) hydrogenated castor oil, as well as alkyl ethers and alkyl phenyl ethers of polyoxyethylene, for example, octoxynol 10, octoxynol 40. Sometimes surfactants are included to increase physical stability or for other purposes.

[00510] Viscosity enhancing agents include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxypropylmethylcellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia gum, chitosans, and combinations thereof.

[00511] Wetting agents include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium docusate, triacetin, Tween 80, vitamin E TPGS, ammonium salts, etc.

Therapeutic regimens

[00512] In some embodiments, the pharmaceutical compositions described herein are administered for therapeutic uses. In some embodiments, the pharmaceutical composition is administered once a day, twice a day, three times a day, or more. The pharmaceutical composition is administered daily, every day, every other day, five days a week, once a week, every other week, two weeks a month, three weeks a month, once a month, twice a month, three times a month, or more. The pharmaceutical composition is administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or more.

[00513] In the event that the patient's condition does improve, at the discretion of the physician, the composition may be administered continuously, alternatively, the dosage of the composition to be administered may be temporarily reduced or temporarily suspended for a specified period of time (i.e., a "drug break"). In some cases, the duration of the drug break varies from 2 days to 1 year, including, by way of example, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during the drug interruption period is 10-100%, including by way of example only 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[00514] In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once per week, once every two weeks, once every three weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once every 13 weeks, once every 14 weeks, once every 15 weeks, once every 16 weeks, once every 17 weeks, once every 18 weeks, once every 19 weeks, once every 20 weeks, once every 21 weeks, once every 22 weeks, once every 23 weeks, once every 24 weeks, once every 25 weeks, once every 26 weeks, once every 27 weeks, or once every 28 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once a week. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every two weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every three weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 4 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 5 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 6 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 7 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 8 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 9 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 10 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 11 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 12 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 13 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 14 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 15 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 16 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 17 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 18 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 19 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 20 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 21 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 22 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 23 weeks. In some embodiments, an effective amount of an IL-2 conjugate is administered to a subject in need thereof once every 24 weeks.

[00515] In some embodiments, the amount of the agent that corresponds to such an amount varies depending on factors such as the particular compound, the severity of the disease, the individual characteristics (e.g., weight) of the subject or host in need of treatment, but is nonetheless typically determined in a manner known in the art in accordance with the particular circumstances relating to a particular case, including, for example, the particular agent to be administered, the route of administration, and the subject or host to be treated. In some cases, the required dose is typically presented as a single dose or divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, such as two, three, four or more sub-doses per day.

[00516] In some embodiments, the methods comprise administering to a subject in need thereof a dose of an IL-2 conjugate in the range of 1 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or about 2 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or about 4 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or about 6 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or about 8 μg of IL-2 conjugate per kg of subject body weight of the subject to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 10 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 12 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight¸ or from about 14 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 16 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 18 μg of IL-2 conjugate per kg of subject body weight of the subject to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 20 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 22 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 24 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 26 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 28 μg of IL-2 conjugate per kg of subject body weight subject to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 32 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 34 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 36 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight¸ or from about 40 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 45 μg of IL-2 conjugate per kg of subject body weight subject to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 50 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 55 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 60 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 65 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 70 μg of IL-2 conjugate per kg of subject body weight of the subject to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 75 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 80 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight¸ or from about 85 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 90 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 95 μg of IL-2 conjugate per kg of subject body weight of the subject to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 100 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 110 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 120 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 130 μg of IL-2 conjugate per kg of subject body weight to about 200 μg of IL-2 conjugate per kg of subject body weight, or from about 140 μg of IL-2 per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 150 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 160 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 170 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 180 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight, or from about 190 μg IL-2 conjugate per kg subject body weight to about 200 μg IL-2 conjugate per kg subject body weight. The above ranges are suggestive only, as the number of variables with respect to an individual treatment regimen is large, and significant deviations from these recommended values are not uncommon. Such dosages vary depending on a number of variables, including, but not limited to, the activity of the compound being administered, the disease or condition being treated, the route of administration, the needs of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner. In some embodiments, the toxicity and therapeutic efficacy of such treatment regimens are determined by standard pharmaceutical procedures using cell culture or experimental animals, including, but not limited to, determination of LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index and is expressed as the ratio between LD50 and ED50. Compounds exhibiting high therapeutic indices are preferred. Data from cell culture assays and animal studies are used to establish the dosage range for human use. The dosage of such compounds is preferably within the range of circulating concentrations that includes the ED50 with minimal toxicity. The dosage varies within this range depending on the dosage form and route of administration used.

[00517] In some embodiments, the methods comprise administering a dose of an IL-2 conjugate to a subject in need thereof at a dose of about 1 μg of IL-2 conjugate per kg of subject body weight, or about 2 μg of IL-2 conjugate per kg of subject body weight, about 4 μg of IL-2 conjugate per kg of subject body weight, about 6 μg of IL-2 conjugate per kg of subject body weight, about 8 μg of IL-2 conjugate per kg of subject body weight, about 10 μg of IL-2 conjugate per kg of subject body weight, about 12 μg of IL-2 conjugate per kg of subject body weight, about 14 μg of IL-2 conjugate per kg of subject body weight, about 16 μg of IL-2 conjugate per kg of subject body weight, about 18 μg IL-2 conjugate per kg subject body weight, approximately 20 μg IL-2 conjugate per kg subject body weight, approximately 22 μg IL-2 conjugate per kg subject body weight, approximately 24 μg IL-2 conjugate per kg subject body weight, approximately 26 μg IL-2 conjugate per kg subject body weight, approximately 28 μg IL-2 conjugate per kg subject body weight, approximately 30 μg IL-2 conjugate per kg subject body weight, approximately 32 μg IL-2 conjugate per kg subject body weight, approximately 34 μg IL-2 conjugate per kg subject body weight, approximately 36 μg IL-2 conjugate per kg subject body weight, approximately 38 μg IL-2 conjugate per kg subject body weight, approximately 40 μg IL-2 conjugate per kg subject body weight, approximately 42 μg IL-2 conjugate per kg subject body weight, approximately 44 μg IL-2 conjugate per kg subject body weight, approximately 46 μg IL-2 conjugate per kg subject body weight, approximately 48 μg IL-2 conjugate per kg subject body weight, approximately 50 μg IL-2 conjugate per kg subject body weight, approximately 55 μg IL-2 conjugate per kg subject body weight, approximately 60 μg IL-2 conjugate per kg subject body weight, approximately 65 μg IL-2 conjugate per kg subject body weight, approximately 70 μg IL-2 conjugate per kg subject body weight, approximately 75 μg IL-2 conjugate per kg subject body weight, approximately 80 μg IL-2 conjugate per kg subject body weight, approximately 85 μg IL-2 conjugate per kg subject body weight, approximately 90 μg IL-2 conjugate per kg subject body weight, approximately 95 μg IL-2 conjugate per kg subject body weight, approximately 100 μg IL-2 conjugate per kg subject body weight, approximately 110 μg IL-2 conjugate per kg subject body weight, approximately 120 μg IL-2 conjugate per kg subject body weight, approximately 130 μg IL-2 conjugate per kg subject body weight, approximately 140 μg IL-2 conjugate per kg subject body weight, approximately 150 μg IL-2 conjugate per kg of subject body weight, approximately 160 μg of IL-2 conjugate per kg of subject body weight, approximately 170 μg of IL-2 conjugate per kg of subject body weight, approximately 180 μg of IL-2 conjugate per kg of subject body weight, approximately 190 μg of IL-2 conjugate per kg of subject body weight, or approximately 200 μg of IL-2 conjugate per kg of subject body weight. The above ranges are only suggestive since the number of variables in an individual treatment regimen is large and significant deviations from these recommended values are not uncommon. Such dosages will vary depending on a number of variables, not limited to, the activity of the compound being administered, the disease or condition being treated, the route of administration, the needs of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner. In some embodiments, the toxicity and therapeutic efficacy of such therapeutic regimens are determined using standard pharmaceutical procedures using cell cultures or experimental animals, including but not limited to determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index and is expressed as the ratio between the LD50 and the ED50. Compounds that exhibit high therapeutic indices are preferred. Data obtained from cell culture assays and animal studies are used to formulate a dosage range for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that includes the ED50 with minimal toxicity. The dosage varies within this range depending on the dosage form used and the route of administration used.

[00518] After the patient's condition has improved, a maintenance dose is administered as needed. Thereafter, the dosage or frequency of administration, or both, may be reduced as symptoms dictate to a level at which improvement in the disease, disorder, or condition is maintained.

[00519] In some embodiments, the amount of the agent that corresponds to such an amount varies depending on factors such as the particular compound, the severity of the disease, the individual characteristics (e.g., weight) of the subject or host in need of treatment, but is nonetheless typically determined in a manner known in the art in accordance with the particular circumstances relating to a particular case, including, for example, the particular agent to be administered, the route of administration, and the subject or host to be treated. In some cases, the required dose is typically presented as a single dose or divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, such as two, three, four or more sub-doses per day.

[00520] The above ranges are only suggestive since the number of variables in an individual treatment regimen is large and significant deviations from these recommended values are not uncommon. Such dosages vary depending on a number of variables, not limited to the activity of the compound employed, the disease or condition being treated, the route of administration, the needs of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[00521] In some embodiments, the toxicity and therapeutic efficacy of such therapeutic regimens are determined using standard pharmaceutical procedures using cell cultures or experimental animals, including, but not limited to, determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective to 50% of the population). The dose ratio of toxic to therapeutic effects is the therapeutic index and is expressed as the ratio between the LD50 and the ED50. Compounds that exhibit high therapeutic indices are preferred. Data obtained from cell culture assays and animal studies are used to develop a dosage range for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that includes the ED50 with minimal toxicity. The dosage varies within this range depending on the dosage form used and the route of administration used.

Sets/product

[00522] Disclosed herein in certain embodiments are kits and articles of manufacture for use with one or more of the methods and compositions described herein. Such kits include a carrier, package, or container that is divided into portions to accommodate one or more containers, such as vials, tubes, and the like, wherein each container contains one of the individual elements to be used in the method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are made of various materials, such as glass or plastic.

[00523] In some embodiments, kits comprise articles of manufacture that are useful for developing adoptive cell therapy agents. In some embodiments, kits comprise one or more of the cytokine (e.g., IL-2) polypeptides or cytokine (e.g., IL-2) conjugates disclosed herein, and optionally one or more pharmaceutical excipients described herein to facilitate delivery of the cytokine (e.g., IL-2) polypeptides or cytokine (e.g., IL-2) conjugates. Such kits may optionally include one or more auxiliary components comprising inducers of T cell receptor signaling or modulation (e.g., antibodies to immune checkpoints, antibodies to CD3/CD28, major histocompatibility complexes (MHC), etc.), or alternative cytokines, or cytokine receptor agonists. Such kits further optionally include an identifying description or label, or instructions relating to their use in the methods described herein. In some embodiments, the kits comprise one or more polynucleic acid sequences encoding the IL-2 conjugates disclosed herein, an activator of a CD4+ helper cell, a CD8+ effector naive and memory cell, a natural killer (NK) cell, or a natural killer T cell (NKT), and/or a pharmaceutical composition thereof.

[00524] Kits and products for the treatment of proliferative and infectious diseases

[00525] Disclosed herein in some embodiments are kits comprising an isolated and modified interleukin 2 (IL-2) polypeptide comprising at least one unnatural amino acid at a position in the polypeptide that reduces the extent of binding between the modified IL-2 polypeptide and interleukin 2 receptor α (IL-2Rα), but retains a significant extent of binding to the interleukin 2 receptor βγ (IL-2Rβγ) signaling complex to form an IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of a wild-type IL-2 polypeptide to IL-2Rα. In some embodiments, the reduction in binding to IL-2Rα comprises a reduction in binding affinity of about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% compared to a wild-type IL-2 polypeptide. In some embodiments, the reduction in binding to IL-2Rα comprises a reduction of 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more compared to a wild-type IL-2 polypeptide.

[00526] Disclosed herein in some embodiments are kits comprising an isolated and modified interleukin 2 (IL-2) polypeptide comprising at least one unnatural amino acid, wherein the isolated and modified IL-2 polypeptide exhibits efficacy in transducing a first receptor signal to an IL-2βγ signaling complex and efficacy in transducing a second receptor signal to an IL-2αβγ signaling complex, and wherein the difference between the efficiency of the first receptor signaling and the efficiency of the second receptor signaling is less than 10-fold. In some embodiments, the difference in efficiency of receptor signaling is less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold.

[00527] Disclosed herein in some embodiments are kits comprising a polynucleotide sequence encoding a modified interleukin 2 (IL-2) polypeptide comprising at least one unnatural amino acid at a position in the polypeptide that reduces the extent of binding between the modified IL-2 polypeptide and interleukin 2 receptor α (IL-2Rα), but retains a significant extent of binding to the interleukin 2 receptor βγ (IL-2Rβγ) signaling complex to form an IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of a wild-type IL-2 polypeptide to IL-2Rα.

[00528] Disclosed herein in some embodiments are kits comprising a polynucleotide sequence encoding a modified interleukin 2 (IL-2) polypeptide comprising at least one unnatural amino acid, wherein the isolated and modified IL-2 polypeptide exhibits efficacy in transducing a first receptor signal to an IL-2βγ signaling complex and efficacy in transducing a second receptor signal to an IL-2αβγ signaling complex, and wherein the difference between the efficacy in transducing the first receptor signal and the efficacy in transducing the second receptor signal is less than 10-fold. In some embodiments, the difference in receptor signaling efficiency is less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold.

[00529] Kits and products for the treatment of autoimmune diseases

[00530] Disclosed herein in some embodiments are kits comprising an isolated and modified interleukin 2 (IL-2) polypeptide comprising at least one unnatural amino acid at a position in the polypeptide that reduces the efficiency of receptor signaling to the interleukin 2 receptor βγ (IL-2Rβγ) or reduces the recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex but maintains significant activation of the interleukin 2 receptor αβγ (IL-2Rαβγ), wherein the reduced efficiency of receptor signaling is compared to the efficiency of receptor signaling between a wild-type IL-2 polypeptide and IL-2Rβγ, and wherein the recruitment is compared to the recruitment of the IL-2Rγ subunit by the wild-type IL-2 polypeptide.

[00531] Disclosed herein in some embodiments are kits comprising an isolated and modified interleukin 2 (IL-2) polypeptide comprising at least one unnatural amino acid at a position in the polypeptide that increases recruitment of an IL-2Rα subunit to the IL-2 polypeptide, resulting in activation of the interleukin 2 receptor αβγ (IL-2Rαβγ), wherein the increase in recruitment is compared to recruitment of the IL-2Rα subunit by a wild-type IL-2 polypeptide.

[00532] In some embodiments, a modified IL-2 polypeptide with reduced efficiency of receptor signaling to IL-2Rβγ is capable of promoting expansion of CD4+ regulatory T cells (Treg). In some embodiments, the conjugation moiety impairs or blocks the efficiency of IL-2 receptor signaling to IL-2Rβγ or reduces recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex. In some embodiments, the proliferation of CD4+ Treg cells by the modified IL-2/IL-2Rαβγ complex is equivalent to or greater than that of a wild-type IL-2 polypeptide. In some embodiments, the modified IL-2/IL-2Rαβγ complex induces proliferation of CD4+ Treg cells into a population that is sufficient to modulate disease progression in an animal model. In some embodiments, the modified IL-2 polypeptide exhibits first receptor signaling efficiency to IL-2Rβγ and second receptor signaling efficiency to IL-2Rαβγ, wherein the first receptor signaling efficiency is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or less than the second receptor signaling efficiency. In some embodiments, the first receptor signaling efficiency of the modified IL-2 polypeptide is lower than the receptor signaling efficiency of the wild-type IL-2 polypeptide to IL-2Rβγ. In some embodiments, the efficiency of the modified IL-2 polypeptide in second receptor signaling is lower than the efficiency of receptor signaling of the wild-type IL-2 polypeptide to IL-2Rαβγ. In some embodiments, the modified IL-2 polypeptide further provides for an increase in recruitment of the IL-2Rα subunit to the IL-2 polypeptide, resulting in activation of the interleukin 2 receptor αβγ (IL-2Rαβγ), wherein the increase in recruitment is compared to recruitment of the IL-2Rα subunit by the wild-type IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide further provides for a reduction in the recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex, wherein the reduced recruitment is compared to the recruitment of the IL-2Rβ subunit and/or the IL-2Rγ subunit by a wild-type IL-2 polypeptide.

[00533] In some embodiments, the position of the at least one non-naturally occurring amino acid in the aforementioned modified IL-2 polypeptides is selected from K35, T37, R38, T41, F42, K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105, and Y107, wherein the residue positions correspond to positions 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 69, 71, 72, 104, 105, and 107 set forth in SEQ ID NO: 1. In some embodiments, the position of the at least one non-naturally occurring amino acid is selected from T37, R38, T41, F42, K43, F44, Y45, E61, E62, P65, E68, and L72, wherein the residue positions correspond to positions 37, 38, 41, 42, 43, 44, 45, 61, 62, 65, 68, and 72 as set forth in SEQ ID NO: 1. In some embodiments, the position of the at least one non-natural amino acid is selected from K35, K64, V69, N71, M104, C105, and Y107, wherein the residue positions correspond to positions 35, 64, 69, 71, 104, 105, and 107 as set forth in SEQ ID NO: 1. In some embodiments, the position of the at least one non-natural amino acid is selected from T37, R38, T41, Y45, E61, E68, and L72, wherein the residue positions correspond to positions 37, 38, 41, 45, 61, 68, and 72 as set forth in SEQ ID NO: 1. In some embodiments, the position of the at least one unnatural amino acid is selected from F42, K43, F44, E62, and P65, wherein the residue positions correspond to positions 42, 43, 44, 62, and 65 as set forth in SEQ ID NO: 1. In some embodiments, the position of the at least one unnatural amino acid is selected from P2, T3, S4, S5, S6, T7, K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, R81, P82, R83, D84, S87, N88, N89, V91, I92, L94, E95, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, T113, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, where the numbering amino acid residues correspond to the numbering in SEQ ID NO: 1. In some embodiments, the position of the at least one non-natural amino acid is selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, I92, L94, E95, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, wherein the numbering of the amino acid residues corresponds to the numbering in SEQ ID NO: 1. In some embodiments, the position of the at least one non-natural amino acid is selected from P2, T3, S4, S5, S6, T7, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, P82, R83, N89, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, and T113, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some embodiments, the position of the at least one non-naturally occurring amino acid is selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95, and Q126, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some embodiments, the position of the at least one non-naturally occurring amino acid is selected from K8, K9, and H16, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some embodiments, the position of the at least one non-naturally occurring amino acid is selected from Q22, N26, N88, and Q126, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some embodiments, the position of the at least one non-naturally occurring amino acid is selected from E15, D20, D84, and E95, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some embodiments, the position of the at least one non-naturally occurring amino acid is selected from L12, L19, and M23, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some embodiments, the position of the at least one non-naturally occurring amino acid is selected from at least one unnatural amino acid is selected from Q22 and N26, where the numbering of the amino acid residues corresponds to the numbering in SEQ ID NO: 1.

[00534] Kits and products in general

[00535] In some embodiments, the at least one unnatural amino acid is a lysine analog, comprises an aromatic side chain, comprises an azido group, comprises an alkyne group, or comprises an aldehyde or ketone group. In some embodiments, the at least one unnatural amino acid does not comprise an aromatic side chain. In some embodiments, the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK; the chemical structure of which is shown as compound 90 in Figure 3C), N6-(propargyloxy)-carbonyl)-L-lysine (PraK; the chemical structure of which is shown as compound 112 in Figure 3C), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-Propargyloxyphenylalanine, p-Propargyl-phenylalanine, 3-methyl-phenylalanine, L-Dopa, Fluorinated Phenylalanine, Isopropyl-L-phenylalanine, p-Azido-L-phenylalanine, p-Acyl-L-phenylalanine, p-Benzoyl-L-phenylalanine, p-Bromophenylalanine, p-Amino-L-phenylalanine, Isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, Phosphonotyrosine, Tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-copropyl)amino)ethyl)selanin)propanoic acid, 2-amino-3-(phenylselanyl)propanoic acid, or selenocysteine. In some embodiments, the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK; the chemical structure of which is shown as compound 90 in Figure 3C) or N6-(propargyloxy)-carbonyl)-L-lysine (PraK; the chemical structure of which is shown as compound 112 in Figure 3C). In some embodiments, the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK; the chemical structure of which is shown as compound 90 in Figure 3C). In some embodiments, the at least one unnatural amino acid comprises N6-(propargyloxy)-carbonyl)-L-lysine (PraK; the chemical structure of which is shown as compound 112 in Figure 3C).

[00536] In some embodiments, the at least one unnatural amino acid comprises an alkyne that is reacted with a conjugate moiety that comprises a water-soluble polymer comprising polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or a combination thereof. In some embodiments, the water-soluble polymer comprises a PEG molecule

[00537] In some embodiments, the modified IL-2 polypeptide comprises a conjugation moiety. In some embodiments, the conjugation moiety comprises a water-soluble polymer, a lipid, a protein, and/or a peptide. In some embodiments, the water-soluble polymer comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or a combination thereof. In some embodiments, the water-soluble polymer comprises a PEG molecule.

[00538] In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the conjugation moiety comprises a PEG molecule that is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide than the in vivo plasma half-life of a PEG that is smaller in size than the conjugation moiety. In some cases, the conjugation moiety comprises a PEG molecule that is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide than the in vivo plasma half-life of a PEG that is larger in size than the conjugation moiety.

[00539] In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of a wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of a modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of a wild-type IL-2 polypeptide and IL-2Rα.

[00540] In some embodiments, the PEG molecule is a linear PEG. In some embodiments, the PEG molecule is a branched PEG. In some embodiments, the PEG has a molecular weight of about 2,000 to 50,000 daltons (Da). In some embodiments, the PEG has a molecular weight of about 5,000 Da, 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some cases, the PEG has a molecular weight of 5,000 Da. In some cases, the PEG has a mass of 10,000 Da. In some cases, the PEG has a mass of 15,000 Da. In some cases, the PEG has a mass of 20,000 Da. In some cases, the PEG has a mass of 25,000 Da. In some cases, the mass of PEG is 30,000 Da. In some cases, the mass of PEG is 35,000 Da. In some cases, the mass of PEG is 40,000 Da. In some cases, the mass of PEG is 45,000 Da. In some cases, the mass of PEG is 50,000 Da.

[00541] The articles provided herein comprise packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, vials, bags, containers, bottles, and any packaging material suitable for the selected formulation and the intended route of administration and treatment.

[00542] For example, the container(s) comprises one or more modified IL-2 polypeptides comprising K35, T37, R38, T41, F42, K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105, or Y107, wherein the residue positions correspond to 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 69, 71, 72, 104, 105, and 107 as set forth in SEQ ID NO: 1. In some embodiments, a modified IL-2 polypeptide comprising T37 with residue positions corresponding to positions 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 69, 71, 72, 104, 105 and 107 as set forth in SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2000-50000 Da. In some embodiments, the molecular weight is 5000 Da. In some embodiments, the molecular weight is 10000 Da. In some embodiments, the molecular weight is 15000 Da. In some embodiments, the molecular weight is 20000 Da. In some embodiments, the molecular weight is 25000 Da. In some embodiments, the molecular weight is 30000 Da. In some embodiments, the molecular weight is 35000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00543] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue R38, corresponding to position 38 of SEQ ID NO: 1, comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00544] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue T41 corresponding to position 41 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00545] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue F42 corresponding to position 42 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00546] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue K43 corresponding to position 43 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00547] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue F44 corresponding to position 44 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00548] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue Y45 corresponding to position 45 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00549] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue E60 corresponding to position 60 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00550] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue E61 corresponding to position 61 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00551] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue E62 corresponding to position 62 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00552] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue K64, corresponding to position 64 of SEQ ID NO: 1, comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00553] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue P65 corresponding to position 65 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00554] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue E68 corresponding to position 68 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00555] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue V69 corresponding to position 69 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00556] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue N71, corresponding to position 71 of SEQ ID NO: 1, comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00557] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue L72 corresponding to position 72 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00558] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue M104 corresponding to position 104 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has a minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00559] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue C105 corresponding to position 105 of SEQ ID NO: 1 comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00560] In some embodiments, a modified IL-2 polypeptide comprising a mutation at residue Y107, corresponding to position 107 of SEQ ID NO: 1, comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-2 polypeptide. In some cases, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, the PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the molecular weight of the PEG does not affect formation of the modified IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding of the wild-type IL-2 polypeptide and IL-2Rα.

[00561] The kit typically includes labels with a list of contents and/or instructions for use, as well as package inserts with instructions for use. A set of instructions is also typically included.

[00562] In one embodiment, the label is on or associated with the container. In one embodiment, the label is on the container when the letters, numbers, or other symbols that form the label are attached, molded, or etched onto the container itself, and the label is associated with the container when it is within a device or holder that also holds the container, such as a package insert. In one embodiment, the label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for using the contents, such as in the methods described herein.

[00563] In certain embodiments, the pharmaceutical compositions are provided in a package or dispenser device that contains one or more unit dosage forms containing a compound as provided herein. The package, for example, comprises a metal or plastic foil, such as a blister pack. In one embodiment, the package or dispenser is accompanied by instructions for administration. In one embodiment, the package or dispenser is also accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, the notice reflecting approval by the agency of the drug form for human or veterinary administration. Such a notice is, for example, a label approved by the U.S. Food and Drug Administration for a drug or a package insert for an approved product. In one embodiment, compositions containing a compound as provided herein, formulated in a compatible pharmaceutical carrier, are also prepared, placed in an appropriate container, and labeled for treatment of a specified condition.

Implementation options

[00564] In certain embodiments, disclosed herein are cytokine conjugates and their use in treating one or more indications. Also described herein in some embodiments are interleukin 2 (IL-2) conjugates and their use in treating one or more indications. In some cases, the one or more indications include cancer, a pathogenic infection, or an autoimmune disease. In some cases, described herein are methods of modulating the interaction between IL-2 and an IL-2 receptor to stimulate or expand certain populations of T cells, natural killer (NK) cells, and/or natural killer T (NKT) cells. In some embodiments, the IL-2 conjugates comprise conjugation moieties (e.g., PEG), which contribute to the increase or decreasein vivoplasma half-life without affecting pharmacokinetics, including desired cytokine-receptor interactions and immune cell expansion. Additionally, in additional instances, described herein are pharmaceutical compositions and kits that contain one or more interleukin conjugates (e.g., IL-2 conjugates) described herein that may be useful as reagents for developing adoptive cell therapies intended to treat one or more of the indications described herein.

[00565] In aspects disclosed herein, isolated and modified interleukin 2 (IL-2) polypeptides are provided that comprise at least one unnatural amino acid at a position in the polypeptide that reduces the extent of binding between the modified IL-2 polypeptide and interleukin 2 receptor α (IL-2Rα), but maintains a significant extent of binding to the interleukin 2 receptor βγ (IL-2Rβγ) signaling complex to form an IL-2/IL-2Rβγ complex, wherein the reduced binding to IL-2Rα is compared to binding between a wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the difference in receptor signaling efficiency is less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold. In some embodiments, the position of the at least one unnatural amino acid is selected from K35, T37, R38, T41, F42, K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105, Y107, wherein the residue positions correspond to positions 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 69, 71, 72, 104, 105, and 107 set forth in SEQ ID NO: 1. In some embodiments, the position of the at least one unnatural amino acid is selected from T37, R38, T41, F42, K43, F44, Y45, E61, E62, P65, E68, and L72, wherein the residue positions correspond to positions 37, 38, 41, 42, 43, 44, 45, 61, 62, 65, 68, and 72 as set forth in SEQ ID NO: 1. In some embodiments, the position of the at least one non-naturally occurring amino acid is selected from K35, K64, V69, N71, M104, C105, and Y107, wherein the residue positions correspond to positions 35, 64, 69, 71, 104, 105, and 107 as set forth in SEQ ID NO: 1. In some embodiments, the position of the at least one non-naturally occurring amino acid is selected from T37, R38, T41, Y45, E61, E68, and L72, wherein the residue positions correspond to positions 37, 38, 41, 45, 61, 68, and 72 as set forth in SEQ ID NO: 1. In some embodiments, the position of the at least one unnatural amino acid is selected from F42, K43, F44, E62, and P65, wherein the residue positions correspond to positions 42, 43, 44, 62, and 65 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid is a lysine analog, comprises an aromatic side chain, comprises an azido group, comprises an alkyne group, or comprises an aldehyde or ketone group. In some embodiments, the at least one unnatural amino acid does not comprise an aromatic side chain. In some embodiments, the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK; the chemical structure of which is shown as compound 90 in Figure 3C), N6-(propargyloxy)-carbonyl-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-copropyl)amino)ethyl)selanine)propanoic acid, 2-amino-3-(phenylselanyl)propanoic acid or selenocysteine. In some embodiments, at least one unnatural amino acid is introduced into the modified IL-2 polypeptide via a tRNA synthetase/tRNA orthogonal pair. In some embodiments, the orthogonal tRNA of the orthogonal synthetase/tRNA pair comprises at least one unnatural nucleobase. In some embodiments, the modified IL-2 polypeptide is covalently attached to the conjugation moiety via at least one unnatural amino acid. In some embodiments, the conjugation moiety comprises a water-soluble polymer, a lipid, a protein, and/or a peptide. In some embodiments, the water-soluble polymer comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or a combination thereof. In some embodiments, the water-soluble polymer comprises a PEG molecule. In some embodiments, the PEG molecule is linear PEG. In some embodiments, the PEG molecule is branched PEG. In some embodiments, the PEG has a mass of about 2,000 to 50,000 daltons (Da). In some embodiments, the PEG has a molecular weight of about 5,000 Da, 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the molecular weight determines, at least in part, the in vivo plasma half-life of the IL-2 polypeptide. In some embodiments, the water-soluble polymer comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, the lipid comprises a fatty acid. In some embodiments, the fatty acid comprises from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms. In some embodiments, the fatty acid is a saturated fatty acid. In some embodiments, the protein comprises albumin, transferrin, or transthyretin. In some embodiments, the conjugation moiety comprises a TLR agonist. In some embodiments, the protein comprises an antibody or binding fragments thereof. In some embodiments, the antibody or binding fragments thereof provides an Fc portion of an antibody. In some embodiments, the peptide comprises an XTEN peptide, a glycine-rich amino acid homopolymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein polymer (GLK). In some embodiments, the conjugation moiety is indirectly linked to at least one non-natural amino acid of the modified IL-2 via a linker. In some embodiments, the linker comprises a homobifunctional linker, a heterobifunctional linker, a zero-length linker, a cleavable or non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof. In some embodiments, the decrease in affinity is about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the decrease in binding affinity to IL-2Rα compared to a wild-type IL-2 polypeptide. In some embodiments, the decrease in binding affinity to IL-2Rα is about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more compared to a wild-type IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide is a functionally active fragment of a full-length IL-2 polypeptide, a recombinant IL-2 polypeptide, or a recombinant human IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide comprises an N-terminal deletion, a C-terminal deletion, or a combination thereof. In some embodiments, the N-terminal deletion comprises a deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 residues from the N-terminus, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some embodiments, the C-terminal deletion comprises a deletion of the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more residues from the C-terminus, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some embodiments, the functionally active fragment comprises a region of IL-2 10-133, 20-133, 30-133, 10-130, 20-130, 30-130, 10-125, 20-125, 30-125, 1-130, or 1-125, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some embodiments, the modified IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments, the modified IL-2 polypeptide with reduced binding affinity for IL-2Rα is capable of providing for expansion of populations of CD4+ helper cells, CD8+ effector naive and memory cells, CD8+ T cells, natural killer (NK) cells, or natural killer T (NKT) cells, or a combination thereof. In some embodiments, the conjugate moiety or unnatural amino acid disrupts or blocks the binding of IL-2 to IL-2Rα. In some embodiments, activation of a population of CD4+ helper cells, CD8+ effector naive and memory cells, natural killer (NK) cells, or natural killer T cells (NKT) by the IL-2Rβγ complex with a modified IL-2 polypeptide maintains significant efficiency of activation of said cell population compared to a wild-type IL-2 polypeptide. In some embodiments, the efficiency of receptor signaling of the modified IL-2 polypeptide to the IL-2Rβγ complex is greater than the efficiency of receptor signaling of the wild-type IL-2 polypeptide to the IL-2Rβγ complex. In some embodiments, the efficiency of receptor signaling of the modified IL-2 polypeptide to the IL-2Rβγ complex is lower than the efficiency of receptor signaling of the wild-type IL-2 polypeptide to the IL-2Rβγ complex. In some embodiments, the modified IL-2 polypeptide exhibits first receptor signaling efficiency to IL-2Rβγ and second receptor signaling efficiency to IL-2Rαβγ, and wherein the first receptor signaling efficiency is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 500-fold higher than the second receptor signaling efficiency, wherein the first receptor signaling efficiency of the modified IL-2 polypeptide is higher than the receptor signaling efficiency of the wild-type IL-2 polypeptide to IL-2Rβγ, and the second receptor signaling efficiency of the modified IL-2 polypeptide is lower than the receptor signaling efficiency of the wild-type IL-2 polypeptide to IL-2Rαβγ. In some embodiments, the first receptor signaling efficiency of the modified IL-2 polypeptide is at least 1-fold lower than the receptor signaling efficiency of the wild-type IL-2 polypeptide.

[00566] In aspects disclosed herein, isolated and modified interleukin 2 (IL-2) polypeptides comprising at least one unnatural amino acid are provided, wherein the isolated and modified IL-2 polypeptide exhibits effectiveness in transducing a first receptor signal to an IL-2βγ signaling complex and effectiveness in transducing a second receptor signal to an IL-2αβγ signaling complex, and wherein the difference between the efficiency of the first receptor signaling and the efficiency of the second receptor signaling is less than 10-fold. In some embodiments, the difference in receptor signaling efficiency is less than 5-fold, less than 4-fold, less than 3-fold, less than 2-fold, or less than 1-fold. In some embodiments, the position of the at least one unnatural amino acid is selected from K35, T37, R38, T41, F42, K43, F44, Y45, E60, E61, E62, K64, P65, E68, V69, N71, L72, M104, C105, Y107, wherein the residue positions correspond to positions 35, 37, 38, 41, 42, 43, 44, 45, 61, 62, 64, 65, 68, 69, 71, 72, 104, 105, and 107 set forth in SEQ ID NO: 1. In some embodiments, the position of the at least one unnatural amino acid is selected from T37, R38, T41, F42, K43, F44, Y45, E61, E62, P65, E68, and L72, wherein the residue positions correspond to positions 37, 38, 41, 42, 43, 44, 45, 61, 62, 65, 68, and 72 as set forth in SEQ ID NO: 1. In some embodiments, the position of the at least one non-naturally occurring amino acid is selected from K35, K64, V69, N71, M104, C105, and Y107, wherein the residue positions correspond to positions 35, 64, 69, 71, 104, 105, and 107 as set forth in SEQ ID NO: 1. In some embodiments, the position of the at least one non-naturally occurring amino acid is selected from T37, R38, T41, Y45, E61, E68, and L72, wherein the residue positions correspond to positions 37, 38, 41, 45, 61, 68, and 72 as set forth in SEQ ID NO: 1. In some embodiments, the position of the at least one unnatural amino acid is selected from F42, K43, F44, E62, and P65, wherein the residue positions correspond to positions 42, 43, 44, 62, and 65 as set forth in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid is a lysine analog, comprises an aromatic side chain, comprises an azido group, comprises an alkyne group, or comprises an aldehyde or ketone group. In some embodiments, the at least one unnatural amino acid does not comprise an aromatic side chain. In some embodiments, the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK; the chemical structure of which is shown as compound 90 in Figure 3C), N6-(propargyloxy)-carbonyl-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-copropyl)amino)ethyl)selanine)propanoic acid, 2-amino-3-(phenylselanyl)propanoic acid or selenocysteine. In some embodiments, at least one unnatural amino acid is introduced into the modified IL-2 polypeptide via a tRNA synthetase/tRNA orthogonal pair. In some embodiments, the orthogonal tRNA of the orthogonal synthetase/tRNA pair comprises at least one unnatural nucleobase. In some embodiments, the modified IL-2 polypeptide is covalently attached to the conjugation moiety via at least one unnatural amino acid. In some embodiments, the conjugation moiety comprises a water-soluble polymer, a lipid, a protein, and/or a peptide. In some embodiments, the water-soluble polymer comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or a combination thereof. In some embodiments, the water-soluble polymer comprises a PEG molecule. In some embodiments, the PEG molecule is linear PEG. In some embodiments, the PEG molecule is branched PEG. In some embodiments, the PEG has a mass of about 2,000 to 50,000 daltons (Da). In some embodiments, the PEG has a molecular weight of about 5,000 Da, 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the molecular weight determines, at least in part, the in vivo plasma half-life of the IL-2 polypeptide. In some embodiments, the water-soluble polymer comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, the lipid comprises a fatty acid. In some embodiments, the fatty acid comprises from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms. In some embodiments, the fatty acid is a saturated fatty acid. In some embodiments, the protein comprises albumin, transferrin, or transthyretin. In some embodiments, the conjugation moiety comprises a TLR agonist. In some embodiments, the protein comprises an antibody or binding fragments thereof. In some embodiments, the antibody or binding fragments thereof provides an Fc portion of an antibody. In some embodiments, the peptide comprises an XTEN peptide, a glycine-rich amino acid homopolymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein polymer (GLK). In some embodiments, the conjugation moiety is indirectly linked to at least one non-natural amino acid of the modified IL-2 via a linker. In some embodiments, the linker comprises a homobifunctional linker, a heterobifunctional linker, a zero-length linker, a cleavable or non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof. In some embodiments, the decrease in affinity is about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the decrease in binding affinity to IL-2Rα compared to a wild-type IL-2 polypeptide. In some embodiments, the decrease in binding affinity to IL-2Rα is about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more compared to a wild-type IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide is a functionally active fragment of a full-length IL-2 polypeptide, a recombinant IL-2 polypeptide, or a recombinant human IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide comprises an N-terminal deletion, a C-terminal deletion, or a combination thereof. In some embodiments, the N-terminal deletion comprises a deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 residues from the N-terminus, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some embodiments, the C-terminal deletion comprises a deletion of the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more residues from the C-terminus, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some embodiments, the functionally active fragment comprises a region of IL-2 10-133, 20-133, 30-133, 10-130, 20-130, 30-130, 10-125, 20-125, 30-125, 1-130, or 1-125, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some embodiments, the modified IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments, the modified IL-2 polypeptide with reduced binding affinity for IL-2Rα is capable of providing for expansion of populations of CD4+ helper cells, CD8+ effector naive and memory cells, CD8+ T cells, natural killer (NK) cells, or natural killer T (NKT) cells, or a combination thereof. In some embodiments, the conjugate moiety or unnatural amino acid disrupts or blocks the binding of IL-2 to IL-2Rα. In some embodiments, activation of a population of CD4+ helper cells, CD8+ effector naive and memory cells, natural killer (NK) cells, or natural killer T cells (NKT) by the IL-2Rβγ complex with a modified IL-2 polypeptide maintains significant efficiency of activation of said cell population compared to a wild-type IL-2 polypeptide. In some embodiments, the efficiency of receptor signaling of the modified IL-2 polypeptide to the IL-2Rβγ complex is greater than the efficiency of receptor signaling of the wild-type IL-2 polypeptide to the IL-2Rβγ complex. In some embodiments, the efficiency of receptor signaling of the modified IL-2 polypeptide to the IL-2Rβγ complex is lower than the efficiency of receptor signaling of the wild-type IL-2 polypeptide to the IL-2Rβγ complex. In some embodiments, the modified IL-2 polypeptide exhibits first receptor signaling efficiency to IL-2Rβγ and second receptor signaling efficiency to IL-2Rαβγ, and wherein the first receptor signaling efficiency is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 500-fold higher than the second receptor signaling efficiency, wherein the first receptor signaling efficiency of the modified IL-2 polypeptide is higher than the receptor signaling efficiency of the wild-type IL-2 polypeptide to IL-2Rβγ, and the second receptor signaling efficiency of the modified IL-2 polypeptide is lower than the receptor signaling efficiency of the wild-type IL-2 polypeptide to IL-2Rαβγ. In some embodiments, the first receptor signaling efficiency of the modified IL-2 polypeptide is at least 1-fold lower than the receptor signaling efficiency of the wild-type IL-2 polypeptide.

[00567] In aspects disclosed herein, interleukin 2 (IL-2) conjugates are provided comprising a non-natural amino acid covalently attached to a conjugate moiety, wherein the non-natural amino acid is located in region 35-107, and wherein region 35-107 corresponds to residues K35-Y107 in the sequence of SEQ ID NO: 1. In some embodiments, the non-natural amino acid is located in region 42, and wherein region 42 corresponds to residue F42. In some embodiments, the conjugate moiety comprises a water-soluble polymer with a molecular weight of 2,000 to 50,000 daltons (Da). In some embodiments, the conjugate moiety comprises a water-soluble polymer having a molecular weight of 5,000 Da, 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the water-soluble polymer comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or a combination thereof. In some embodiments, the molecular weight determines, at least in part, the in vivo plasma half-life of the IL-2 conjugate. In some embodiments, the IL-2 conjugate comprises a mutation at residue P65 corresponding to residue position 65 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the IL-2 conjugate comprises a mutation at residue E61, corresponding to residue position 61 of SEQ ID NO: 1, and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the IL-2 conjugate comprises a mutation at residue F42, corresponding to residue position 42 of SEQ ID NO: 1, and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da.

[00568] Aspects disclosed herein provide interleukin 2 receptor βγ (IL-2Rβγ) binding proteins, wherein the binding protein has a binding affinity for interleukin 2 receptor α (IL-2Rα) less than wild-type human IL-2 (hIL-2), and wherein the binding protein comprises at least one unnatural amino acid. In some embodiments, the binding protein is a modified IL-2 polypeptide or a functionally active fragment thereof, wherein the modified IL-2 polypeptide comprises at least one unnatural amino acid. In some embodiments, the at least one unnatural amino acid is located within region 35-107, and wherein region 35-107 corresponds to residues K35-Y107 of SEQ ID NO: 1.

[00569] In aspects disclosed herein, IL-2/IL-2Rβγ complexes are provided comprising a modified IL-2 polypeptide comprising a non-natural amino acid and IL-2Rβγ, wherein the modified IL-2 polypeptide has a reduced binding affinity for IL-2Rα, and wherein the reduced binding affinity is compared to the binding affinity between a wild-type IL-2 polypeptide and IL-2Rα. In some embodiments, the modified IL-2 polypeptide further comprises a conjugation moiety covalently attached to the non-natural amino acid.

[00570] In aspects disclosed herein, there are provided CD4+ helper cell, CD8+ effector naive and memory cell, CD8+ T cell, natural killer (NK) cell, or natural killer T cell (NKT) activators that selectively allow expansion of CD4+ helper cells, CD8+ effector naive and memory cells, natural killer (NK) cells, or natural killer T cells (NKT) in a cell population, wherein said activator comprises a modified interleukin 2 (IL-2) polypeptide comprising at least one non-natural amino acid. In some embodiments, said activator allows expansion of CD4+ regulatory (Treg) T cells by less than 20%, 15%, 10%, 5%, 1%, or 0.1% upon contact of said activator with said cell population. In some embodiments, said activator does not provide expansion of Treg cells in said cell population. In some embodiments, said cell population is an in vivo cell population. In some embodiments, said cell population is an in vitro cell population. In some embodiments, said cell population is an ex vivo cell population.

[00571] In aspects disclosed herein, a pharmaceutical composition is provided comprising an isolated and modified IL-2 polypeptide described herein, an IL-2 conjugate described herein, an IL-2Rβγ binding protein described herein, or a CD4+ helper cell, CD8+ effector naive and memory cell, natural killer (NK) cell, or natural killer T (NKT) cell activator described herein, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for systemic delivery. In some embodiments, the pharmaceutical composition is formulated for parenteral administration. In some embodiments, the IL-2 polypeptide or IL-2 conjugate comprises a mutation at residue F42 corresponding to residue position 42 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da). In some embodiments, the IL-2 polypeptide or IL-2 conjugate comprises a mutation at residue F42 corresponding to residue position 42 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 10,000 daltons (Da). In some embodiments, the IL-2 polypeptide or IL-2 conjugate comprises a mutation at residue F42 corresponding to residue position 42 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 15,000 daltons (Da). In some embodiments, the IL-2 polypeptide or IL-2 conjugate comprises a mutation at residue F42 corresponding to residue position 42 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 20,000 daltons (Da). In some embodiments, the IL-2 polypeptide or IL-2 conjugate comprises a mutation at residue F42 corresponding to residue position 42 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 25,000 daltons (Da). In some embodiments, the IL-2 polypeptide or IL-2 conjugate comprises a mutation at residue F42 corresponding to residue position 42 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 30,000 daltons (Da). In some embodiments, the IL-2 polypeptide or IL-2 conjugate comprises a mutation at the P65 residue corresponding to residue position 65 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da). In some embodiments, the IL-2 polypeptide or IL-2 conjugate comprises a mutation at the P65 residue corresponding to residue position 65 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 10,000 daltons (Da). In some embodiments, the IL-2 polypeptide or IL-2 conjugate comprises a mutation at the P65 residue corresponding to residue position 65 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 15,000 daltons (Da). In some embodiments, the IL-2 polypeptide or IL-2 conjugate comprises a mutation at the P65 residue corresponding to residue position 65 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 20,000 daltons (Da). In some embodiments, the IL-2 polypeptide or IL-2 conjugate comprises a mutation at the P65 residue corresponding to residue position 65 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 25,000 daltons (Da). In some embodiments, the IL-2 polypeptide or IL-2 conjugate comprises a mutation at the P65 residue corresponding to residue position 65 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 30,000 daltons (Da). In some embodiments, the IL-2 polypeptide or IL-2 conjugate comprises a mutation at residue E62 corresponding to residue position 62 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da). In some embodiments, the IL-2 polypeptide or IL-2 conjugate comprises a mutation at residue E62 corresponding to residue position 62 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 10,000 daltons (Da). In some embodiments, the IL-2 polypeptide or IL-2 conjugate comprises a mutation at residue E62 corresponding to residue position 62 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 15,000 daltons (Da). In some embodiments, the IL-2 polypeptide or IL-2 conjugate comprises a mutation at residue E62 corresponding to residue position 62 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 20,000 daltons (Da). In some embodiments, the IL-2 polypeptide or IL-2 conjugate comprises a mutation at residue E62 corresponding to residue position 62 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 25,000 daltons (Da). In some embodiments, the IL-2 polypeptide or IL-2 conjugate comprises a mutation at residue E62 corresponding to residue position 62 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 30,000 daltons (Da).

[00572] In aspects disclosed herein, a method of treating a disease or condition in a subject in need thereof is provided, comprising administering to the subject a therapeutically effective amount of an isolated and modified IL-2 polypeptide described herein, an IL-2 conjugate described herein, an IL-2Rβγ binding protein described herein, an activator of a CD4+ helper cell, a CD8+ effector naive and memory cell, a natural killer (NK) cell, or a natural killer T (NKT) cell described herein, or a pharmaceutical composition described herein. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is a solid tumor cancer. In some embodiments, the solid tumor cancer is bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the hematological malignancy is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, high-grade B-cell non-Burkitt's lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-cell lymphoblastic lymphoma, prolymphocytic B-cell leukemia, lymphoplasmacytic lymphoma, marginal zone cell lymphoma spleen, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. In some embodiments, the disease or condition comprises a pathogenic infection. In some embodiments, the pathogenic infection is caused by a retrovirus, a virus, a deoxyribonucleic acid (DNA) virus, or a ribonucleic acid (RNA) virus, or a combination thereof. In some embodiments, the RNA virus includes a negative-sense single-stranded (ss) RNA virus, a positive-sense sRNA virus, or a double-stranded (ds) RNA virus. In some embodiments, the DNA virus includes a single-stranded (ss) DNA virus or a double-stranded (ds) DNA virus. In some embodiments, the disease or condition comprises an autoimmune disease. In some embodiments, the disease or condition comprises alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes, juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, multiple sclerosis, pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis, vitiligo, or Wegener's granulomatosis, or a combination thereof. In some embodiments, the methods further comprise administering an additional therapeutic agent. In some embodiments, the isolated and modified IL-2 polypeptide, IL-2 conjugate, IL-2Rβγ binding protein, CD4+ helper cell, CD8+ effector naive and memory cell, natural killer (NK) cell or natural killer T cell (NKT) activator, or pharmaceutical composition, and an additional therapeutic agent are administered simultaneously. In some embodiments, the isolated and modified IL-2 polypeptide, IL-2 conjugate, IL-2Rβγ binding protein, CD4+ helper cell, CD8+ effector naive and memory cell, natural killer (NK) cell or natural killer T cell (NKT) activator, or pharmaceutical composition, and an additional therapeutic agent are administered sequentially. In some embodiments, the isolated and modified IL-2 polypeptide, IL-2 conjugate, IL-2Rβγ binding protein, CD4+ helper cell, CD8+ effector naive and memory cell, natural killer (NK) cell or natural killer T cell (NKT) activator, or pharmaceutical composition is administered prior to an additional therapeutic agent. In some embodiments, the isolated and modified IL-2 polypeptide, IL-2 conjugate, IL-2Rβγ binding protein, CD4+ helper cell, CD8+ effector naive and memory cell, natural killer (NK) cell or natural killer T cell (NKT) activator, or pharmaceutical composition is administered after an additional therapeutic agent is administered.

[00573] In aspects disclosed herein, methods are provided for expanding a population of CD4+ helper cells, CD8+ effector naive and memory cells, natural killer (NK) cells, or natural killer T cells (NKT cells), comprising contacting the cell population with an isolated and modified IL-2 polypeptide described herein, any IL-2 conjugate described herein, an IL-2 conjugate described herein, an IL-2Rβγ binding protein described herein, a CD4+ helper cell, CD8+ effector naive and memory cell, CD8+ T cell, natural killer (NK) cell, or natural killer T cell (NKT) cell activator described herein, or a pharmaceutical composition described herein for a time sufficient to induce complex formation with IL-2Rβγ, thereby stimulating the expansion of a population of Teff-, CD8+ T-cells and/or NK-cells. In some embodiments, the isolated and modified IL-2 polypeptide, IL-2 conjugate, IL-2Rβγ binding protein, CD4+ helper cell, CD8+ effector naive and memory cell, natural killer (NK) cell or natural killer T-cell (NKT) activator or pharmaceutical composition provides for the expansion of CD4+ regulatory (Treg) T-cells by less than 20%, 15%, 10%, 5% or 1% in a population of CD3+ cells compared to the expansion of CD4+ Treg-cells in a population of CD3+ cells upon contact with a wild-type IL-2 polypeptide. In some embodiments, the isolated and modified IL-2 polypeptide, IL-2 conjugate, IL-2Rβγ binding protein, CD4+ helper cell, CD8+ effector naive and memory cell, natural killer (NK) cell, or natural killer T (NKT) cell activator, or pharmaceutical composition does not provide for expansion of CD4+ Treg cells in the cell population. In some embodiments, the ratio of Teff cells to Treg cells in the cell population following incubation with the isolated and modified IL-2 polypeptide, IL-2 conjugate, IL-2Rβγ binding protein, CD4+ helper cell, CD8+ effector naive and memory cell, natural killer (NK) cell, or natural killer T (NKT) cell activator, or pharmaceutical composition is about or at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 50:1, or 100:1. In some embodiments, the method is an in vivo method. In some embodiments, the method is an in vitro method. In some embodiments, the method is an ex vivo method. In some embodiments, the subject is a human.

[00574] In aspects disclosed herein, kits are provided comprising one or more of an isolated and modified IL-2 polypeptide described herein, an IL-2 conjugate described herein, an IL-2Rβγ binding protein described herein, an activator of a CD4+ helper cell, a CD8+ effector naive and memory cell, a natural killer (NK) cell, or a natural killer T (NKT) cell described herein, and/or a pharmaceutical composition described herein. In some embodiments, the modified IL-2 polypeptide comprises a mutation at residue F42, corresponding to residue position 42 of SEQ ID NO: 1, and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some embodiments, PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of PEG does not affect the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or the desired maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of a wild-type IL-2 polypeptide to IL-2Rα. In some embodiments, the molecular weight of PEG does not affect the formation of a modified IL-2/IL-2Rβγ polypeptide complex. In some embodiments, the IL-2 conjugate comprises a mutation at residue F42, corresponding to residue position 42 of SEQ ID NO: 1, and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some embodiments, PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of PEG does not affect the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or the desired maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of a wild-type IL-2 polypeptide to IL-2Rα. In some embodiments, the molecular weight of PEG does not affect the formation of a modified IL-2/IL-2Rβγ polypeptide complex. In some embodiments, the modified IL-2 polypeptide comprises a mutation at residue E62, corresponding to residue position 62 of SEQ ID NO: 1, and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some embodiments, PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of PEG does not affect the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or the desired maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of a wild-type IL-2 polypeptide to IL-2Rα. In some embodiments, the molecular weight of PEG does not affect the formation of a modified IL-2/IL-2Rβγ polypeptide complex. In some embodiments, the IL-2 conjugate comprises a mutation at residue E62, corresponding to residue position 62 of SEQ ID NO: 1, and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some embodiments, PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of PEG does not affect the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or the desired maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of a wild-type IL-2 polypeptide to IL-2Rα. In some embodiments, the molecular weight of PEG does not affect the formation of a modified IL-2/IL-2Rβγ polypeptide complex. In some embodiments, the modified IL-2 polypeptide comprises a mutation at residue P65 corresponding to residue position 65 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some embodiments, PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of PEG does not affect the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or the desired maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of a wild-type IL-2 polypeptide to IL-2Rα. In some embodiments, the molecular weight of PEG does not affect the formation of a modified IL-2/IL-2Rβγ polypeptide complex. In some embodiments, the IL-2 conjugate comprises a mutation at residue P65 corresponding to residue position 65 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some embodiments, PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of PEG does not affect the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or the desired maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of a wild-type IL-2 polypeptide to IL-2Rα. In some embodiments, the molecular weight of PEG does not affect formation of the modified IL-2/IL-2Rβγ polypeptide complex.

[00575] In aspects disclosed herein, a kit is provided comprising one or more polynucleic acid sequences encoding an IL-2 polypeptide described herein, an IL-2 conjugate described herein, an IL-2Rβγ binding protein described herein, a CD4+ helper cell, CD8+ effector naive and memory cell, natural killer (NK) cell, or natural killer T (NKT) cell activator described herein, and/or a pharmaceutical composition described herein. In some embodiments, the modified IL-2 polypeptide comprises a mutation at residue F42, corresponding to residue position 42 of SEQ ID NO: 1, and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some embodiments, PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of PEG does not affect the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or the desired maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of a wild-type IL-2 polypeptide to IL-2Rα. In some embodiments, the molecular weight of PEG does not affect the formation of a modified IL-2/IL-2Rβγ polypeptide complex. In some embodiments, the IL-2 conjugate comprises a mutation at residue F42, corresponding to residue position 42 of SEQ ID NO: 1, and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some embodiments, PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of PEG does not affect the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or the desired maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of a wild-type IL-2 polypeptide to IL-2Rα. In some embodiments, the molecular weight of PEG does not affect the formation of a modified IL-2/IL-2Rβγ polypeptide complex. In some embodiments, the modified IL-2 polypeptide comprises a mutation at residue E62, corresponding to residue position 62 of SEQ ID NO: 1, and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da, e.g., 5 kDa or 5 kDa), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some embodiments, PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of PEG does not affect the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or the desired maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of a wild-type IL-2 polypeptide to IL-2Rα. In some embodiments, the molecular weight of PEG does not affect the formation of a modified IL-2/IL-2Rβγ polypeptide complex. In some embodiments, the IL-2 conjugate comprises a mutation at residue E62, corresponding to residue position 62 of SEQ ID NO: 1, and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some embodiments, PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of PEG does not affect the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or the desired maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of a wild-type IL-2 polypeptide to IL-2Rα. In some embodiments, the molecular weight of PEG does not affect the formation of a modified IL-2/IL-2Rβγ polypeptide complex. In some embodiments, the modified IL-2 polypeptide comprises a mutation at residue P65 corresponding to residue position 65 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some embodiments, PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of PEG does not affect the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or the desired maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of a wild-type IL-2 polypeptide to IL-2Rα. In some embodiments, the molecular weight of PEG does not affect the formation of a modified IL-2/IL-2Rβγ polypeptide complex. In some embodiments, the IL-2 conjugate comprises a mutation at residue P65 corresponding to residue position 65 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some embodiments, PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of PEG does not affect the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rα or the desired maintained binding to the IL-2Rβγ signaling complex, wherein the reduced binding to IL-2Rα is compared to binding of a wild-type IL-2 polypeptide to IL-2Rα. In some embodiments, the molecular weight of PEG does not affect the formation of a modified IL-2/IL-2Rβγ polypeptide complex.

[00576] Aspects disclosed herein feature a cell culture system comprising an immune cell and an isolated and modified IL-2 polypeptide described herein and/or an IL-2 conjugate described herein. In some embodiments, the immune cell comprises a CD4+ helper cell, a CD8+ effector naive and memory T cell, a CD8+ cell, a natural killer (NK) cell, or a natural killer T (NKT) cell. In some embodiments, the modified IL-2 polypeptide or IL-2 conjugate is effective to enhance the expansion of the immune cell.

[00577] Aspects disclosed herein provide reagents for making an adoptive cell therapy agent comprising an isolated and modified IL-2 polypeptide described herein and/or an IL-2 conjugate described herein. In some embodiments, the isolated and modified IL-2 polypeptide and/or IL-2 conjugate comprises a mutation at residue P65 corresponding to residue position 65 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the isolated and modified IL-2 polypeptide and/or IL-2 conjugate comprises a mutation at residue E61, corresponding to residue position 61 of SEQ ID NO: 1, and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the isolated and modified IL-2 polypeptide and/or IL-2 conjugate comprises a mutation at residue F42, corresponding to residue position 42 of SEQ ID NO: 1, and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the molecular weight of the PEG is effective to improve the solubility of the isolated and modified IL-2 polypeptide and/or IL-2 conjugate. In some embodiments, the molecular weight of the PEG is effective to enhance the stability of the isolated and modified IL-2 polypeptide and/or IL-2 conjugate. In some embodiments, the molecular weight of the PEG is effective to improve the manufacturing efficiency of an adoptive cell therapy agent comprising the isolated and modified IL-2 polypeptide and/or IL-2 conjugate.

[00578] In aspects disclosed herein, an isolated and modified interleukin 2 (IL-2) polypeptide is provided that comprises at least one unnatural amino acid at a particular position that reduces the efficiency of receptor signaling to the interleukin 2 receptor βγ (IL-2Rβγ) or reduces the recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex but maintains significant activation of the interleukin 2 receptor αβγ (IL-2Rαβγ), wherein the reduced efficiency of receptor signaling is compared to the efficiency of receptor signaling between a wild-type IL-2 polypeptide and IL-2Rβγ, and wherein the recruitment is compared to the recruitment of the IL-2Rγ subunit by the wild-type IL-2 polypeptide.

[00579] In aspects disclosed herein, an isolated and modified interleukin 2 (IL-2) polypeptide is provided, comprising at least one unnatural amino acid at a particular position, which increases recruitment of an IL-2Rα subunit to the IL-2 polypeptide, resulting in activation of the interleukin 2 receptor αβγ (IL-2Rαβγ), wherein the increase in recruitment is compared to recruitment of the IL-2Rα subunit by a wild-type IL-2 polypeptide. In some embodiments, the position of the at least one non-naturally occurring amino acid is selected from A108, D109, E110, T111, A112, T113, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some embodiments, the position of the at least one non-naturally occurring amino acid is selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, I92, L94, E95, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some embodiments, the position of the at least one unnatural amino acid is selected from P2, T3, S4, S5, S6, T7, K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, R81, P82, R83, D84, S87, N88, N89, V91, I92, L94, E95, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107,. In some embodiments, the position of the at least one unnatural amino acid is selected from K8, K9, Q11, L12, E15, H16, L18, L19, D20, Q22, M23, N26, R81, D84, S87, N88, V91, I92, L94, E95, E116, N119, R120, T123, A125, Q126, S127, S130, T131, L132, and T133, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some embodiments, the position of the at least one unnatural amino acid is selected from P2, T3, S4, S5, S6, T7, G27, N29, N30, Y31, K32, K35, T37, M46, K47, K48, A50, T51, E52, K53, H55, Q57, E60, E67, N71, Q74, S75, K76, N77, F78, H79, P82, R83, N89, K97, G98, S99, E100, T101, T102, F103, M104, C105, E106, Y107, A108, D109, E110, T111, A112, and T113, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some embodiments, the position of the at least one non-naturally occurring amino acid is selected from K8, K9, L12, E15, H16, L19, D20, Q22, M23, N26, D84, N88, E95 and Q126, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some embodiments, the position of the at least one non-naturally occurring amino acid is selected from K8, K9, and H16, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some embodiments, the position of the at least one non-naturally occurring amino acid is selected from Q22, N26, N88, and Q126, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some embodiments, the position of the at least one non-naturally occurring amino acid is selected from E15, D20, D84, and E95, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some embodiments, the position of the at least one non-naturally occurring amino acid is selected from L12, L19, and M23, wherein the amino acid residue numbering corresponds to the numbering in SEQ ID NO: 1. In some embodiments, the position of the at least one non-naturally occurring amino acid is selected from Q22 and N26, wherein the numbering of the amino acid residues corresponds to the numbering in SEQ ID NO: 1. In some embodiments, the at least one unnatural amino acid is a lysine analog, is a cysteine analog, or is a histidine analog, has an aromatic side chain, has an azido group, has an alkyne group, or has an aldehyde or ketone group. In some embodiments, the at least one unnatural amino acid does not have an aromatic side chain. In some embodiments, the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK; the chemical structure of which is shown as compound 90 in Figure 3C), N6-(propargyloxy)-carbonyl-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (AMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-copropyl)amino)ethyl)selanine)propanoic acid, 2-amino-3-(phenylselanyl)propanoic acid or selenocysteine. In some embodiments, at least one unnatural amino acid is incorporated into the modified IL-2 polypeptide via an orthogonal tRNA. In some embodiments, the orthogonal tRNA disclosed herein comprises at least one unnatural nucleobase. In some embodiments, the modified IL-2 polypeptide is covalently attached to the conjugation moiety via at least one unnatural amino acid. In some embodiments, the conjugation moiety comprises a water-soluble polymer, lipid, protein, or peptide. In some embodiments, the water-soluble polymer comprises polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or a combination thereof. In some embodiments, the water-soluble polymer comprises a PEG molecule. In some embodiments, the PEG molecule is linear PEG. In some embodiments, the PEG molecule is branched PEG. In some embodiments, the PEG has a mass of about 2,000 to 50,000 daltons (Da). In some embodiments, the PEG has a molecular weight of about 5,000 Da, 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the unnatural amino acid comprises F42 at residue position 42 of SEQ ID NO: 1. In some embodiments, the unnatural amino acid comprises P65 at residue position 65 of SEQ: 1. In some embodiments, the unnatural amino acid comprises E62 at residue position 62 of SEQ ID NO: 1. In some embodiments herein, molecular weight determines, at least in part, the in vivo plasma half-life of the IL-2 polypeptide. In some embodiments, PEG is associated with a longer in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some embodiments, PEG is associated with a shorter in vivo plasma half-life of the modified IL-2 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of PEG does not affect the efficiency of receptor signaling of the modified IL-2 polypeptide to IL-2βγ or IL-2αβγ signaling complexes. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced binding of the modified IL-2 polypeptide to IL-2Rβγ or the desired maintained binding to the IL-2Rβγα signaling complex, wherein the reduced binding to IL-2Rβγ is compared to the binding of a wild-type IL-2 polypeptide and IL-2Rβγα. In some embodiments, the molecular weight of the PEG does not affect the formation of the modified IL-2/IL-2Rβγ polypeptide complex. In some embodiments, the water-soluble polymer comprises a polysaccharide. In some embodiments, the polysaccharide comprises dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some embodiments, the lipid comprises a fatty acid. In some embodiments, the fatty acid comprises from about 6 to about 26 carbon atoms, from about 6 to about 24 carbon atoms, from about 6 to about 22 carbon atoms, from about 6 to about 20 carbon atoms, from about 6 to about 18 carbon atoms, from about 20 to about 26 carbon atoms, from about 12 to about 26 carbon atoms, from about 12 to about 24 carbon atoms, from about 12 to about 22 carbon atoms, from about 12 to about 20 carbon atoms, or from about 12 to about 18 carbon atoms. In some embodiments, the fatty acid is a saturated fatty acid. In some embodiments, the protein comprises albumin, transferrin, or transthyretin. In some embodiments, the protein comprises an antibody or binding fragments thereof. In some embodiments herein, the antibody or binding fragments thereof provide an Fc portion of an antibody. In some embodiments, the peptide comprises an XTEN peptide, a glycine-rich amino acid homopolymer (HAP), a PAS polypeptide, an elastin-like polypeptide (ELP), a CTP peptide, or a gelatin-like protein polymer (GLK). In some embodiments, the conjugation moiety is indirectly linked to at least one non-natural amino acid of the modified IL-2 via a linker. In some embodiments, the linker comprises a homobifunctional linker, a heterobifunctional linker, a zero-length linker, a cleavable or non-cleavable dipeptide linker, a maleimide group, a spacer, or a combination thereof. In some embodiments, the isolated and modified IL-2 polypeptide is characterized by a decrease in the efficiency of receptor signaling to IL-2Rβγ, and wherein the decrease in the efficiency of receptor signaling to IL-2Rβγ is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold or more compared to a wild-type IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide is a functionally active fragment of a full-length IL-2 polypeptide, a recombinant IL-2 polypeptide, or a recombinant human IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide comprises an N-terminal deletion, a C-terminal deletion, or a combination thereof. In some embodiments, the N-terminal deletion comprises a deletion of the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or 30 residues from the N-terminus, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some embodiments, the C-terminal deletion comprises a deletion of the last 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20 or more residues from the C-terminus, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some embodiments, the functionally active fragment comprises a region of IL-2 10-133, 20-133, 30-133, 10-130, 20-130, 30-130, 10-125, 20-125, 30-125, 1-130, or 1-125, wherein the residue positions are relative to the positions of SEQ ID NO: 1. In some embodiments, the modified IL-2 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of SEQ ID NO: 1. In some embodiments, the modified IL-2 polypeptide with decreased efficiency of IL-2Rβγ receptor signaling is capable of promoting expansion of CD4+ regulatory T cells (Treg). In some embodiments, the conjugation moiety impairs or blocks the efficiency of IL-2 receptor signaling with IL-2Rβγ or reduces the recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex. In some embodiments, the proliferation of CD4+ Treg cells by the modified IL-2/IL-2Rαβγ complex is equivalent to or greater than that of a wild-type IL-2 polypeptide. In some embodiments, the modified IL-2/IL-2Rαβγ complex induces proliferation of CD4+ Treg cells into a population that is sufficient to modulate disease progression in an animal model. In some embodiments, the modified IL-2 polypeptide exhibits first receptor signaling efficiency to IL-2Rβγ and second receptor signaling efficiency to IL-2Rαβγ, wherein the first receptor signaling efficiency is at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or less than the second receptor signaling efficiency. In some embodiments, the first receptor signaling efficiency of the modified IL-2 polypeptide is lower than the receptor signaling efficiency of the wild-type IL-2 polypeptide to IL-2Rβγ. In some embodiments, the efficiency of the modified IL-2 polypeptide in second receptor signaling is lower than the efficiency of receptor signaling of the wild-type IL-2 polypeptide to IL-2Rαβγ. In some embodiments, the modified IL-2 polypeptide further provides for an increase in recruitment of the IL-2Rα subunit to the IL-2 polypeptide, resulting in activation of the interleukin 2 receptor αβγ (IL-2Rαβγ), wherein the increase in recruitment is compared to recruitment of the IL-2Rα subunit by the wild-type IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide further provides for a reduction in the recruitment of the IL-2Rγ subunit to the IL-2/IL-2Rβ complex, wherein the reduced recruitment is compared to the recruitment of the IL-2Rβ subunit and/or the IL-2Rγ subunit by a wild-type IL-2 polypeptide.

[00580] In aspects disclosed herein, there is provided an interleukin 2 receptor αβγ (IL-2Rαβγ) binding protein, wherein the receptor signaling efficiency for the interleukin 2 receptor βγ (IL-2Rβγ) of said binding protein is less than that of wild-type human IL-2 (hIL-2), and wherein said binding protein comprises at least one unnatural amino acid.

[00581] In aspects disclosed herein, an interleukin 2 receptor αβγ (IL-2Rαβγ) binding protein is provided, wherein recruitment of an IL-2Rγ subunit to an IL-2/IL-2Rβ complex by said binding protein is less than that of wild-type human IL-2 (hIL-2), and wherein said binding protein comprises at least one unnatural amino acid. In some embodiments, said binding protein is a modified IL-2 polypeptide or a functionally active fragment thereof, wherein the modified IL-2 polypeptide comprises at least one unnatural amino acid.

[00582] In aspects disclosed herein, IL-2/IL-2Rαβγ complexes are provided comprising a modified IL-2 polypeptide comprising a non-natural amino acid and IL-2Rαβγ, wherein the modified IL-2 polypeptide is characterized by reduced efficiency of receptor signaling to IL-2Rβγ, and wherein the reduced efficiency of receptor signaling is compared to the binding affinity between the wild-type IL-2 polypeptide and IL-2Rβγ. In some embodiments, said activator provides for expansion of CD8+ effector T cell and/or natural killer cells by less than 20%, 15%, 10%, 5%, 1%, or 0.1% in a population of CD3+ cells upon contact of said activator with said population of CD3+ cells, compared to expansion of CD8+ effector T cell and/or natural killer cells in a population of CD3+ cells upon contact with a wild-type IL-2 polypeptide. In some embodiments, said activator does not provide for expansion of CD8+ effector T cell and/or natural killer cells. In some embodiments, said cell population is an in vivo cell population. In some embodiments, said cell population is an in vitro cell population. In some embodiments, said cell population is an ex vivo cell population.

[00583] In aspects disclosed herein, IL-2/IL-2Rαβγ complexes are provided comprising a modified IL-2 polypeptide comprising a non-natural amino acid and IL-2Rαβγ, wherein recruitment of an IL-2Rγ subunit to the IL-2/IL-2Rβ complex by said modified IL-2 polypeptide is less than that of a wild-type IL-2 polypeptide. In some embodiments, the modified IL-2 polypeptide further comprises a conjugation moiety covalently attached to the non-natural amino acid. In some embodiments, said activator provides for expansion of CD8+ effector T cell and/or natural killer cells by less than 20%, 15%, 10%, 5%, 1%, or 0.1% in a population of CD3+ cells upon contact of said activator with said population of CD3+ cells, compared to expansion of CD8+ effector T cell and/or natural killer cells in a population of CD3+ cells upon contact with a wild-type IL-2 polypeptide. In some embodiments, said activator does not provide for expansion of CD8+ effector T cell and/or natural killer cells. In some embodiments, said cell population is an in vivo cell population. In some embodiments, said cell population is an in vitro cell population. In some embodiments, said cell population is an ex vivo cell population.

[00584] In aspects disclosed herein, a CD4+ Treg cell activator is provided that selectively provides for the expansion of CD4+ Treg cells in a cell population, wherein said activator comprises a modified IL-2 polypeptide comprising at least one unnatural amino acid. In some embodiments, said activator provides for the expansion of a CD8+ effector T cell and/or natural killer cells by less than 20%, 15%, 10%, 5%, 1%, or 0.1% in a CD3+ cell population when said activator is contacted with said CD3+ cell population, as compared to the expansion of a CD8+ effector T cell and/or natural killer cells in a CD3+ cell population when contacted with a wild-type IL-2 polypeptide. In some embodiments, said activator does not provide for the expansion of a CD8+ effector T cell and/or natural killer cells. In some embodiments, said cell population is an in vivo cell population. In some embodiments, said cell population is an in vitro cell population. In some embodiments, said cell population is an ex vivo cell population.

[00585] In aspects disclosed herein, a pharmaceutical composition is provided comprising an isolated and modified IL-2 polypeptide described herein, an IL-2Rαβγ binding protein described herein, or a CD4 ⁺ Treg described herein, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is formulated for systemic delivery. In some embodiments, the pharmaceutical composition is formulated for parenteral administration. In some embodiments, the autoimmune disease or disorder comprises alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes, juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, multiple sclerosis, pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis, vitiligo, or Wegener's granulomatosis. In some embodiments, the methods further comprise administering an additional therapeutic agent. In some embodiments, the IL-2 conjugate and the additional therapeutic agent are administered simultaneously. In some embodiments, the IL-2 conjugate and the additional therapeutic agent are administered sequentially. In some embodiments, the IL-2 conjugate is administered before the additional therapeutic agent. In some embodiments, the IL-2 conjugate is administered after the additional therapeutic agent is administered. In some embodiments, the subject is a human.

[00586] In aspects disclosed herein, methods are provided for treating an autoimmune disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an isolated and modified IL-2 polypeptide described herein, an IL-2Rαβγ binding protein described herein, a CD4 ⁺ Treg cell described herein, or a pharmaceutical composition described herein. In some embodiments, the autoimmune disease or disorder comprises alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes, juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, multiple sclerosis, pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis, vitiligo, or Wegener's granulomatosis. In some embodiments, the methods further comprise administering an additional therapeutic agent. In some embodiments, the IL-2 conjugate and the additional therapeutic agent are administered simultaneously. In some embodiments, the IL-2 conjugate and the additional therapeutic agent are administered sequentially. In some embodiments, the IL-2 conjugate is administered before the additional therapeutic agent. In some embodiments, the IL-2 conjugate is administered after the additional therapeutic agent is administered. In some embodiments, the subject is a human.

[00587] In aspects disclosed herein, a method for expanding a population of CD4+ regulatory T cells (Treg) is provided, comprising contacting the cell with an isolated and modified IL-2 polypeptide described herein, an IL-2Rαβγ binding protein described herein, a CD4 ⁺ Treg cell described herein, or a pharmaceutical composition described herein for a time sufficient to induce formation of a complex with IL-2Rαβγ, thereby stimulating expansion of the Treg cell population. In some embodiments, the method is an in vivo method. In some embodiments, the method is an in vitro method. In some embodiments, the method is an ex vivo method.

[00588] In aspects disclosed herein, kits are provided comprising an isolated and modified IL-2 polypeptide described herein, an IL-2Rαβγ binding protein described herein, a CD4 ⁺ Treg cell described herein, or a pharmaceutical composition described herein. In some embodiments, the isolated and modified IL-2 polypeptide comprises a mutation at residue P65 corresponding to residue position 65 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the isolated and modified IL-2 polypeptide comprises a mutation at residue E61, corresponding to residue position 61 of SEQ ID NO: 1, and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the isolated and modified IL-2 polypeptide comprises a mutation at residue F42, corresponding to residue position 42 of SEQ ID NO: 1, and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the molecular weight of the PEG is effective to improve the solubility of the isolated and modified IL-2 polypeptide and/or IL-2 conjugate. In some embodiments, the molecular weight of the PEG is effective to enhance the stability of the isolated and modified IL-2 polypeptide and/or IL-2 conjugate. In some embodiments, the molecular weight of the PEG is effective to improve the manufacturing efficiency of an adoptive cell therapy agent comprising an isolated and modified IL-2 polypeptide and/or IL-2 conjugate.

[00589] In aspects disclosed herein, reagents for making an adoptive cell therapy agent comprising an isolated and modified IL-2 polypeptide described herein, an IL-2Rαβγ binding protein described herein, a CD4 ⁺ Treg cell described herein, or a pharmaceutical composition described herein are provided. In some embodiments, the isolated and modified IL-2 polypeptide comprises a mutation at residue P65 corresponding to residue position 65 of SEQ ID NO: 1 and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the isolated and modified IL-2 polypeptide comprises a mutation at residue E61, corresponding to residue position 61 of SEQ ID NO: 1, and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the isolated and modified IL-2 polypeptide comprises a mutation at residue F42, corresponding to residue position 42 of SEQ ID NO: 1, and a conjugation moiety comprising PEG, wherein the PEG has a molecular weight of about 5,000 daltons (Da), 10,000 Da, 15,000 Da, 20,000 Da, 25,000 Da, 30,000 Da, 35,000 Da, 40,000 Da, 45,000 Da, or 50,000 Da. In some embodiments, the molecular weight of the PEG is effective to improve the solubility of the isolated and modified IL-2 polypeptide and/or IL-2 conjugate. In some embodiments, the molecular weight of the PEG is effective to enhance the stability of the isolated and modified IL-2 polypeptide and/or IL-2 conjugate. In some embodiments, the molecular weight of the PEG is effective to improve the manufacturing efficiency of an adoptive cell therapy agent comprising an isolated and modified IL-2 polypeptide and/or IL-2 conjugate.

Defined Terminology

[00590] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It should be understood that the detailed description is merely illustrative and explanatory and is not limiting of any claimed subject matter. In this application, the use of the singular includes the plural unless specifically stated otherwise. It should be noted that the singular forms used in this specification include plural references unless the context clearly dictates otherwise. In this application, the use of "or" means "and/or" unless otherwise stated. In addition, the use of the term "including", as well as other forms such as "include", "includes" and "included" is not limiting.

[00591] Although various features of the present invention may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the present invention may be described herein in the context of separate embodiments for clarity, the present invention may also be implemented in a single embodiment.

[00592] Reference in this specification to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present invention.

[00593] As used herein, ranges and amounts may be expressed as "about" a particular value or range. "About" also includes an exact amount. Thus, "about 5 μL" means "about 5 μL" as well as "5 μL." Generally, the term "about" includes an amount that is expected to be within experimental error, such as within 15%, 10%, or 5%.

[00594] The section headings used in this document are for organizational purposes only and are not to be construed as limiting the subject matter of the invention described.

[00595] As used herein, the terms "individual(s)", "subject(s)", and "patient(s)" mean any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is non-human. None of the terms require or are limited to situations characterized by the supervision (e.g., continuous or intermittent) of a healthcare professional (e.g., a physician, registered nurse, nurse practitioner, physician assistant, orderly, or hospice worker).

[00596] As used herein, the term "significant" or "substantially" when applied to binding affinity means a change in the binding affinity of a cytokine (e.g., an IL-2 polypeptide) that is sufficient to affect the binding of the cytokine (e.g., an IL-2 polypeptide) to a target receptor. In some cases, the term refers to a change of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some cases, the term refers to a change of at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or more.

[00597] In some cases, the term "significant" or "to a significant extent" when applied to the activation of one or more cell populations by a cytokine signaling complex means a change sufficient to activate the cell population. In some cases, the change to activate the cell population is measured as the efficiency of receptor signaling. In such cases, an EC50 value may be provided. In other cases, an ED50 value may be provided. In additional cases, a concentration or dosage of the cytokine may be provided.

[00598] As used herein, the term "potency" refers to the amount of a cytokine (e.g., an IL-2 polypeptide) required to achieve a target effect. In some cases, the term "potency" refers to the amount of a cytokine (e.g., an IL-2 polypeptide) required to activate a target cytokine receptor (e.g., an IL-2 receptor). In other cases, the term "efficacy" refers to the amount of a cytokine (e.g., an IL-2 polypeptide) required to activate a target cell population. In some cases, potency is measured as the ED50 (effective dose 50), or the dose required to achieve 50% of the maximal effect. In other cases, potency is measured as the EC50 (effective concentration 50), or the dose required to achieve the target effect in 50% of the population.

Numbered embodiments

[00599] The present invention provides the following non-limiting numbered embodiments.

Embodiment 1. An IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (I),

formula (I),

Where

Z is CH ₂ and Y is ;

Y is CH ₂ , and Z is ;

Z is CH ₂ and Y is ; or

Y is CH ₂ , and Z is ;

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has the structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 2. An IL-2 conjugate comprising the amino acid sequence of SEQ ID NO: 4, wherein at least one amino acid residue in the IL-2 conjugate is replaced by a structure of formula (I),

formula (I),

Where

Z is CH ₂ and Y is ;

Y is CH ₂ , and Z is ;

Z is CH ₂ and Y is ; or

Y is CH ₂ , and Z is ;

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa;

X has the structure

; And

wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate refers to the positions in SEQ ID NO: 1; or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 3. The IL-2 conjugate of embodiment 1 or 2, wherein Z is CH ₂ and Y is , or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 4. The IL-2 conjugate of embodiment 1 or 2, wherein Y is CH ₂ and Z is , or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 5. The IL-2 conjugate of embodiment 1 or 2, wherein Z is CH ₂ and Y is , or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 6. The IL-2 conjugate of embodiment 1 or 2, wherein Z is CH ₂ and Y is , or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 7. The IL-2 conjugate of embodiment 1 or 2, wherein Y is CH ₂ and Z is , or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 8. The IL-2 conjugate of embodiment 1 or 2, wherein the PEG group has an average molecular weight selected from 5 kDa, 10 kDa, 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 9. The IL-2 conjugate of embodiment 8, wherein the PEG group has an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 10. The IL-2 conjugate of embodiment 8, wherein the PEG group has an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 11. The IL-2 conjugate of embodiment 1 or 2, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is selected from K35, F42, F44, K43, E62, P65, R38, T41, E68, Y45, V69 and L72, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate refers to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 12. An IL-2 conjugate according to embodiment 11, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate is selected from F42, E62 and P65, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-2 conjugate refers to the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 13. An IL-2 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 15-19, wherein [AzK_PEG] has a structure of formula (II), or formula (III), or a mixture of formula (II) and formula (III),

formula (II),

formula (III),

Where

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has the structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 14. The IL-2 conjugate of embodiment 13, wherein [AzK_PEG] is a mixture of formula (II) and formula (III), or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 15. The IL-2 conjugate of embodiment 13, wherein [AzK_PEG] has the structure of formula (II),

formula (II),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 16. The IL-2 conjugate of embodiment 15, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 15, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 17. The IL-2 conjugate of embodiment 16, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 18. The IL-2 conjugate of embodiment 17, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 19. The IL-2 conjugate of embodiment 17, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 20. The IL-2 conjugate of embodiment 17, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 21. The IL-2 conjugate of embodiment 15, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 16, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 22. The IL-2 conjugate of embodiment 21, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 23. The IL-2 conjugate of embodiment 22, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 24. The IL-2 conjugate of embodiment 23, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 25. The IL-2 conjugate of embodiment 23, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 26. The IL-2 conjugate of embodiment 15, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 17, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 27. The IL-2 conjugate of embodiment 26, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 28. The IL-2 conjugate of embodiment 27, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 29. The IL-2 conjugate of embodiment 27, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 30. The IL-2 conjugate of embodiment 27, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 31. The IL-2 conjugate of embodiment 15, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 18, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 32. The IL-2 conjugate of embodiment 31, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 33. The IL-2 conjugate of embodiment 32, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 34. The IL-2 conjugate of embodiment 33, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 35. The IL-2 conjugate of embodiment 33, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 36. The IL-2 conjugate of embodiment 15, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 19, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 37. The IL-2 conjugate of embodiment 36, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 38. The IL-2 conjugate of embodiment 37, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 39. The IL-2 conjugate of embodiment 38, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 40. The IL-2 conjugate of embodiment 38, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 41. The IL-2 conjugate of embodiment 13, wherein [AzK_PEG] has the structure of formula (III),

formula (III),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 42. The IL-2 conjugate of embodiment 41, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 15, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 43. The IL-2 conjugate of embodiment 42, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 44. The IL-2 conjugate of embodiment 43, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 45. The IL-2 conjugate of embodiment 44, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 46. The IL-2 conjugate of embodiment 44, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 47. The IL-2 conjugate of embodiment 41, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 16, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 48. The IL-2 conjugate of embodiment 47, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 49. The IL-2 conjugate of embodiment 48, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 50. The IL-2 conjugate of embodiment 49, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 51. The IL-2 conjugate of embodiment 49, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 52. The IL-2 conjugate of embodiment 41, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 17, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 53. The IL-2 conjugate of embodiment 52, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 54. The IL-2 conjugate of embodiment 53, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 55. The IL-2 conjugate of embodiment 54, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 56. The IL-2 conjugate of embodiment 54, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 57. The IL-2 conjugate of embodiment 41, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 18, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 58. The IL-2 conjugate of embodiment 57, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 59. The IL-2 conjugate of embodiment 58, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 60. The IL-2 conjugate of embodiment 59, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 61. The IL-2 conjugate of embodiment 59, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 62. The IL-2 conjugate of embodiment 41, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 19, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 63. The IL-2 conjugate of embodiment 62, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 64. The IL-2 conjugate of embodiment 63, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 65. The IL-2 conjugate of embodiment 64, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 66. The IL-2 conjugate of embodiment 64, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 67. The IL-2 conjugate of any one of embodiments 1-66, wherein W is a linear or branched PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 68. The IL-2 conjugate of any one of embodiments 1-66, wherein W is a linear PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 69. The IL-2 conjugate of any one of embodiments 1-66, wherein W is a branched PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 70. The IL-2 conjugate of any one of embodiments 1-66, wherein W is a methoxy-PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 71. The IL-2 conjugate of embodiment 70, wherein the methoxy-PEG group is linear or branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 72. The IL-2 conjugate of embodiment 71, wherein the methoxy-PEG group is linear, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 73. The IL-2 conjugate of embodiment 71, wherein the methoxy-PEG group is branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 74. An IL-2 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 20-24, wherein [AzK_PEG5kD] has a structure of formula (II), or formula (III), or a mixture of formula (II) and formula (III),

formula (II),

formula (III),

Where

W is a PEG group having an average molecular weight of 5 kDa; and

X has the structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 75. The IL-2 conjugate of embodiment 74, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 20, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 76. The IL-2 conjugate of embodiment 74, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 21, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 77. The IL-2 conjugate of embodiment 74, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 22, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 78. The IL-2 conjugate of embodiment 74, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 23, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 79. The IL-2 conjugate of embodiment 74, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 24, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 80. The IL-2 conjugate of embodiment 74, wherein [AzK_PEG5kD] has the structure of formula (II),

formula (II),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 81. The IL-2 conjugate of embodiment 80, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 20, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 82. The IL-2 conjugate of embodiment 80, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 21, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 83. The IL-2 conjugate of embodiment 80, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 22, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 84. The IL-2 conjugate of embodiment 80, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 23, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 85. The IL-2 conjugate of embodiment 80, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 24, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 86. The IL-2 conjugate of embodiment 74, wherein [AzK_PEG5kD] has the structure of formula (III),

formula (III),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 87. The IL-2 conjugate of embodiment 86, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 20, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 88. The IL-2 conjugate of embodiment 86, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 21, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 89. The IL-2 conjugate of embodiment 86, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 22, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 90. The IL-2 conjugate of embodiment 86, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 23, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 91. The IL-2 conjugate of embodiment 86, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 24, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 92. An IL-2 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 25-29, wherein [AzK_PEG30kD] has a structure of formula (II) or formula (III) or is a mixture of structures of formula (II) and formula (III),

formula (II),

formula (III),

Where

W is a PEG group having an average molecular weight of 30 kDa; and

X has a structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 93. The IL-2 conjugate of embodiment 92, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 25, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 94. The IL-2 conjugate of embodiment 92, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 26, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 95. The IL-2 conjugate of embodiment 92, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 27, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 96. The IL-2 conjugate of embodiment 92, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 28, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 97. The IL-2 conjugate of embodiment 92, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 29, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 98. The IL-2 conjugate of embodiment 92, wherein [AzK_PEG30kD] has the structure of formula (II),

formula (II),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 99. The IL-2 conjugate of embodiment 98, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 25, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 100. The IL-2 conjugate of embodiment 98, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 26, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 101. The IL-2 conjugate of embodiment 98, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 27, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 102. The IL-2 conjugate of embodiment 98, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 28, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 103. The IL-2 conjugate of embodiment 98, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 29, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 104. The IL-2 conjugate of embodiment 92, wherein [AzK_PEG30kD] has the structure of formula (III),

formula (III),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 105. The IL-2 conjugate of embodiment 104, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 25, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 106. The IL-2 conjugate of embodiment 104, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 26, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 107. The IL-2 conjugate of embodiment 104, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 27, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 108. The IL-2 conjugate of embodiment 104, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 28, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 109. The IL-2 conjugate of embodiment 104, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 29, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 110. An IL-2 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 15-19, wherein [AzK_PEG] is a mixture of structures of formula (II) and formula (III),

formula (II),

formula (III),

Where

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has a structure

; or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 111. The IL-2 conjugate of embodiment 110, wherein the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) contained in the total amount of [AzK_PEG] in the IL-2 conjugate is approximately 1:1.

Embodiment 112. The IL-2 conjugate of embodiment 110, wherein the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) contained in the total amount of [AzK_PEG] in the IL-2 conjugate is greater than 1:1.

Embodiment 113. The IL-2 conjugate of embodiment 110, wherein the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) contained in the total amount of [AzK_PEG] in the IL-2 conjugate is less than 1:1.

Embodiment 114. The IL-2 conjugate of any one of embodiments 110-113, wherein W is a linear or branched PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 115. The IL-2 conjugate of any one of embodiments 110-113, wherein W is a linear PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 116. The IL-2 conjugate of any one of embodiments 110-113, wherein W is a branched PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 117. The IL-2 conjugate of any one of embodiments 110-113, wherein W is a methoxy-PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 118. The IL-2 conjugate of embodiment 117, wherein the methoxy-PEG group is linear or branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 119. The IL-2 conjugate of embodiment 118, wherein the methoxy-PEG group is linear, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 120. The IL-2 conjugate of embodiment 118, wherein the methoxy-PEG group is branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 121. An IL-2 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 20-24, wherein [AzK_PEG5kD] is a mixture of structures of formula (II) and formula (III),

formula (II),

formula (III),

Where

W is a PEG group having an average molecular weight of 5 kDa; and

X has a structure

; or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 122. The IL-2 conjugate of embodiment 121, wherein the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) contained in the total amount of [AzK_PEG5kD] in the IL-2 conjugate is approximately 1:1.

Embodiment 123. The IL-2 conjugate of embodiment 121, wherein the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) contained in the total amount of [AzK_PEG5kD] in the IL-2 conjugate is greater than 1:1.

Embodiment 124. The IL-2 conjugate of embodiment 121, wherein the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) contained in the total amount of [AzK_PEG5kD] in the IL-2 conjugate is less than 1:1.

Embodiment 125. An IL-2 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 25-29, wherein [AzK_PEG30kD] is a mixture of structures of formula (II) and formula (III),

formula (II),

formula (III),

Where

W is a PEG group having an average molecular weight of 30 kDa; and

X has a structure

; or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 126. The IL-2 conjugate of embodiment 125, wherein the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) contained in the total amount of [AzK_PEG30kD] in the IL-2 conjugate is approximately 1:1.

Embodiment 127. The IL-2 conjugate of embodiment 125, wherein the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) contained in the total amount of [AzK_PEG30kD] in the IL-2 conjugate is greater than 1:1.

Embodiment 128. The IL-2 conjugate of embodiment 125, wherein the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) contained in the total amount of [AzK_PEG30kD] in the IL-2 conjugate is less than 1:1.

Embodiment 129. An IL-2 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 40-44, wherein [AzK_L1_PEG] has a structure of formula (IV), or formula (V), or a mixture of formula (IV) and formula (V),

formula (IV),

formula (V),

Where

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has a structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 130. The IL-2 conjugate of embodiment 129, wherein [AzK_L1_PEG] is a mixture of formula (IV) and formula (V), or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 131. The IL-2 conjugate of embodiment 129, wherein [AzK_L1_PEG] has the structure of formula (IV),

formula (IV),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 132. The IL-2 conjugate of embodiment 131, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 40, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 133. The IL-2 conjugate of embodiment 132, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 134. The IL-2 conjugate of embodiment 133, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 135. The IL-2 conjugate of embodiment 133, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 136. The IL-2 conjugate of embodiment 133, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 137. The IL-2 conjugate of embodiment 131, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 41, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 138. The IL-2 conjugate of embodiment 137, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 139. The IL-2 conjugate of embodiment 138, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 140. The IL-2 conjugate of embodiment 139, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 141. The IL-2 conjugate of embodiment 139, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 142. The IL-2 conjugate of embodiment 129, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 42, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 143. The IL-2 conjugate of embodiment 142, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 144. The IL-2 conjugate of embodiment 143, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 145. The IL-2 conjugate of embodiment 144, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 146. The IL-2 conjugate of embodiment 144, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 147. The IL-2 conjugate of embodiment 129, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 43, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 148. The IL-2 conjugate of embodiment 147, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 149. The IL-2 conjugate of embodiment 148, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 150. The IL-2 conjugate of embodiment 149, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 151. The IL-2 conjugate of embodiment 149, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 152. The IL-2 conjugate of embodiment 129, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 44, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 153. The IL-2 conjugate of embodiment 152, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 154. The IL-2 conjugate of embodiment 153, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 155. The IL-2 conjugate of embodiment 154, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 156. The IL-2 conjugate of embodiment 155, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 157. The IL-2 conjugate of embodiment 129, wherein [AzK_L1_PEG] has the structure of formula (V),

formula (V),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 158. The IL-2 conjugate of embodiment 157, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 40, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 159. The IL-2 conjugate of embodiment 158, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 160. The IL-2 conjugate of embodiment 159, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 161. The IL-2 conjugate of embodiment 160, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 162. The IL-2 conjugate of embodiment 160, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 163. The IL-2 conjugate of embodiment 157, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 41, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 164. The IL-2 conjugate of embodiment 163, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 165. The IL-2 conjugate of embodiment 164, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 166. The IL-2 conjugate of embodiment 165, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 167. The IL-2 conjugate of embodiment 165, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 168. The IL-2 conjugate of embodiment 157, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 42, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 169. The IL-2 conjugate of embodiment 168, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 170. The IL-2 conjugate of embodiment 169, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 171. The IL-2 conjugate of embodiment 170, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 172. The IL-2 conjugate of embodiment 170, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 173. The IL-2 conjugate of embodiment 157, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 43, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 174. The IL-2 conjugate of embodiment 173, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 175. The IL-2 conjugate of embodiment 174, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 176. The IL-2 conjugate of embodiment 175, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 177. The IL-2 conjugate of embodiment 175, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 178. The IL-2 conjugate of embodiment 157, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 44, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 179. The IL-2 conjugate of embodiment 178, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 180. The IL-2 conjugate of embodiment 179, wherein W is a PEG group having an average molecular weight selected from 5 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 181. The IL-2 conjugate of embodiment 180, wherein W is a PEG group having an average molecular weight of 5 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 182. The IL-2 conjugate of embodiment 180, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 183. The IL-2 conjugate of any one of embodiments 129-182, wherein W is a linear or branched PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 184. The IL-2 conjugate of any one of embodiments 129-182, wherein W is a linear PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 185. The IL-2 conjugate of any one of embodiments 129-182, wherein W is a branched PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 186. The IL-2 conjugate of any one of embodiments 129-182, wherein W is a methoxy-PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 187. The IL-2 conjugate of embodiment 186, wherein the methoxy-PEG group is linear or branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 188. The IL-2 conjugate of embodiment 187, wherein the methoxy-PEG group is linear, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 189. The IL-2 conjugate of embodiment 187, wherein the methoxy-PEG group is branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 190. An IL-2 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 45-49, wherein [AzK_L1_PEG5kD] has a structure of formula (IV), or formula (V), or a mixture of formula (IV) and formula (V),

formula (IV),

formula (V),

Where

W is a PEG group having an average molecular weight of 5 kDa; and

X has a structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 191. The IL-2 conjugate of embodiment 190, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 45, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 192. The IL-2 conjugate of embodiment 190, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 46, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 193. The IL-2 conjugate of embodiment 190, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 47, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 194. The IL-2 conjugate of embodiment 190, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 48, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 195. The IL-2 conjugate of embodiment 190, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 49, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 196. The IL-2 conjugate of embodiment 190, wherein [AzK_L1_PEG5kD] has a structure of formula (IV),

formula (IV),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 197. The IL-2 conjugate of embodiment 196, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 45, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 198. The IL-2 conjugate of embodiment 196, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 46, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 199. The IL-2 conjugate of embodiment 196, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 47, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 200. The IL-2 conjugate of embodiment 196, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 48, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 201. The IL-2 conjugate of embodiment 196, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 49, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 202. The IL-2 conjugate of embodiment 190, wherein [AzK_L1_PEG5kD] has a structure of formula (V),

formula (V),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 203. The IL-2 conjugate of embodiment 202, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 45, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 204. The IL-2 conjugate of embodiment 202, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 46, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 205. The IL-2 conjugate of embodiment 202, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 47, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 206. The IL-2 conjugate of embodiment 202, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 48, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 207. The IL-2 conjugate of embodiment 202, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 49, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 208. An IL-2 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 50-54, wherein [AzK_L1_PEG30kD] has a structure of formula (IV) or formula (V) or is a mixture of structures of formula (IV) and formula (V),

formula (IV),

formula (V),

Where

W is a PEG group having an average molecular weight of 30 kDa; and

X has the structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 209. The IL-2 conjugate of embodiment 208, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 50, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 210. The IL-2 conjugate of embodiment 208, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 51, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 211. The IL-2 conjugate of embodiment 208, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 52, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 212. The IL-2 conjugate of embodiment 208, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 53, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 213. The IL-2 conjugate of embodiment 208, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 54, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 214. The IL-2 conjugate of embodiment 208, wherein [AzK_L1_PEG30kD] has the structure of formula (IV),

formula (IV),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 215. The IL-2 conjugate of embodiment 214, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 50, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 216. The IL-2 conjugate of embodiment 214, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 51, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 217. The IL-2 conjugate of embodiment 214, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 52, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 218. The IL-2 conjugate of embodiment 214, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 53, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 219. The IL-2 conjugate of embodiment 214, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 54, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 220. The IL-2 conjugate of embodiment 214, wherein [AzK_L1_PEG30kD] has a structure of formula (V),

formula (V),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 221. The IL-2 conjugate of embodiment 220, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 50, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 222. The IL-2 conjugate of embodiment 220, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 51, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 223. The IL-2 conjugate of embodiment 220, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 52, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 224. The IL-2 conjugate of embodiment 220, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 53, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 225. The IL-2 conjugate of embodiment 220, wherein the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 54, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 226. An IL-2 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 40-44, wherein [Azk_L1_PEG] is a mixture of structures of formula (IV) and formula (V),

formula (IV),

formula (V),

Where

W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa and 60 kDa; and

X has a structure

; or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 227. The IL-2 conjugate of embodiment 226, wherein the ratio of the amount of structure of formula (IV) to the amount of structure of formula (V) contained in the total amount of [AzK_L1_PEG] in the IL-2 conjugate is approximately 1:1.

Embodiment 228. The IL-2 conjugate of embodiment 226, wherein the ratio of the amount of structure of formula (IV) to the amount of structure of formula (V) contained in the total amount of [AzK_L1_PEG] in the IL-2 conjugate is greater than 1:1.

Embodiment 229. The IL-2 conjugate of embodiment 226, wherein the ratio of the amount of structure of formula (IV) to the amount of structure of formula (V) contained in the total amount of [AzK_L1_PEG] in the IL-2 conjugate is less than 1:1.

Embodiment 230. The IL-2 conjugate of any one of embodiments 226-229, wherein W is a linear or branched PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 231. The IL-2 conjugate of any one of embodiments 226-229, wherein W is a linear PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 232. The IL-2 conjugate of any one of embodiments 226-229, wherein W is a branched PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 233. The IL-2 conjugate of any one of embodiments 226-229, wherein W is a methoxy-PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 234. The IL-2 conjugate of embodiment 233, wherein the methoxy-PEG group is linear or branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 235. The IL-2 conjugate of embodiment 234, wherein the methoxy-PEG group is linear, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 236. The IL-2 conjugate of embodiment 234, wherein the methoxy-PEG group is branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

Embodiment 237. An IL-2 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 45-49, wherein [AzK_L1_PEG5kD] is a mixture of structures of formula (IV) and formula (V),

formula (IV),

formula (V),

Where

W is a PEG group having an average molecular weight of 5 kDa; and

X has a structure

; or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 238. The IL-2 conjugate of embodiment 237, wherein the ratio of the amount of structure of formula (IV) to the amount of structure of formula (V) contained in the total amount of [AzK_L1_PEG5kD] in the IL-2 conjugate is approximately 1:1.

Embodiment 239. The IL-2 conjugate of embodiment 237, wherein the ratio of the amount of structure of formula (IV) to the amount of structure of formula (V) contained in the total amount of [AzK_L1_PEG5kD] in the IL-2 conjugate is greater than 1:1.

Embodiment 240. The IL-2 conjugate of embodiment 237, wherein the ratio of the amount of structure of formula (IV) to the amount of structure of formula (V) contained in the total amount of [AzK_L1_PEG5kD] in the IL-2 conjugate is less than 1:1.

Embodiment 241. An IL-2 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 50-54, wherein [AzK_L1 PEG30kD] is a mixture of structures of formula (IV) and formula (V),

formula (IV),

formula (V),

Where

W is a PEG group having an average molecular weight of 30 kDa; and

X has a structure

; or a pharmaceutically acceptable salt, solvate or hydrate thereof.

Embodiment 242. The IL-2 conjugate of embodiment 241, wherein the ratio of the amount of structure of formula (IV) to the amount of structure of formula (V) contained in the total amount of [AzK_L1_PEG30kD] in the IL-2 conjugate is approximately 1:1.

Embodiment 243. The IL-2 conjugate of embodiment 241, wherein the ratio of the amount of structure of formula (IV) to the amount of structure of formula (V) contained in the total amount of [AzK_L1_PEG30kD] in the IL-2 conjugate is greater than 1:1.

Embodiment 244. The IL-2 conjugate of embodiment 241, wherein the ratio of the amount of structure of formula (IV) to the amount of structure of formula (V) contained in the total amount of [AzK_L1_PEG30kD] in the IL-2 conjugate is less than 1:1.

Embodiment 245. A method of treating cancer in a subject comprising administering to the subject in need thereof an effective amount of an IL-2 conjugate according to any one of embodiments 1-244.

Embodiment 246. The method of treating a cancer in a subject according to embodiment 245, wherein the cancer in the subject is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck squamous cell cancer (HNSCC), classical Hodgkin's lymphoma (cHL), primary mediastinal large B cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability cancer, microsatellite stable cancer, gastric cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), melanoma, small cell lung cancer (SCLC), esophageal, glioblastoma, mesothelioma, breast cancer, triple negative breast cancer, prostate cancer, bladder cancer, ovarian cancer, tumors with intermediate or low mutational burden, squamous cell carcinoma of the skin (CSCC), cutaneous squamous cell carcinoma (SCSC), tumors with low or no PD-L1 expression, tumors that spread systemically to the liver and CNS beyond their primary anatomical site of origin, and diffuse large B-cell lymphoma.

Embodiment 247. The method of treating cancer in a subject according to embodiment 246, wherein the cancer in the subject is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), urothelial carcinoma, and melanoma.

Embodiment 248. A method of treating cancer in a subject according to any one of embodiments 245-247, wherein the IL-2 conjugate is administered to the subject in need thereof once every two weeks, once every three weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, or once every 8 weeks.

Embodiment 249. The method of treating cancer in a subject according to embodiment 248, wherein the IL-2 conjugate is administered to the subject in need thereof once weekly or once every two weeks.

Embodiment 250. A method of treating cancer in a subject according to embodiment 249, wherein the IL-2 conjugate is administered to the subject in need thereof once per week.

Embodiment 251. A method of treating cancer in a subject according to embodiment 249, wherein the IL-2 conjugate is administered to the subject in need thereof once every two weeks.

Embodiment 252. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not result in vascular leak syndrome in the subject.

Embodiment 253. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not result in the development of grade 2, grade 3, or grade 4 vascular leak syndrome in the subject.

Embodiment 254. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not result in the subject developing grade 2 vascular leak syndrome.

Embodiment 255. The method of treating cancer in a subject according to embodiment 253, wherein administering an effective amount of an IL-2 conjugate to the subject does not result in the development of grade 3 vascular leak syndrome in the subject.

Embodiment 256. The method of treating cancer in a subject according to embodiment 253, wherein administering an effective amount of the IL-2 conjugate to the subject does not result in the development of grade 4 vascular leak syndrome in the subject.

Embodiment 257. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not result in a loss of vascular tone in the subject.

Embodiment 258. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not result in extravasation of blood plasma proteins and fluid into the extravascular space of the subject.

Embodiment 259. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not result in the development of hypotension and decreased organ perfusion in the subject.

Embodiment 260. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not result in impairment of neutrophil function in the subject.

Embodiment 261. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not result in a decrease in chemotaxis in the subject.

Embodiment 262. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject is not associated with an increased risk of disseminated infection in the subject.

Embodiment 263. The method of embodiment 262, wherein the disseminated infection is sepsis or bacterial endocarditis.

Embodiment 264. The method of embodiment 263, wherein the disseminated infection is sepsis.

Embodiment 265. The method of embodiment 262, wherein the disseminated infection is bacterial endocarditis.

Embodiment 266. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein the subject is treated for any previous bacterial infections prior to administration of the IL-2 conjugate.

Embodiment 267. The method of embodiment 266, wherein the subject is treated with an antibacterial agent selected from oxacillin, nafcillin, ciprofloxacin, and vancomycin prior to administration of the IL-2 conjugate.

Embodiment 268. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not worsen the manifestations of a pre-existing or new-onset autoimmune disease or inflammatory disorder in the subject.

Embodiment 269. The method of embodiment 268, wherein administering an effective amount of the IL-2 conjugate to the subject does not worsen the manifestations of a pre-existing or new-onset autoimmune disease in the subject.

Embodiment 270. The method of embodiment 268, wherein administering an effective amount of the IL-2 conjugate to the subject does not worsen the manifestations of a pre-existing or new inflammatory disorder in the subject.

Embodiment 271. The method of embodiment 268, wherein the autoimmune disease or inflammatory disorder in the subject is selected from Crohn's disease, scleroderma, thyroiditis, inflammatory arthritis, diabetes mellitus, oculobulbar myasthenia gravis, crescentic IgA glomerulonephritis, cholecystitis, cerebral vasculitis, Stevens-Johnson syndrome, and bullous pemphigoid.

Embodiment 272. The method of embodiment 271, wherein the autoimmune disease or inflammatory disorder in the subject is Crohn's disease.

Embodiment 273. The method of embodiment 271, wherein the autoimmune disease or inflammatory disorder in the subject is scleroderma.

Embodiment 274. The method of embodiment 271, wherein the autoimmune disease or inflammatory disorder in the subject is thyroiditis.

Embodiment 275. The method of embodiment 271, wherein the autoimmune disease or inflammatory disorder in the subject is inflammatory arthritis.

Embodiment 276. The method of embodiment 271, wherein the autoimmune disease or inflammatory disorder in the subject is diabetes mellitus.

Embodiment 277. The method of embodiment 271, wherein the autoimmune disease or inflammatory disorder in the subject is oculobulbar myasthenia gravis.

Embodiment 278. The method of embodiment 271, wherein the autoimmune disease or inflammatory disorder in the subject is IgA crescentic glomerulonephritis.

Embodiment 279. The method of embodiment 271, wherein the autoimmune disease or inflammatory disorder in the subject is cholecystitis.

Embodiment 280. The method of embodiment 271, wherein the autoimmune disease or inflammatory disorder in the subject is cerebral vasculitis.

Embodiment 281. The method of embodiment 271, wherein the autoimmune disease or inflammatory disorder in the subject is Stevens-Johnson syndrome.

Embodiment 282. The method of embodiment 271, wherein the autoimmune disease or inflammatory disorder in the subject is bullous pemphigoid.

Embodiment 283. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not result in mental status changes, speech difficulties, cortical blindness, limb or gait ataxia, hallucinations, agitation, lethargy, or coma in the subject.

Embodiment 284. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not result in seizures in the subject.

Embodiment 285. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject is not contraindicated in subjects having a confirmed seizure disorder.

Embodiment 286. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not result in the development of capillary leak syndrome in the subject.

Embodiment 287. The method of embodiment 286, wherein administering an effective amount of the IL-2 conjugate to the subject does not result in grade 2, grade 3, or grade 4 capillary leak syndrome in the subject.

Embodiment 288. The method of embodiment 287, wherein administering an effective amount of the IL-2 conjugate to the subject does not result in the subject developing grade 2 capillary leak syndrome.

Embodiment 289. The method of embodiment 287, wherein administering an effective amount of the IL-2 conjugate to the subject does not result in the subject developing grade 3 capillary leak syndrome.

Embodiment 290. The method of embodiment 287, wherein administering an effective amount of the IL-2 conjugate to the subject does not result in the subject developing grade 4 capillary leak syndrome.

Embodiment 291. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not result in a decrease in mean arterial blood pressure in the subject after administering the IL-2 conjugate to the subject.

Embodiment 292. The method of embodiment 291, wherein administering an effective amount of the IL-2 conjugate to the subject does not result in the development of hypotension in the subject after administering the IL-2 conjugate to the subject.

Embodiment 293. The method of embodiment 292, wherein administering an effective amount of the IL-2 conjugate to the subject does not result in the subject having a decrease in systolic blood pressure below 90 mmHg or a decrease of 20 mmHg from the baseline systolic pressure after administering the IL-2 conjugate to the subject.

Embodiment 294. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not result in the development of edema in the subject after administering the IL-2 conjugate to the subject.

Embodiment 295. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not result in impairment of renal or hepatic function in the subject after administering the IL-2 conjugate to the subject.

Embodiment 296. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not result in eosinophilia in the subject after administering the IL-2 conjugate to the subject.

Embodiment 297. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not result in an increase in the number of eosinophils in the subject's peripheral blood to a value greater than 500 per μL after administering the IL-2 conjugate to the subject.

Embodiment 298. The method of embodiment 297, wherein administering an effective amount of an IL-2 conjugate to a subject does not result in an increase in the number of eosinophils in the subject's peripheral blood to a value greater than 500 per μL to 1500 per μL after administering the IL-2 conjugate to the subject.

Embodiment 299. The method of embodiment 297, wherein administering an effective amount of an IL-2 conjugate to a subject does not result in an increase in the peripheral blood eosinophil count in the subject to a value greater than 1500 per μL to 5000 per μL after administering the IL-2 conjugate to the subject.

Embodiment 300. The method of embodiment 297, wherein administering an effective amount of an IL-2 conjugate to a subject does not result in an increase in the peripheral blood eosinophil count in the subject to a value greater than 5,000 per μL after administering the IL-2 conjugate to the subject.

Embodiment 301. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject is not contraindicated in subjects who are already undergoing treatment with psychotropic drugs.

Embodiment 302. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject is not contraindicated in subjects who are already undergoing treatment with nephrotoxic, myelotoxic, cardiotoxic, or hepatotoxic drugs.

Embodiment 303. The method of embodiment 302, wherein administering an effective amount of the IL-2 conjugate to a subject is not contraindicated in subjects who are already undergoing treatment with aminoglycosides, cytotoxic chemotherapy, doxorubicin, methotrexate, or asparaginase.

Embodiment 304. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject is not contraindicated in subjects who are receiving treatment courses with combination therapies containing antineoplastic agents.

Embodiment 305. The method of embodiment 304, wherein the antineoplastic agent is selected from dacarbazine, cisplatin, tamoxifen, and interferon-alpha.

Embodiment 306. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not result in one or more grade 4 adverse events in the subject after administering the IL-2 conjugate to the subject.

Embodiment 307. The method of embodiment 306, wherein the one or more grade 4 adverse events are selected from hypothermia, shock, bradycardia, ventricular extrasystole, myocardial ischemia, syncope, hemorrhage, atrial arrhythmia, phlebitis, second degree atrioventricular block, endocarditis, pericardial effusion, peripheral gangrene, thrombosis, coronary artery disorder, stomatitis, nausea and vomiting, abnormal liver function tests, gastrointestinal hemorrhage, hematemesis, hemorrhagic diarrhea, gastrointestinal disorder, intestinal perforation, pancreatitis, anemia, leukopenia, leukocytosis, hypocalcemia, elevated alkaline phosphatase, elevated blood urea nitrogen (BUN), hyperuricemia, elevated non-protein nitrogen (NPN), respiratory acidosis, somnolence, agitation, neuropathy, paranoid reaction, seizure, grand mal seizure, delirium, asthma, pulmonary edema, hyperventilation, hypoxia, hemoptysis, hypoventilation, pneumothorax, mydriasis, pupillary dysfunction, renal dysfunction, renal failure, and acute tubular necrosis.

Embodiment 308. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to a group of subjects does not result in one or more grade 4 adverse events occurring in more than 1% of the subjects after administering the IL-2 conjugate to the subjects.

Embodiment 309. The method of embodiment 307, wherein the one or more grade 4 adverse events are selected from hypothermia, shock, bradycardia, ventricular extrasystole, myocardial ischemia, syncope, hemorrhage, atrial arrhythmia, phlebitis, second degree atrioventricular block, endocarditis, pericardial effusion, peripheral gangrene, thrombosis, coronary artery disorder, stomatitis, nausea and vomiting, abnormal liver function tests, gastrointestinal hemorrhage, hematemesis, hemorrhagic diarrhea, gastrointestinal disorder, intestinal perforation, pancreatitis, anemia, leukopenia, leukocytosis, hypocalcemia, elevated alkaline phosphatase, elevated blood urea nitrogen (BUN), hyperuricemia, elevated non-protein nitrogen (NPN), respiratory acidosis, somnolence, agitation, neuropathy, paranoid reaction, seizure, grand mal seizure, delirium, asthma, pulmonary edema, hyperventilation, hypoxia, hemoptysis, hypoventilation, pneumothorax, mydriasis, pupillary dysfunction, renal dysfunction, renal failure, and acute tubular necrosis.

Embodiment 310. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to a group of subjects does not result in one or more adverse events in more than 1% of the subjects after administering the IL-2 conjugate to the subjects, wherein the one or more adverse events are selected from duodenal ulcer, intestinal necrosis, myocarditis, supraventricular tachycardia, permanent or transient blindness secondary to optic neuritis, transient ischemic attack, meningitis, cerebral edema, pericarditis, allergic interstitial nephritis, and tracheoesophageal fistula.

Embodiment 311. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to a group of subjects does not result in one or more adverse events occurring in more than 1% of the subjects after administering the IL-2 conjugate to the subjects, wherein the one or more adverse events are selected from malignant hyperthermia, cardiac arrest, myocardial infarction, pulmonary embolism, stroke, intestinal perforation, liver or kidney failure, severe depression leading to suicide, pulmonary edema, respiratory arrest, respiratory failure.

Embodiment 312. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not result in the production of neutralizing antibodies to the IL-2 conjugate.

Embodiment 313. A method of treating cancer in a subject according to any one of embodiments 245-256, wherein administering the IL-2 conjugate to the subject provides an increase in the number of peripheral CD8+ T and NK cells in the subject without an increase in the number of peripheral CD4+ regulatory T cells in the subject.

Embodiment 314. A method of treating cancer in a subject according to any one of embodiments 245-256, wherein administering the IL-2 conjugate to the subject provides an increase in the number of peripheral CD8+ T and NK cells in the subject without increasing the number of peripheral eosinophils in the subject.

Embodiment 315. A method of treating cancer in a subject according to any one of embodiments 245-256, wherein administering the IL-2 conjugate to the subject increases the number of intratumoral CD8+ T and NK cells in the subject without increasing the number of intratumoral CD4+ regulatory T cells in the subject.

Embodiment 316. A pharmaceutical composition comprising an effective amount of an IL conjugate according to any one of embodiments 1-244 and one or more pharmaceutically acceptable excipients.

Embodiment 317. A method of treating cancer in a subject according to any one of embodiments 245-251, wherein administering an effective amount of an IL-2 conjugate to the subject does not require an intensive care unit or skilled cardiopulmonary or critical care physicians.

Embodiment 318. The method of embodiment 317, wherein administering an effective amount of the IL-2 conjugate to the subject does not require an intensive care unit.

Embodiment 319. The method of embodiment 317, wherein administering an effective amount of the IL-2 conjugate to the subject does not require skilled cardiopulmonary or critical care physicians.

EXAMPLES

[00600] These examples are provided for illustrative purposes only and do not limit the scope of the claims presented herein.

[00601] Each of the compounds disclosed in Examples 2-12 utilizes SEQ ID NO: 4 and an [AzK_PEG] moiety, wherein the position of the substituted amino acid in the IL-2 conjugate is within the positions in the sequence of SEQ ID NO: 4.

[00602] For example, the compound designated as "P65_5kD" in Tables 2A and 2B was prepared using methods similar to those described in Example 2, wherein a protein having the sequence of SEQ ID NO: 4 was first prepared in which the proline at position 65 was replaced with AzK. The AzK-containing protein was then reacted under click chemistry conditions with DBCO containing a methoxy, linear PEG group having an average molecular weight of 5 kDa to yield a product having the sequence of SEQ ID NO: 20, comprising a structure of formula (II), a structure of formula (III), or a mixture of structures of formulas (II) and (III), wherein W is a methoxy, linear PEG group having an average molecular weight of 5 kDa.

[00603] In another example, the compound designated "P65_30kD" in Tables 2A and 2B was prepared by first preparing a protein having the sequence of SEQ ID NO: 4, in which the proline at position 65 was replaced with AzK. The AzK-containing protein was then reacted under click chemistry conditions with DBCO containing a methoxy, linear PEG group having an average molecular weight of 30 kDa to obtain a product having the sequence of SEQ ID NO: 25, comprising a structure of formula (II), a structure of formula (III), or a mixture of structures of formulas (II) and (III), wherein W is a methoxy, linear PEG group having an average molecular weight of 30 kDa.

[00604] Similarly, a compound designated as "P65_30kD" was prepared by first preparing a protein having the sequence of SEQ ID NO: 4, in which the proline at position 65 was replaced with N 6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) (SEQ ID NO: 10). The compound can also be defined as a compound comprising the amino acid sequence of SEQ ID NO: 4, in which the proline at position 65 (P65) is replaced with a structure of formula (VI) or a structure of formula (VII) or a mixture of structures of formulas (VI) and (VII), and wherein n is an integer such that the PEG group has a molecular weight of approximately 30 kDa. The compound can also be defined as a compound comprising the amino acid sequence of SEQ ID NO: 4, in which proline at position 65 (P65) is replaced by a structure of formula (X) or a structure of formula (XI) or a mixture of structures of formulas (X) and (XI), and where n is an integer such that the PEG group has a molecular weight of approximately 30 kDa. Compound P65_30kD was used in Examples 5, 6, 7 and 8.

[00605] In another example, the compound designated as "E62_5kD" in Tables 2A and 2B was prepared by first preparing a protein having the sequence of SEQ ID NO: 4, in which the glutamic acid at position 62 was replaced with AzK. The AzK-containing protein was then reacted under click chemistry conditions with DBCO containing a methoxy, linear PEG group having an average molecular weight of 5 kDa to obtain a product having the sequence of SEQ ID NO: 21, comprising a structure of formula (II), a structure of formula (III), or a mixture of structures of formulas (II) and (III), wherein W is a methoxy, linear PEG group having an average molecular weight of 5 kDa.

[00606] Similarly, a compound designated "E62_5kD" was prepared by first preparing a protein having the sequence of SEQ ID NO: 4, in which the glutamic acid at position 62 (E62) was replaced with N 6-((2-azidoethoxy)-carbonyl)-L-lysine AzK (SEQ ID NO: 11). The AzK-containing protein was then reacted under click chemistry conditions with DBCO containing a methoxy, linear PEG group having an average molecular weight of 5 kDa to obtain a product having the sequence of SEQ ID NO: 21, comprising a structure of formula (II), a structure of formula (III), or a mixture of structures of formulas (II) and (III), wherein W is a methoxy, linear PEG group having an average molecular weight of 5 kDa. Compound E62_5kD was used in Example 9.

[00607] In another example, the compound designated as "E62_30kD" in Tables 2A and 2B was prepared by first preparing a protein having the sequence of SEQ ID NO: 4, in which the glutamic acid at position 62 was replaced with AzK. The AzK-containing protein was then reacted under click chemistry conditions with DBCO containing a methoxy, linear PEG group having an average molecular weight of 30 kDa to obtain a product having the sequence of SEQ ID NO: 26, comprising a structure of formula (II), a structure of formula (III), or a mixture of structures of formulas (II) and (III), wherein W is a methoxy, linear PEG group having an average molecular weight of 30 kDa.

[00608] Similarly, a compound designated "E62_30kD" was prepared by first preparing a protein having the sequence of SEQ ID NO: 4, in which the glutamic acid at position 62 (E62) was replaced with N 6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) (SEQ ID NO: 11). The AzK-containing protein was then reacted under click chemistry conditions with DBCO containing a methoxy, linear PEG group having an average molecular weight of 30 kDa to obtain a product having the sequence of SEQ ID NO: 26, comprising a structure of formula (II), a structure of formula (III), or a mixture of structures of formulas (II) and (III), wherein W is a methoxy, linear PEG group having an average molecular weight of 30 kDa. The compound can also be defined as a compound comprising the amino acid sequence of SEQ ID NO: 4, in which the glutamic acid at position 62 (E62) is replaced by a structure of formula (VI) or a structure of formula (VII) or a mixture of structures of formulas (VI) and (VII), and wherein n is an integer such that the PEG group has a molecular weight of about 30 kDa. The compound can also be defined as a compound comprising the amino acid sequence of SEQ ID NO: 4, in which the glutamic acid at position 62 (E62) is replaced by a structure of formula (X) or a structure of formula (XI) or a mixture of structures of formulas (X) and (XI), and wherein n is an integer such that the PEG group has a molecular weight of about 30 kDa. Compound E62_30kD was used in Examples 3, 5 and 9.

[00609] In another example, the compound designated "K35_30kD" and used in Example 12 was prepared by first preparing a protein having the sequence of SEQ ID NO: 4, in which the lysine at position 35 was replaced with N 6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) (SEQ ID NO: 14). The AzK-containing protein was then reacted under click chemistry conditions with DBCO containing a methoxy, linear PEG group having an average molecular weight of 30 kDa to obtain a product having the sequence of SEQ ID NO: 29, comprising a structure of formula (II), a structure of formula (III), or a mixture of structures of formulas (II) and (III), wherein W is a methoxy, linear PEG group having an average molecular weight of 30 kDa. The compound can also be defined as a compound comprising the amino acid sequence of SEQ ID NO: 4, in which lysine at position 35 (K35) is replaced by a structure of formula (VI) or a structure of formula (VII) or a mixture of structures of formulas (VI) and (VII), and where n is an integer such that the PEG group has a molecular weight of about 30 kDa. The compound can also be defined as a compound comprising the amino acid sequence of SEQ ID NO: 4, in which lysine at position 35 (K35) is replaced by a structure of formula (X) or a structure of formula (XI) or a mixture of structures of formulas (X) and (XI), and where n is an integer such that the PEG group has a molecular weight of about 30 kDa.

[00610] The compound disclosed in Examples 13 and 14, designated "IL2_P65_[AzK_L1_PEG30kD]-1", was prepared using methods similar to those described in Example 2, wherein a protein having the sequence of SEQ ID NO 3 was first prepared in which the proline at position 64 was replaced with AzK. The AzK-containing protein was then reacted under click chemistry conditions with DBCO containing a methoxy, linear PEG group having an average molecular weight of 30 kDa to obtain a product having the sequence of SEQ ID NO: 50, comprising a structure of formula (IV), a structure of formula (V), or a mixture of structures of formulas (IV) and (V), wherein W is a methoxy, linear PEG group having an average molecular weight of 30 kDa.

[00611] Similarly, a compound designated "IL2_P65_[AzK_L1_PEG30kD]-1" was prepared using methods similar to those described in Example 2, wherein a protein having the sequence of SEQ ID NO: 3 was first prepared in which the proline at position 64 was replaced with N 6-((2-azidoethoxy)-carbonyl)-L-lysine AzK (SEQ ID NO: 35). The AzK-containing protein was then reacted under click chemistry conditions with DBCO containing a methoxy, linear PEG group having an average molecular weight of 30 kDa. The compound IL-2_P65[AzK_L1_PEG30kD]-1 was also defined as a compound having the sequence under SEQ ID NO: 3, in which the proline residue at position 64 (P64) is replaced by a structure of formula (VIII) or a structure of formula (IX) or a mixture of structures of formulas (VIII) and (IX), and where n is an integer such that the PEG group has a molecular weight of approximately 30 kDa. The compound IL-2_P65[AzK_L1_PEG30kD]-1 was also defined as a compound having the sequence under SEQ ID NO: 3, in which the proline residue at position 64 (P64) is replaced by a structure of formula (XII) or a structure of formula (XIII) or a mixture of structures of formulas (XII) and (XIII), and where n is an integer such that the PEG group has a molecular weight of approximately 30 kDa. Compound IL2_P65_[AzK_L1_PEG30kD]-1 was used in Examples 13, 14 and 15.

EXAMPLE 1

[00612] Kinase and Cytokine Receptor Dimerization Assays

[00613] Cell processing

[00614] PathHunter cell lines were expanded from frozen stock cultures according to standard procedures. Cells were seeded in a total volume of 20 µl into 384-well white-walled microplates and incubated for the appropriate time period prior to analysis.

[00615] Agonist format

[00616] To determine the agonist, cells were incubated with sample to induce a response. Stock cultures were intermediately diluted to produce 5X sample in assay buffer. Approximately 5 μl of 5X sample was added to cells and incubated at 37°C for 6-16 hours depending on the assay. The concentration of carrier medium was 1%.

[00617] Signal detection

[00618] The signal in the assay was generated by a single addition of 12.5 or 15 μL (50% v/v) of PathHunter detection reagent cocktail for the agonist and antagonist assays, respectively, followed by incubation for one hour at room temperature. For some assays, activity was detected using a highly sensitive detection reagent (PathHunter Flash Kit) to improve assay performance. For these assays, an equal volume of detection reagent (25 or 30 μL) was added to the wells, followed by incubation for one hour at room temperature. Microplates were read after signal generation using a PerkinElmer EnvisionTM chemiluminescent signal detection instrument.

[00619] Data Analysis

[00620] Compound activity was analyzed using the CBIS data analysis package (ChemInnovation, CA). For agonist mode assays, percent activity was calculated using the following formula:

% activity = 100% x (average RLU of test sample - average RLU of control vehicle) / (average MAX RLU of control ligand - average RLU of control vehicle).

[00621] For antagonist mode assays, the percentage of inhibition was calculated using the following formula:

% inhibition = 100% x (1 - (mean RLU of test sample - mean RLU of vehicle control) / (mean RLU of EC80 control - mean RLU of vehicle control)).

EXAMPLE 2

[00622] Cell-based screening for identification of PEGylated IL-2 compounds without involvement of IL-2R α

[00623] Structural data for the IL-2/heterotrimeric receptor signaling complex (PDB: 2ERJ) were used to guide the design of nAA PEGylation sites to specifically abolish the interaction of IL-2 and the IL-2 receptor α subunit (IL-2Rα). Functional analysis of exemplary IL-2 conjugates was performed: K35, F42, K43, E62, and P65. IL-2 conjugates were expressed as inclusion bodies in E. coli , purified, and refolded using standard procedures prior to site-specific PEGylation of the IL-2 product using copper-free DBCO-mediated click chemistry to stably covalently attach mPEG moieties to AzK ( Scheme 1 ).

[00624] Scheme 1. Illustrative synthesis of AzK_PEG interleukin variants (where n denotes the number of repeating PEG units). Regioisomers

[00625] Scheme 2. Illustrative synthesis of AzK_PEG interleukin variants (where n denotes the number of repeating PEG units). A mixture of regioisomers can be obtained.

[00626] IL-2 conjugates were screened for functional activity at Discoverx (Fremont, CA) using the PathHunter IL-2 Cytokine Receptor Assay. This assay utilized the recombinant human cell line U2OS, which expresses the IL-2 receptor β (IL-2Rβ) and γ (IL-2Rγ) subunits, each fused to half of the β-galactosidase split reporter enzyme. Additionally, a second cell line was constructed to express the IL-2Rα subunit. Parallel analysis with these two cell lines allows for the assessment of activation of the IL-2 receptor αβγ variant as well as the basal βγ complex. IL-2 agonist activity at the IL-2 βγ receptor complex stimulates receptor dimerization and restoration of the β-galactosidase reporter, yielding a chemiluminescent signal. The assay was run in agonist mode to determine the _EC50 of each assay, and comparison of dose-response profiles between IL2Rα positive and negative cell types allows the contribution of IL2Rα to the observed activity to be determined.

[00627] Table 1 shows EC50 data for IL-2 receptor agonism in a cell-based screen of 10 kDa PEGylated IL-2 conjugates (except where noted)

Website βγ
EC50 (nM) αβγ
EC50 (nM) βγ / αβγ ratio Native 1.68 0.074 23 Optimal 1.68 1.68 1 K35 6.75 0.15 45 F42 6.09 0.515 12 K43 9.84 0.131 75 E62 3 1.5 2 P65* 23.8 4.44 5 R38 4.16 0.165 25 T41 6.37 0.0489 130 E68 7.70 0.0893 86 Y45* 9.06 0,110 83 V69* 9.99 0.083 121 *denotes pegylated IL-2 conjugate 30 kDa.

[00628] Biochemical interactions of pegylated IL-2 with human IL-2 receptor subunits

[00629] The kinetics of interactions of the pegylated IL-2 compound with human IL-2 receptor subunits were measured using surface plasmon resonance (SPR) at Biosensor Tools LLC (Salt Lake City, UT). For these studies, the human IgG1 Fc-fused extracellular domains of IL-2 Rα (Sino Biological, catalog #10165-H02H) and β (Sino Biological, catalog #10696-H02H) were immobilized on the surface of a Biacore protein A-coated CM4 sensor chip. These surfaces were analyzed in duplicate with a 2-fold dilution series starting with 2 μM native IL-2 (wild-type IL-2; Thermo #PHC0021), P65_30kD, P65_5kD, E62_30kD, or E62_5kD using a Biacore 2000 SPR instrument. Samples were injected for 60 s to allow association measurements, followed by buffer alone (wash) for 30 s to measure dissociation. Response units (RU, y-axis) were plotted against time (s, x-axis).

[00630] To assess the effect of the IL-2 receptor alpha subunit on IL-2 binding to β, α was immobilized in approximately two-fold excess over β. Native IL-2 (wild-type IL-2), P65_30kD, P65_5kD, E62_30kD, or E62_5kD were coated onto these surfaces in a three-fold dilution series starting at 2.5 μM. The binding data were consistent with a 1:1 interaction model that included array shifting, and the extracted kinetic parameters are provided in Table 2A and Table 2B . Table 2A and Table 2B and Figures 4A-4B show that small PEGs abolish IL2R-alpha engagement but have a less non-specific effect on IL2R-beta engagement.

Table 2A. Kinetic parameters for interactions of IL-2 variant with individual IL-2 receptor subunit surfaces with the IL-2 receptor α surface

k _a (M ^-1 With ^-1 ) k _d (With ^-1 ) K _D (μM) Native IL-2 4.5 ± 0.3 x10 ⁷ 0.410 ± 0.01 0.009 ± 0.002 P65_30kD 114 ± 36 x10 ⁷ 0.018 ± 0.008 158 ± 21 P65_5kD 797 ± 226 x10 ⁷ 0.033 ± 0.004 42 ± 7 E62_30kD 333 ± 88 x10 ⁷ 0.050 ± 0.01 162 ±7 E62_5kD 1010 ± 41 x10 ⁷ 0.035 ± 0.002 34.4 ± 0.3

Table 2B. Kinetic parameters for interactions of IL-2 variant with individual IL-2 receptor subunit surfaces - with the IL-2 receptor β surface

k _a (M ^-1 With ^-1 ) k _d (With ^-1 ) K _D (μM) Native IL-2 1.3 ± 0.2×10 ⁶ 0.185 ± 0.009 0.145 ± 0.005 P65_30kD 1.8 ± 0.2×10 ⁵ 0.370 ± 0.01 2.09 ± 0.09 P65_5kD 9.0 ± 0.4×10 ⁵ 0.270 ± 0.01 0.305 ± 0.002 E62_30kD 1.8 ± 0.4×10 ⁵ 0.208 ± 0.006 1.14 ± 0.01 E62_5kD 6.6 ± 0.8×10 ⁵ 0.281 ± 0.004 0.428 ± 0.00

[00631] On sensor surfaces containing immobilized IL-2Rα, native IL-2 showed rapid association and slow dissociation kinetics, demonstrating high affinity binding ( Figure 4A ). In contrast, neither P65_30kD nor E62_30kD interacted with the IL-2Rα surface even at the highest assayed concentration of 2 μM ( Figure 4A ). Similarly, F42_30kD did not interact with the IL-2Rα surface ( Figure 4C ). Surfaces containing immobilized IL-2 Rβ showed comparable association and dissociation responses with both native IL-2 (wild-type IL-2) and IL-2 P65_30kD ( Fig. 4B ), as well as with F42_30kD ( Fig. 4C ). The minor difference in _KD observed between the compounds for the β-subunit is due to a decrease in the association rate of IL-2 P65_30kD compared to native IL-2, which is expected based on the change in the hydrodynamic radius of this PEGylated compound (lower diffusion coefficient) as well as non-specific shielding effects of the large PEG moiety on distant binding surfaces. These results suggest that P65_30kD, E62_30kD, and F42_30kD are defective in interactions with IL-2Rα while retaining significant binding to IL-2Rβ.

[00632] Ex-vivo profiling of IL-2 immune response in PBMC samples generated using the human primary leukocyte reduction system (LRS)

[00633] To determine how the differential receptor specificities of IL-2 P65_30kD, K64_30kD, K43_30kD, K35_30kD, and F42_30kD influence the activation of primary immune cell subsets, concentration-response profiling of lymphocyte activation was performed in human peripheral blood mononuclear cell (PBMC) samples generated by LRS using multicolor flow cytometry. These studies were performed at PrimityBio LLC (Fremont, CA). Fresh LRS samples were treated with native IL-2, L-2 P65_30kD, K64_30kD, K43_30kD, K35_30kD, and F42_30kD in a 5-fold dilution series starting at a maximum concentration of 30 μg/mL. After 45 min of incubation, samples were fixed and stained with antibodies to detect the phosphorylated form of the transcription factor STAT5 (pSTAT5), a marker of upstream recruitment and activation of IL-2 receptor signaling complexes, and a panel of surface markers for subsequent pSTAT5 formation in specific T cell and natural killer (NK) cell subsets. The staining panel for flow cytometric examination of LRS-derived PBMC samples included markers for effector T cells (Teff: CD3+, CD4+, CD8+, CD127+), NK cells (CD3-, CD16+), and regulatory T cells (Treg: CD3+, CD4+, CD8-, IL-2Rα+, CD127-1).

[00634] Flow cytometry data were analyzed for the activation of different T and NK cell subsets in a concentration-response fashion by reading pSTAT5 accumulation following treatment with native IL-2 ( Figures 5A and 5G ), E62_30kD ( Figure 5H ), P65_30kD ( Figures 5B and 5I ), K64_30kD ( Figure 5C ), K43_30kD ( Figure 5D ), K35_kDa ( Figure 5E ), and F42_30kDa ( Figure 5F ). Consistent with the Treg-specific expression of IL-2 Rα, native IL-2 demonstrated increased efficiency in stimulating pSTAT5 in Tregs compared to CD8 Teff and NK cells. Compared to the native compound, the PEGylated variants shown in Figure 5B–5F demonstrate moderately reduced efficacy against CD8 T cell and NK cell populations, but show differential reduction in efficacy against IL-2-expressing Rα Treg cells compared to native IL-2.

[00635] Table 3 shows the EC50 dependence dose-response of pSTAT5 signaling (EC₅₀) upon proliferation of human LRS or CTLL-2 samples when treated with the indicated IL-2 variant.

Table 3.EC ₅₀ dose-response relationships for pSTAT5 signaling (EC ₅₀ ) in the proliferation of human LRS or CTLL-2 samples when treated with the indicated IL-2 variant

Processing NK cells Treg cells CD8+ T cells CD8/Treg ratio CTLL-2 Native IL-2 5150,5 62.5 25703,5 411.3 846 E62_30kD 12834 37213 66644 1.8 398,012 E62_5kD 5327,5 18146 41552,5 2,3 275,590 E62K 10305 11086 64037 5.8 58,213 P65_30kD 15741 40740,5 113638 2.8 677,198 P65_5kD 1920 6324,5 13769,5 2,2 194,924 K35_30kD 14021 358 63023 176.0 BUT. F42_30kD 16397 36856 107944 2.9 123,936 K43_30kD 9004 4797 50504 10.5 BUT.

[00636] EC ₅₀ values (pg/mL) were calculated from dose-response curves obtained from MFI plots.

EXAMPLE 3

[00637] PEG and residue substitution promote non-alpha pharmacological properties of variants

[00638] To determine how PEG and residue substitution affect the non-alpha pharmacological properties of IL-2 E62, concentration-response profiling of lymphocyte activation was performed in human peripheral blood mononuclear cell (PBMC) samples generated by LRS using multicolor flow cytometry. These studies were performed at PrimityBio LLC (Fremont, CA). Fresh LRS samples were treated with either native IL-2, E62K, or E62_30kD in a 5-fold dilution series starting at a maximum concentration of 30 μg/mL. After 45 min of incubation, samples were fixed and stained with antibodies to detect the phosphorylated form of the transcription factor STAT5 (pSTAT5), a marker of upstream recruitment and activation of IL-2 receptor signaling complexes, and a panel of surface markers for subsequent pSTAT5 formation in specific T cell and natural killer (NK) cell subsets. The staining panel for flow cytometric examination of LRS-derived PBMC samples includes markers for CD4, CD4+ central memory cells, CD4+ effector memory cells, CD4+ memory T cells, CD4+ naive T cells, CD4+ T cells, CD8, CD8+ central memory cells, CD8+ effector memory cells, CD8+ memory T cells, CD8+ naive T cells, CD8+ T cells, NK cells, and regulatory T cells.

[00639] Flow cytometry data were analyzed for activation of different T and NK cell subsets by IL-2 ( Fig. 6A ), E62K ( Fig. 6B ), or E62_30kD ( Fig. 6C ). These results indicate that the amino acid substitution at position 62, as well as AzK PEGylation at the modified position, may contribute to reduced IL-2 Rα engagement and, as a result, differential efficacy against primary lymphocytes that express these receptors.

Table 4.EC ₅₀ dose-response relationships for pSTAT5 signaling (EC ₅₀ ) in the proliferation of human LRS samples treated with the indicated IL-2 variant

Processing Treg cells CD8+ T cells CD8/Treg ratio Native IL-2 17 6092 358 E62_30kD 33580 64134 1.9 E62K 11086 64037 5.8

EXAMPLE 4

[00640] PEG size dependence of non-alpha pharmacological properties of variants

[00641] IL-2 conjugates were screened for functional activity at Discoverx (Fremont, CA) using the PathHunter IL-2 Cytokine Receptor Assay. This assay utilized the recombinant human cell line U2OS, which expresses the IL-2 receptor β (IL-2Rβ) and γ (IL-2Rγ) subunits, each fused to half of the β-galactosidase split reporter enzyme. Additionally, a second cell line was constructed to express the IL-2Rα subunit. Parallel analysis with these two cell lines allows for the assessment of activation of the IL-2 receptor αβγ variant as well as the basal βγ complex. IL-2 agonist activity at the IL-2 βγ receptor complex stimulates receptor dimerization and restoration of the β-galactosidase reporter, yielding a chemiluminescent signal. The assay was run in agonist mode to determine the _EC50 of each assay, and comparison of dose-response profiles between IL2Rα positive and negative cell types allows the contribution of IL2Rα to the observed activity to be determined.

[00642] Figure 7 shows that the observed potency of compounds pegylated with 5 kDa and 30 kDa PEG was similar in cells expressing IL-2 Rα as well as in cells expressing IL-2 Rβγ alone. These results suggest that the reduction in IL-2 Rα engagement by these compounds is not sensitive to the molecular weight of the PEG in the conjugate.

[00643] EXAMPLE 5

[00644] PK/PD studies in naive (E3826-U1704) and B16-F10 tumor-bearing (E3826-U1803) C57BL/6 mice

[00645] The study schedules are presented in Tables 5 and 6, with the dose calculated based on the mass of the protein component, excluding the mass of the PEG moiety. Blood samples were collected by cardiac puncture at the indicated time points at sacrifice. Study E3826-U1704 included 13 time points (0.13, 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72, 96, and 120 hours) with 3 mice sacrificed at each time point, and Study E3826-U1803 included 9 time points (2, 8, 12, 24, 48, 72, 120, 168, and 240 hours) with 4-7 mice sacrificed at each time point. Plasma and blood cells (in both studies) and tumors in study E3826-U1803 were collected for PK and PD analyses.

[00646] Bioassay of plasma samples was performed using a dedicated human IL-2 ELISA assay (Abcam, Cambridge, UK). Aldesleukin, E62_30kD, P65_30kD, and internal standard concentrations in plasma-derived samples were determined by ELISA. PK data analysis was performed at NW Solutions (Seattle, WA). PK data were imported into Phoenix WinNonlin v6.4 (Certara/Pharsight, Princeton, NJ) for analysis. Mean group plasma concentrations over time were analyzed by non-compartmental methods using an i.v. bolus model.

Table 5. PK/PD Study No. E3826-U1704 - Control and Treatment Groups in Naïve C57/BL6 Mice

Processing Dose*(mg/kg) Route of administration, scheme Time points N Control 0 IV, single dose 13 3 Aldesleukin 0.3 IV, single dose 13 3 P65_30kD 0.3 IV, single dose 13 3 E62_30kD 0.3 IV, single dose 13 3 *Dose refers to the amount of IL-2 P65_30kD polypeptide, and the dose was calculated based on the mass of the protein component, excluding the mass of the PEG fragment.

Table 6. PK/PD study #E3826-U1803 - control group and experimentally treated B16F-10 melanoma mice ( with the dose calculated based on the mass of the protein component, excluding the mass of the PEG fragment)

Processing Dose (mg/kg) Route of administration, scheme Time point N Absent (before dose) 0 mg/kg Absent 1 6 Control medium carrier 0 mg/kg IV, single dose 9 3 P65_30kD 1 mg/kg IV, single dose 9 4 P65_30kD 3 mg/kg IV, single dose 9 4

[00647] Plasma concentration profiles of P65_30kD, E62_30kD, E62_5kD, and aldesleukin at 0.3 mg/kg are plotted in Fig. 8 .

[00648] In study E3826-U1704, both P65_30kD and E62_30kD demonstrated superior PK profiles compared to aldesleukin, as shown in Table 5. Following a single IV bolus dose of aldesleukin, Tmax was observed at 0.03 hours post-dose (first post-dose time point), and mean plasma concentrations were measured up to 4 hours post-dose. Following a single IV bolus dose of P65_30kD and E62_30kD, Tmax was observed at 0.03 hours post-dose, and mean plasma concentrations were measured up to 120 hours post-dose (last time point). In a separate study following IV dosing of E62_5kD, T _max was observed at 0.133 hours post-dose, and mean plasma concentrations were measured up to 12 hours post-dose.

[00649] The exposure based on C _max and AUC _0-t was as follows: P65_30kD > E62_30kD >> E62_5kD > aldesleukin. E62_5kD, with the smaller PEG, had a PK profile similar to rIL-2. P65_30kD exposure was 5.5-fold and 200-fold greater than aldesleukin exposure based on C max and AUC 0-t , respectively. Additionally, P65_30kD exhibited a 23-fold increase in t1/2 (13.3 hours vs. 0.57 hours) and an approximately 198-fold decrease in CL (6.58 vs. 1300 mL/h/kg) compared to aldesleukin. For both P65_30kD and E62_30kD, the volume of distribution (82.4 and 92.3 ml/kg, respectively) was approximately 4.2-4.7-fold smaller than that of aldesleukin and similar to the mouse blood volume (85 ml/kg [Boersen 2013]). This indicates that P65_30kD and E62_30kD are distributed primarily into the systemic circulation.

Table 7. PK parameters of P65_30kD in female C57BL/6 mice

Parameter Units of measurement P65_30kD E62_30kD E62_5kD Aldesleukin T _max h 0,030 0,030 0.133 0,030 C _max ng/ml 4,870 4,230 936 884 AUC _0-t h*ng/ml 45,600 37,100 798 229 R ² 0.992 0.986 0.851 0,900 AUC _INF h*ng/ml 45,600 37,100 807 230 t _1/2 h 13,300 14,500 2.56 0.573 CL ml/h/kg 6,580 8.07 372 1300 V _ss ml/kg 82.4 92.3 404 390

Note: R ² is the goodness-of-fit parameter for the final phase of each concentration-time profile.

All parameters are given with an accuracy of up to 3 significant digits.

EXAMPLE 6

[00650] Pharmacodynamic studies in the peripheral blood compartment

[00651] STAT5 phosphorylation and induction of cell proliferation (early molecular marker Ki-67 and cell number) were used as pharmacodynamic endpoints to evaluate the pharmacological profile of P65_30kD relative to its pharmacokinetics. The pSTAT5 PD marker showed optimal correlation with PK for both P65_30kD and aldesleukin in CD8+ effector T cells (Table 7). A sustained increase in pSTAT5 was observed in both NK and CD8+ T cells for up to 72 hours and for up to 24 hours in Tregs. pSTAT5 induction returned to baseline levels only after 2 hours in aldesleukin-treated mice (Fig. 8). STAT5 phosphorylation translated into proliferative responses (72-120 h) of CD8+ effector T cells and NK cells, but not Tregs (Figs. 9A-9C). Phenotypic analysis of CD8+ effector T cells revealed a significant expansion of CD44+ memory cells in this population ( Figs. 10A-10B ).

[00652] Pharmacodynamic studies in the tumor compartment in C57BL/6 mice bearing B16-F10 tumor (E3826-U1803)

[00653] Table 8 shows plasma and tumor drug concentrations following a single 3 mg/kg dose of P65_30kD in B16-F10 tumor-bearing mice, with the dose calculated based on the mass of the protein component and excluding the mass of the PEG moiety. The tumor half-life was twice that of plasma (24.4 vs. 12.6), indicating that P65_30kD penetrates the tumor and remains in the tumor. The ends of the curves intersect, indicating that plasma delivers faster than tumor (data not shown). The tumor:plasma AUC ratios were 9.7% and 8.4% for the 1 and 3 mg/kg doses, respectively.

Table 8. PK parameters of P65_30kD in blood plasma and tumor in female C57BL/6 mice with B16-F10 tumor

Parameter P65_30kD (3 mg/kg) Blood plasma Tumor T _max (h) *2.00 8 C _max (ng/ml) 40000 1550 t1/2 (h) 12.60 24.4 AUC _0-t (h*ng/ml) 656,000 55200 R ² 0.974 0.988 AUC _INF (h*ng/ml) 656,000 55200

[00654] Figures 11A-11B show P65_30kD-mediated NK and CD+ T cell expansion in B16F10 tumors. Figure 11A shows the percentage of NK cells, CD8+ cells, and Tregs in the tumor CD3+ T cell population following treatment with a single 3 g/kg i.v. bolus dose of P65_30kD. Tumor samples were analyzed for immune cell populations by flow cytometry 5 days post-treatment. Each data point represents the mean of 3 replicates at each time point ± SEM. Cell population data shown are from day 5 tumor samples, and the CD8/Treg ratio was calculated for day 7 samples . 11B shows the ratio of CD8+ effector cells to CD4+ regulatory T cells 7 days after treatment with a single 3 mg/kg i.v. bolus dose of P65_30kD. Each data point represents the mean of 3 replicates at each time point ± SEM.

[00655] MTD Assay in Balb/c Mice E3826-U1802

[00656] A dose-ranging study of P65_30kD was performed in naive female Balb/c mice at Crown Biosciences, Inc. (San Diego, CA). The study design is shown in Table 9 , with the dose calculated based on the mass of the protein component, excluding the mass of the PEG moiety. Blood samples were collected from the submandibular vein at 7 time points (0.25, 1, 4, 12, 24, 34, 48, and 72 hours). Both plasma and blood cells were collected for PK and PD analyses.

[00657] All plasma samples were analyzed for human IL-2, as well as the murine cytokines IL-2, TNF-α, IFNγ, IL-5, and IL-6 using commercially available ELISA kits.

Table 9. PK/PD and MTD Study #E3826-U1802 - Control and Treatment Groups in Naïve Balb/C Mice

Processing Dose (mg/kg) Route of administration, scheme Time point N Previously untreated 0 mg/kg 0 3 Control medium carrier 0 mg/kg i/v, BID x 3 3 3 Aldesleukin 0.01 mg/kg i/v, BID x 3 3 3 Aldesleukin 0.03 mg/kg i/v, BID x 3 3 3 Aldesleukin 0.1 mg/kg i/v, BID x 3 3 3 Aldesleukin 1.0 mg/kg i/v, BID x 3 3 3 Aldesleukin 3.0 mg/kg i/v, BID x 3 3 3 Aldesleukin 5.0 mg/kg i/v, BID x 3 3 3 P65_30kD 0.01 mg/kg IV, single dose 3 3 P65_30kD 0.03 mg/kg IV, single dose 3 3 P65_30kD 0.1 mg/kg IV, single dose 3 3 P65_30kD 1.0 mg/kg IV, single dose 3 3 P65_30kD 3.0 mg/kg IV, single dose 3 3 P65_30kD 5.0 mg/kg IV, single dose 3 3 #P65_30kD 0.3 mg/kg IV, single dose 8 3

* At all time points except the 72-hour time point, blood was collected from the submandibular vein. At the 72-hour time point, blood collection was performed with sacrifice.

# Only the 0.3 mg/kg dose of P65_30kD was used for PK/PD assessment.

[00658] Toxicology studies in the MTD study using Balb/c mice

[00659] The major toxicity associated with high doses of aldesleukin is vascular leak syndrome and associated cytokine release syndrome (CRS). To assess the potential for this effect in mice, single intravenous doses of P65_30kD were administered at doses ranging from 0.01 to 5.0 mg/kg ( Table 10 ), with the dose calculated based on the mass of the protein component, excluding the mass of the PEG moiety. Hematology, histopathology, organ weight, and cytokine analysis were performed. No abnormalities were observed in hematology, histopathology, or body weight compared to vehicle control-treated mice in either P65_30kD or aldesleukin-treated mice. Regarding the cytokine analysis, aldesleukin was observed to increase IL-5 levels in plasma starting from 1 mg/kg to 5 mg/kg ( Figure 12A ). With P65_30kD, a moderate increase in IL-5 (but less than aldesleukin) was observed only at 5 mg/kg ( Figure 12B ). A transient increase in systemic IFNγ levels was observed for both aldesleukin and P65_30kD.

EXAMPLE 7

[00660] PK/PD in Cynomolgus Monkeys - Study #20157276

[00661] The pharmacokinetic and pharmacodynamic profiles of P65_30kD were evaluated in previously treated cynomolgus macaques following a single 0.3 mg/kg intravenous dose, with the dose calculated based on the mass of the protein component excluding the mass of the PEG moiety. The study was performed at Charles River Laboratories, Inc. (Reno, NV) and PK data analysis was performed at NW Solutions (Seattle, WA). Blood samples were collected pre-dose and at 21 time points (0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, 120, 144, 168, 192, and 240 hours post-dose). Both plasma and blood cells were collected for PK and PD analyses. Selected time points were used for PK, PD, cell population, and hematology analysis.

[00662] All plasma samples were analyzed for human IL-2 (PK assays) using commercially available ELISA kits.

[00663] Table 10 shows the PK parameters of P65_30kD in the cynomolgus macaque.

Table 10

0.3 mg/kg Animal 2699 Animal 2705 Average value ROA Parameter Units of measurement Grade i/v T _max h 0,500 0,500 0,500 C _max ng/ml 11000 11400 11200 AUC _0-t h*ng/ml 121000 120000 121000 t _1/2 h 13.4 13.9 13.6 CL ml/hour/kg 2.47 2.49 2.48 V _ss ml/kg 29.0 32.1 30.5

[00664] Following a single IV bolus administration, T _max was observed at 0.5 hours post-dose (first post-dose time point) and mean plasma concentrations were measured up to 168 hours post-dose (last measurement). T _1/2 and AUC for P65_30kD were 13.6 hours and 121,000 h*ng/mL, respectively.

[00665] Hematological parameters - cynomolgus macaques - study #20157276

[00666] For hematological parameters, assessment time points correspond to days before dosing on day -1 and 1, 3, 6, 8, 10, 12, 14, 17, 19, 21 days after dosing.

[00667] Fig. 13 shows absolute and differential white blood cell counts. Data represent mean ±SD (N=2 animals/dose group).

[00668] Analysis of white blood cell (WBC) subsets showed that the significant increase in WBC was due to an expansion of the lymphocyte cell population, consistent with a P65_30kD mechanism. No increase in eosinophils was observed.

EXAMPLE 8

[00669] Markers of Treg expansion in primary lymphocytes treated with various PEG IL-2 compounds

[00670] Primary lymphocytes were isolated from fresh human leukocyte reduction system (LRS), pretreated with Ficoll, and stored at 4°C overnight. The following day, FACS was used to sort lymphocyte populations, including CD8-positive T cells, NK cells, and Tregs. The isolated populations were stimulated with plate-coupled anti-CD8/CD28 antibody for two days. Native IL-2 and K9_30kD dose curves were incubated at 37°C. On day 3, the medium was refreshed and compounds were again added according to the dose curves. On day 6, cells were harvested and analyzed using flow cytometry to profile CD25 and Ki-67 levels as an indicator of proliferation. In Fig. 16 shows that PEG IL-2 compounds can specifically expand immune cell populations ex vivo in primary lymphocytes compared to normal IL-2 control. Fig. 16A shows Ki67 immune cell expansion markers after IL-2 (control) treatment. Fig. 16B shows Ki67 immune cell expansion markers after P65_30kD treatment.

EXAMPLE 9

[00671] PEG size can be adjusted to adjust the pharmacokinetics of clearance rate in vivo

[00672] The PK/PD relationship of a pegylated IL-2 variant E62 modified with 5 kDa and 30 kDa mPEG was examined compared to the control compound aldesleukin (Proleukin). Four groups of 6-8 week old C57/Bl6 mice weighing 16 to 22 g were used, with 12 mice per group. Mice were dosed by a single i.v. injection on day 0, time 0. At the indicated time points, approximately 100 μl of whole blood was collected into EDTA tubes for processing: 50 μl was fixed in flow cytometry buffer, and plasma was extracted from the remaining sample for ELISA analysis. For the final blood collection, approximately 300 µl of whole blood was collected, the remaining sample was centrifuged to obtain blood plasma and used for ELISA.

Prior to 10 mL/kg i.v. dosing, the test articles were formulated at 37.5 μg/mL in PBS and 100 μL samples were collected at t=8 min, 15 min, 30 min, and 1, 2, 4, 8, 12, 24, 48, 72, 96, and 120 h post-dose. Table 11 shows the processing schedule, with the dose calculated based on the mass of the protein component, excluding the mass of the PEG moiety. Table 12 illustrates the different in vivo clearance rates.

[00673] Table 11 illustrates in vivo treatments of mice with the IL2 variant

Group Processing Dose (mg/kg) 1 Carrier medium 0,0 2 Aldesleukin 0.3 3 E62 (30 kDa PEG) 0.3 4 E62 (5 kDa PEG) 0.3

[00674] Table 12 illustrates clearance rates for IL variants compared to aldesleukin control.

Processing Aldesleukin E62 (30 kDa PEG) E62 (5 kDa PEG) Cmax, ng/ml 745 8,450 936 AUC _0-t (ng*h/ml) 432 81,900 807 t _1/2 (h) 0.576 10.4 2.56 Vss (ml/kg) 232 48.9 404 CL (ml/g/kg) 694 3.66 373

EXAMPLE 10

[00675] Table 11 illustrates the IL-2 sequences and compounds described herein.

Name Subsequence SEQ ID NO: IL-2
(homo sapiens)
(mature form) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 1 IL-2
(homo sapiens)
(predecessor)
NCBI Accession No: AAB46883.1 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT 2

[00676] EXAMPLE 11

[00677] Ex-vivo determination of the immune response profile of illustrative IL-2 compounds in PBMC samples obtained using the human primary leukocyte reduction system (LRS)

[00678] To determine how the different receptor specificities of exemplary IL-2 compounds affect the activation of primary immune cell subsets, concentration-response profiling of lymphocyte activation was performed in human LRS-derived peripheral blood mononuclear cell (PBMC) samples using multicolor flow cytometry. The conjugates of Table 13 were synthesized by modifying SEQ NO. 1. These studies were performed at PrimityBio LLC (Fremont, CA). Primary lymphocytes derived from human LRS samples were treated with a dilution series of the exemplary IL-2 compounds and quantified based on pSTAT5 signaling in each lymphocyte cell type using the panel shown in Table 12.

[00679] Table 12. Notation indicating cell populations

Marker Cell population CD3 T cells CD4 Th cells CD8 Effector T cells CD45RA Naive T cells CD56 NK cells CD14/19 Monocyte/B cells CD25 Treg or "trained" T cells CD127 Non-Treg cell CD62L Memory T cell vs. effector memory T cell pSTAT5 (Y694) Activation marker

[00680] Flow cytometry data were analyzed for activation of different T and NK cell subsets in a concentration-response fashion, measuring pSTAT5 accumulation following treatment with the exemplary IL-2 variant K9_30kD.

[00681] Fig. 15A - Fig. 15B show dose-response curves for pSTAT5 signaling in primary human cells generated by LRS ( Fig. 15A ) and the proliferation response in mouse CTLL-2 populations ( Fig. 15B ).

[00682] Table 13 shows the EC50 dose-response relationship for pSTAT5 signaling (EC50) in proliferation of human LRS or CTLL-2 samples when treated with the indicated IL-2 variant.

Table 13 .EC ₅₀ dose-response relationships for pSTAT5 signaling (EC ₅₀ ) in the proliferation of human LRS or CTLL-2 samples when treated with the indicated IL-2 variant

Compound NK cells CD8+ T cells Treg cells CD8+/Treg ratio Fold increase in EC50 of Treg compared to native IL-2 Proliferation of CTLL-2 Native IL-2 4586 31024 75 414 1 455.8 K9_30kD 169578 1100679 2217 496 30 504 H16_30kD 2545257 12070108 34976 345 466 80755 L19_30kD 6756768 22436430 93205 241 1243 3510 D20_30kD 2643930 9505217 1129455 8 15059 689939 M23_30kD 143620 539824 1030 524 14 1102 N26_30kD 258531 1188859 2459 483 33 2594 N88_30kD 3298113 11111537 323201 34 4309 66606 E100_30kD 35088 195823 483 405 6 1676 N119_30kD 34010 143380 535 268 11 1215 T123_30kD 33396 152928 269 569 6 255 Q126_30kD 3676807 19722480 29454 670 393 3584 S127_30kD 20210 92190 150 615 3 123 T131_30kD 24207 132922 258 515 3 641 N88R/D109_30kD 2780819 12503386 175805 71 3663 59577 V91K 20537 102255 142 720 3 99.5 N88R 2312847 15025734 11082 1356 148 363

[00683] EC ₅₀ values (pg/mL) were calculated from dose-response curves obtained from MFI plots.

[00684] *Changes in Treg efficiency compared to native IL-2 (wild-type IL-2) were analyzed in each individual experiment.

EXAMPLE 12

[00685] PK study in C57BL/6 mice

[00686] The experimental details are presented in Table 14 , where the dose was calculated based on the mass of the protein component, excluding the mass of the PEG moiety.

[00687] Table 14 .

Group Number of animals Test/control product (dose) Route, scheme of administration End point(s) 1 9 Native IL-2 (wild type) (3.0 mg/kg) Concentration: 0.6 mg/ml IV, single dose on day 0 at T=0 at 5 ml/kg Blood sampling at 0.08, 0.25, 0.5, 1, 2, 4, 8, 12 and 24 hours after dose administration 2 9 K35_30kD (3.0 mg/kg) Concentration: 0.6 mg/ml IV, single dose on day 0 at T=0 at 5 ml/kg 3 9 K35_30kD (0.3 mg/kg) Concentration: 0.6 mg/ml IV, single dose on day 0 at T=0 at 5 ml/kg Additional 6 None None Collection of blank samples (no time limit) Total 33

[00688] The pharmacokinetic properties of the exemplary compound pegylated IL-2 K35_30kD were evaluated at two dose levels. Lyophilized test article was reconstituted in PBS and administered to nine male C57BL/6 mice at doses of 0.3 and 3 mg/kg via intravenous injection into the tail vein for each dose group (see collection details below). Blood samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-dose. The hIL-2 ELISA kit from Abcam (ab100566), which does not cross-react with native mouse IL-2, was used to detect and quantify test articles. To correct for ELISA-specific differences in detection sensitivity between the native and PEGylated compound panels, standard curves of native IL-2 and the K35_30kD test article were generated using the test article dilution buffer, with data analyzed relative to the corresponding standard curves. Data plotted are the mean and SEM of the three individual samples (biological replicates) described above, and PK parameters for the K35_30kD test articles were obtained and are presented in Table 15 .

[00689] Table 15 .

Analyte Parameter Unit of measurement Dose 0.3 mg/kg 3 mg/kg Grade IL-2 K35-mPEG30kD T _max h 0,250 0,250 C _max ng/ml 6080 57700 AUC _ot h*/ng/ml 38500 425000 R ² 0.994 0.947 AUC _½extrap % 35.3 37.4 AUC _0-∞ h*ng/ml 59600 679000 t _1/2 h 18.2 19.5 C _max /D kg*ng/ml/mg 20300 19200 AUC _ot /D h*ng/ml 128000 142000

EXAMPLE 13

[00690] Characterization of binding to human IL-2R alpha and IL-2R beta

[00691] A study was performed to characterize the binding of an exemplary IL-2 conjugate, IL-2_P65[AzK_ L1_PEG30kD]-1, to human IL-2R alpha and IL-2R beta.

[00692] Test article samples binding to IL-2R alpha. Test article samples in solution were assayed for binding to IL-2R alpha receptor surfaces. Response data were processed by subtracting signals from a reference surface without the receptor as well as the average of the buffer injections using Scrubber-2 (Biologic Software Pty Ltd). Responses to a series of rhIL-2 concentrations were generally consistent with a 1:1 interaction model, including a mass transfer step ( Figures 17A-17B ). Summary binding constants are presented in Table 16 .

[00693] Table 16

K _D (nM) IL-2 R alpha IL-2 R beta rhIL-2 11 ± 1 0.7 ± 1 IL-2_P65[AzK_ L1_PEG30kD]-1 N.D. 3.1 ± 0.3

[00694] Capture of Fc-tagged IL-2R beta on a protein-coated CM4 sensor chip. The CM4 sensor chip was inserted into a Biacore 4000 optical biosensor and the instrument was filled three times with HBS-P running buffer (HBS-P is 1X HBS-N supplemented with 0.005% Tween-20). Protein A was coupled using standard NHS/EDC coupling conditions. IL-2R beta Fc was dissolved in water to a concentration of 0.1 mg/mL and then diluted 1/1000 with HBS-P running buffer. IL-2R beta Fc was injected for different periods of time to create 2 receptor surfaces with different densities (~750 RU and 1500 RU, data not shown).

[00695] Characterization of IL-2R beta binding samples. Samples of the test article in solution were assayed for binding to IL-2R beta receptor surfaces. Response data were processed by subtracting signals from a reference surface without the receptor as well as the average of the buffer injections using Scrubber-2 (Biologic Software Pty Ltd). Responses to rhIL-2 (2-fold dilutions of a peak concentration of 4 μM) and IL-2_P65[AzK_ L1_PEG30kD]-1 (2-fold dilutions of a peak concentration of 8 μM) samples, assayed in duplicate, were generally consistent with a 1:1 interaction model, including a mass transfer step, as shown in Figs. 17C-17D . Summary binding constants are presented in Table 16 .

[00696] Results: His-tagged IL-2R alpha was captured at different densities on a nickel-loaded NTA sensor chip in the Biacore SPR biosensor system. Fc-tagged IL-2R beta was captured at different densities on a protein A-coated CM4 sensor chip. The response data were fit to a 1:1 interaction model to determine the binding constants for each interaction. Recombinant human IL-2 (rhIL-2) bound to IL-2R alpha with an affinity of ~11 nM, while no binding of IL-2_P65[AzK_L1_PEG30kD]-1 samples to IL-2R alpha could be detected. RhIL-2 bound to IL-2R beta with an affinity of ~700 nM, and IL-2_P65[AzK_ L1_PEG30kD]-1 bound to IL-2R beta with an affinity of ~3 μM under these assay conditions.

EXAMPLE 14

[00697] A study was performed to determine the efficacy and differential cell type specificity of IL-2_P65[AzK_L1_PEG30kD]-1 compared to recombinant human interleukin-2 (hIL-2) for the efficacy of signaling involving the phosphorylated form of the transcription factor STAT5 (pSTAT5) in human primary immune cell types.

[00698] Human PBMC sample processing methods. Stock solutions of IL-2 (control, 1 mg/mL) and IL-2_P65[AzK_L1_PEG30kD]-1 ("batch 1": 1.27 mg/mL; "batch 2": 2.29 mg/mL) were stored as stock solutions frozen at -20°C.

[00699] Lot 1 IL-2_P65[AzK_L1_PEG30kD]-1 and Lot 2 compounds were diluted with PBS and IL-2 was diluted with PBS+0.1% BSA to generate 10X stock solutions. The concentration of the 10X IL-2 stock solution was 5 μg/mL and the concentration of the GLP-1 and GLP-2 stock solutions ranged from 6 to 300 μg/mL depending on the experiment. The 10X stock solutions were diluted in serial 5-fold dilutions to create a 10-point dose titration. The maximum dose of IL-2 was 5 μg/mL and the Lot 1 and Lot 2 stock solutions ranged from 6 to 300 μg/mL depending on the experiment. 10 µL of each stock solution was added to 90 µL of cell samples to achieve a final maximum dose for IL-2 of 500 ng/mL and 0.6-30 µg/mL for each of Batch 1 and Batch 2.

[00700] Sample stimulation. For stimulation, 10 μl of the above titrated dose was added to 90 μl of blood sample pre-equilibrated to 37°C. Samples were incubated at 37°C for 45 minutes. At the end of the incubation period, red blood cells were lysed and the cells were simultaneously fixed as follows:

[00701] 100 μl of cells were transferred to 900 μl of BD Lyse/Fix buffer (Beckton Dickinson, cat #558049) and immediately vortexed. BD Lyse/Fix was prepared by diluting the stock solution 1:5 with cell culture water immediately prior to addition. Samples were incubated for 10 min at room temperature, then centrifuged at 450 x g for 5 min to pellet the cells. The pelleted cells were washed with PBS+0.5% BSA and stored at -37°C until analysis.

[00702] Staining Protocol

Step 1: Cells were thawed at room temperature. Step 2: Fc Block (TruStain FcX™) was added. Step 3: Incubated at room temperature for 5 minutes. Step 4: The following antibodies from Table 17 were added.

[00703] Table 17

[00704] Antibodies for Human Panel

Fluorophore CD4 BUV737 CD56 BV711 CD16 BV711 CD8 BUV805 CD27 BV786 CD45RA BUV395 CD127 FITC CD25 Biotin

[00705] Step 5. Incubate for 20 minutes at room temperature. Step 6. Cells were washed twice with PBS+0.5% BSA. Step 7. Cells were permeabilized by adding 10 volumes of methanol per cell volume. Step 8. Cells were incubated for 10 minutes at 4°C. Step 9. Washed with PBS. Step 10. Cells were washed with PBS without 0.5% BSA. Step 11. Fc Block (TruStain FcX™) was added. Step 12. The following post-permeabilization staining panel from Table 18 was added.

[00706] Table 18

[00707] Staining reagents

Fluorophore CD3 PE-Cy7 STAT5 Ax647 Streptavidin BV421 FOXp3 P.E.

[00708] Flow cytometry and data analysis. Samples were analyzed on a Becton Dickinson Fortessa and LSR II with five lasers (372 nM, 405 nM, 488 nM, 561 nM, and 640 nM). The instruments were equipped with 20 detectors including scatter parameters. The instruments were routinely calibrated using Becton Dickinson Cytometer Setup & Tracking Beads. 96-well plates containing stained samples were analyzed at less than 8,000 cells/sec using a 96-well high-throughput sampler.

[00709] Data were exported as .fcs files to a network driver and compensated for fluorophore excess, and the .fcs files were annotated. The .fcs files were then gated according to the strategies described in Fig. 18. Cells were first gated on singlets using FSC-A followed by FSC-H to exclude any aggregates or doublets ( Fig. 18, singlets gate, ^1st panel). Within this gate, cells were gated on mid-high forward scatter (FSC-A) and side scatter (SSC-A) to exclude red blood cells, debris, and granulocytes ( Fig. 18, lymphocytes gate, ^2nd panel). T cells were then gated as the CD3+, CD56/16-negative population, ^3rd panel. NK cells were identified as a CD3 negative, CD56/16 high population, panel ^3. T cells were then separated into CD4+ T cells and CD8+ T cells ( Fig. 18, panel ⁴ ). Tregs were then gated from CD4+ T cells as a CD25 ^high x C127 ^low population, Fig. 18, panel ⁵ .

[00710] Statistics and plotting to obtain EC ₅₀ values . The mean fluorescence intensity (MFI) for each cell population, donor, and compound treatment was calculated from the signal in the phosphorylation channel. Statistics were analyzed using Spotfire. Spotfire plotted the data on a logarithmic scale for compound doses and a linear scale for MFI readings. The data were fit using a 4-parameter logistic regression equation. The EC ₅₀ was calculated as the inflection point of the curve.

[00711] Results. Human IL-2 and IL-2_P65[AzK_L1_PEG30kD]-1 samples were diluted and assayed in triplicate against each of three separate donors as described above. The calculated half-maximal effective concentration ( _EC50 ) values are shown in Table 19. The results demonstrate that IL-2_P65[AzK_L1_PEG30kD]-1 is a potent agonist of IL-2 receptor signaling in human lymphocytes. Consistent with previous in vitro binding studies showing that IL-2_P65[AzK_L1_PEG30kD]-1 specifically interacts with the IL-2Rβ subunit and not with IL2Rα, it demonstrated a specific reduction in signaling efficiency in Treg cells, whose efficiency depends on IL-2Rα engagement, compared to Teff and NK cells, which do not constitutively express high levels of IL-2Rα.

[00712] Table 19

[00713] Efficacy characteristics of hIL-2 and IL-2_P65[AzK_ L1_PEG30kD]-1 batches against primary CD8+ T cell, NK cell, and Treg cell subsets from human donors.

EC ₅₀ human cells (ng/ml) Material CD8+ T CD8+ T memory NK Treg CD8/Treg ratio Human IL-2 12.4 ± 1.29 11.3 ± 1.63 2.88 ± 1.63 0.027 ± 0.005 460 Party 1* 224 ± 25.3 240 ± 27.3 54.7 ± 6.55 114 ± 20.3 2.0 Party 2* 265 ± 12.4 284 ± 18.3 55.7 ± 8.62 132 ± 24.0 2.0

Data represent mean ± SEM calculated from triplicate samples from three independent donors. *IL-2_P65[AzK_L1_PEG30kD]-1.

[00714] Example 15. Stability of IL-2_P65[AzK_L1_PEG30kD]-1 in Human Serum

[00715] The study was conducted to evaluate the stability of the compound IL-2_P65[AzK_L1_PEG30kD]-1 in human serum over time.

[00716] The following materials were used for the study:

Reagent Source Catalogue No. Compound IL-2_P65[AzK_L1_PEG30kD]-1 Synthorx Batch 19-1756 HUMAN BLOOD SERUM A/B, pooled Fischer Sci NC9370648 Goat anti-human IL2 antibody Abcam Ab10752 PEG polyclonal antibody ThermoFisher PA5-32247 Mouse Anti-Rabbit IgG-HRP Southern Biotech 4090-05 Casein Blocker™ in PBS, 1% ThermoFisher 37528 1-Step™ Ultra TMB-ELISA Substrate Solution ThermoFisher 34028 Stop solution for TMB substrates ThermoFisher N600 PBS (10X), pH 7.4 ThermoFisher 70011044

[00717] IL-2_P65[AzK_L1_PEG30kD]-1 compound was added to pooled human serum A/B at three (3) concentrations (2590 ng/mL, 847 ng/mL, and 212 ng/mL) in PCR tubes. Each time point concentration was prepared in triplicate. IL-2_P65[AzK_L1_PEG30kD]-1 compound was prepared at stock solution concentrations of 25900 ng/mL, 8470 ng/mL, and 2120 ng/mL, and 20 μL of each stock solution was added to 180 μL of pooled human serum, mixed well, and incubated at 37°C for up to 168 hours. Samples were collected at time points corresponding to 0, 6, 24, 48, 72, 96, 120 and 168 h after incubation, divided into two aliquots and frozen at -80°C for batch analysis. The first set of samples was analyzed on one day and the second set of samples was analyzed on another day.

[00718] On the day of the assay, samples were thawed at room temperature using a customized ELISA format using goat anti-human IL-2 and PEG polyclonal antibody. The ELISA plate was coated overnight at 4°C with 1 μg/mL anti-human IL2 α and washed with 1 x PBS for 3 x 300 μL using a plate washer. The assay plate was blocked for one (1) hour with 1% casein in PBS. Assay samples were pre-diluted with human serum to bring them within the detection range of the ELISA. Calibration standard and QC samples were also prepared with human serum matrix at the exact concentration. All samples were diluted 25-fold with assay buffer (1% casein) to the MRD (minimum required dilution). Samples were added to the plate, sealed for two (2) hours at room temperature on a plate shaker and washed with 1 x PBS for 3 x 300 µL using a plate washer. The secondary detection antibody, α-PEG (rabbit), at 1 µg/mL was added at 100 µL per well, sealed for one (1) hour at room temperature on a plate shaker and washed with 1 x PBS for 3 x 300 µL using a plate washer. The final detection antibody, α-rabbit-HRP, was diluted 1:5000 and added at 100 µL per well, sealed for 45 minutes at room temperature on a plate shaker and washed with 1 x PBS for 3 x 300 µL using a plate washer. The substrate was added at 100 μL per well and placed away from light for 30 minutes without sealing or shaking. Finally, 50 μL of stop solution was added to each well and the plate was immediately read using a Spectramax iD5 plate reader to obtain OD450 values for all samples. Raw values were interpolated to the corresponding concentrations using 4-parameter logistic curve fitting methods to determine sample concentrations. The data presented in Figure 19 are the result of two replicate ELISA runs. These results demonstrate that IL-2_P65[AzK_L1_PEG30kD]-1 is stable in human serum matrix until the last time point analyzed, which was 168 hours.

[00719] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now be apparent to those skilled in the art without departing from the present invention. It will be understood that various alternatives to the embodiments of the present invention described herein may be used in practicing the present invention. It is intended that the following claims define the scope of the present invention and that methods and structures within the scope of the claims and their equivalents are encompassed by them.

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SEQUENCE LIST

<110> SYNTHORX, INC.

<120> IL-2 CONJUGATES AND METHODS OF THEIR APPLICATION

<130> 46085-729.601

<140>

<141>

<150> 62/940,173

<151> 2019-11-25

<150> 62/899,035

<151> 2019-09-11

<150> 62/870,581

<151> 2019-07-03

<150> 62/847,844

<151> 2019-05-14

<150> 62/802,191

<151> 2019-02-06

<160> 87

<170> PatentIn version 3.5

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Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

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Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

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Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

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Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

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Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

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Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

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Ile Ser Thr Leu Thr

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Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

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Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

20 25 30

Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile

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Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe

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Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu

65 70 75 80

Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

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Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile

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Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala

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Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe

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Cys Gln Ser Ile Ile Ser Thr Leu Thr

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<223> Description of the artificial sequence: Synthetic

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Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

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Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

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Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

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Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

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Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

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Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

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Ser Thr Leu Thr

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<223> Description of the artificial sequence: Synthetic

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Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

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Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

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Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

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Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

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Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

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Ile Ser Thr Leu Thr

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<213> Artificial sequence

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<223> Description of the artificial sequence: Synthetic

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<221>MOD_RES

<222> (65)..(65)

<223> Any unnatural amino acid

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Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

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Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

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Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Xaa Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 6

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

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<220>

<221>MOD_RES

<222> (62)..(62)

<223> Any unnatural amino acid

<400> 6

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Xaa Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

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Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

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Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 7

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

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<220>

<221>MOD_RES

<222> (42)..(42)

<223> Any unnatural amino acid

<400> 7

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Xaa Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

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Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

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Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 8

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

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<220>

<221>MOD_RES

<222> (43)..(43)

<223> Any unnatural amino acid

<400> 8

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Xaa Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

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Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 9

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (35)..(35)

<223> Any unnatural amino acid

<400> 9

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Xaa Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 10

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (65)..(65)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<400> 10

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Lys Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 11

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (62)..(62)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<400> 11

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Lys Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

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Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 12

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (42)..(42)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<400> 12

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Lys Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 13

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (43)..(43)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<400> 13

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 14

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (35)..(35)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<400> 14

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 15

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (65)..(65)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 15

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Lys Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 16

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (62)..(62)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 16

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Lys Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 17

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (42)..(42)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 17

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Lys Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 18

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (43)..(43)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 18

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 19

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (35)..(35)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 19

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 20

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (65)..(65)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 20

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Lys Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 21

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (62)..(62)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 21

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Lys Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 22

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (42)..(42)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 22

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Lys Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 23

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (43)..(43)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 23

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 24

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (35)..(35)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 24

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 25

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (65)..(65)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 25

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Lys Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 26

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (62)..(62)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 26

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Lys Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 27

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (42)..(42)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 27

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Lys Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 28

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (43)..(43)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 28

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 29

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (35)..(35)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 29

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 30

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (64)..(64)

<223> Any unnatural amino acid

<400> 30

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Xaa

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 31

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (61)..(61)

<223> Any unnatural amino acid

<400> 31

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Xaa Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 32

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (41)..(41)

<223> Any unnatural amino acid

<400> 32

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Xaa Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 33

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (42)..(42)

<223> Any unnatural amino acid

<400> 33

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Xaa Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 34

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (34)..(34)

<223> Any unnatural amino acid

<400> 34

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Xaa Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 35

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (64)..(64)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<400> 35

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Lys

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 36

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (61)..(61)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<400> 36

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Lys Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 37

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (41)..(41)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<400> 37

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Lys Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 38

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (42)..(42)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<400> 38

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 39

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (34)..(34)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<400> 39

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 40

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (64)..(64)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 40

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Lys

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 41

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (61)..(61)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 41

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Lys Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 42

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (41)..(41)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 42

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Lys Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 43

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (42)..(42)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 43

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 44

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (34)..(34)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 44

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 45

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (64)..(64)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 45

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Lys

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 46

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (61)..(61)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 46

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Lys Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 47

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (41)..(41)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 47

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Lys Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 48

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (42)..(42)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 48

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 49

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (34)..(34)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 49

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 50

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (64)..(64)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 50

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Lys

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 51

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (61)..(61)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 51

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Lys Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 52

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (41)..(41)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 52

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Lys Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 53

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (42)..(42)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 53

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 54

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (34)..(34)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 54

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 55

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (65)..(65)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 55

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Lys Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 56

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (62)..(62)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification for a detailed description of the substitutions.

and preferred embodiments

<400> 56

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Lys Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 57

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (42)..(42)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification for a detailed description of the substitutions.

and preferred embodiments

<400> 57

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Lys Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 58

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (43)..(43)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 58

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 59

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (35)..(35)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 59

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 60

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (65)..(65)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 60

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Lys Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 61

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (62)..(62)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 61

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Lys Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 62

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (42)..(42)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 62

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Lys Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 63

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (43)..(43)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 63

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 64

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (35)..(35)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 64

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 65

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (65)..(65)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 65

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Lys Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 66

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (62)..(62)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 66

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Lys Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 67

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (42)..(42)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 67

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Lys Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 68

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (43)..(43)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 68

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 69

<211> 133

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (35)..(35)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine, stable

PEG conjugated

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 69

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 70

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (64)..(64)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 70

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Lys

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 71

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (61)..(61)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 71

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Lys Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 72

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (41)..(41)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 72

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Lys Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 73

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (42)..(42)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 73

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 74

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (34)..(34)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 74

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 75

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (64)..(64)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 75

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Lys

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 76

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (61)..(61)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 76

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Lys Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 77

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (41)..(41)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 77

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Lys Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 78

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (42)..(42)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 78

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 79

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (34)..(34)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 79

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 80

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (64)..(64)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 80

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Lys

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 81

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (61)..(61)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 81

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Lys Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 82

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (41)..(41)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 82

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Lys Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 83

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (42)..(42)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 83

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 84

<211> 132

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<220>

<221>MOD_RES

<222> (34)..(34)

<223> N6-((2-azidoethoxy)-carbonyl)-L-lysine

<220>

<223> See the filed specification of the invention for a detailed description.

replacements and preferred embodiments

<400> 84

Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu

1 5 10 15

Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn

20 25 30

Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys

35 40 45

Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro

50 55 60

Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg

65 70 75 80

Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys

85 90 95

Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr

100 105 110

Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile

115 120 125

Ser Thr Leu Thr

130

<210> 85

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<400> 85

Gly Gly Gly Gly Ser

1 5

<210> 86

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

peptide

<220>

<221>MOD_RES

<222> (4)..(4)

<223> Any amino acid except Pro

<400> 86

Val Pro Gly Xaa Gly

1 5

<210> 87

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: Synthetic

polypeptide

<400> 87

Phe Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro

1 5 10 15

Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

20 25 30

<---

Claims (52)

1. An IL-2 conjugate comprising an amino acid sequence having at least about 80% identity to SEQ ID NO: 3, wherein at least one amino acid residue at an amino acid position selected from K34, F41, K42, E61, P64, R37, T40, E67, Y44 and V68, related to the amino acid positions in SEQ ID NO: 3, is replaced by a structure of formula (I) Where Z is CH ₂ and Y is ; Y is CH ₂ , and Z is ; Z is CH ₂ and Y is ; or Y is CH ₂ , and Z is ; W is a PEG group having a molecular weight selected from about 5 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, and about 60 kDa; X is an L-amino acid with the structure X-1 indicates the point of attachment of the amino acid residue preceding the substituted amino acid; and X+1 indicates the point of attachment of the amino acid residue following the substituted amino acid. 2. The IL-2 conjugate of claim 1, wherein said conjugate comprises the amino acid sequence of SEQ ID NO: 3, wherein at least one amino acid residue at an amino acid position selected from K34, F41, K42, E61, P64, R37, T40, E67, Y44 and V68, related to the amino acid positions in SEQ ID NO: 3, is replaced by a structure of formula (I). 3. The IL-2 conjugate of claim 1 or 2, wherein said at least one amino acid residue at position P64 is replaced by a structure of formula (I). 4. An IL-2 conjugate according to any one of claims 1-3, wherein said structure of formula (I) has a structure of formula (IV) or formula (V): 5. The IL-2 conjugate of any one of claims 1-4, wherein W is a PEG group having a molecular weight of approximately 30 kDa. 6. The IL-2 conjugate of claim 2, wherein said at least one amino acid residue at position P64 is replaced by a structure of formula (I), which has a structure of formula (XII) or formula (XIII): Where: n is an integer such that the PEG group having the structure -( _OCH2CH2 ) _{n -OCH3 has} a molecular weight of about 5 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, or about 60 kDa; And wavy lines indicate covalent bonds to amino acid residues in SEQ ID NO: 3 that are not substituted. 7. The IL-2 conjugate of claim 6, wherein n is an integer such that the PEG group having the structure -(OCH ₂ CH ₂ ) _n -OCH ₃ has a molecular weight of approximately 30 kDa. 8. A pharmaceutical composition for treating cancer, comprising an effective amount of an IL-2 conjugate according to any one of claims 1-7 and a pharmaceutically acceptable excipient. 9. A pharmaceutical composition for treating cancer, comprising an effective amount of a mixture of IL-2 conjugates according to claim 6 and a pharmaceutically acceptable excipient, wherein said mixture comprises (i) IL-2 conjugates in which at least one amino acid residue at position P64 is replaced by a structure of formula (XII), and (ii) IL-2 conjugates in which at least one amino acid residue at position P64 is replaced by a structure of formula (XIII). 10. A pharmaceutical composition for treating cancer, comprising an effective amount of a mixture of IL-2 conjugates according to claim 4 or 5 and a pharmaceutically acceptable excipient, wherein said mixture comprises (i) IL-2 conjugates in which the structure of Formula (I) has a structure of formula (IV), and (ii) IL-2 conjugates in which the structure of formula (I) has a structure of formula (V). 11. A pharmaceutical composition for treating cancer, comprising an effective amount of a mixture of IL-2 conjugates according to claim 7 and a pharmaceutically acceptable excipient, wherein said mixture comprises (i) IL-2 conjugates in which said at least one amino acid residue at position P64 is replaced by a structure of Formula (XII), and IL-2 conjugates in which amino acid P64 is replaced by a structure of formula (XIII). 12. A method of treating cancer in a subject in need thereof, comprising administering to said subject an effective amount of an IL-2 conjugate according to any one of claims 1-7 or a pharmaceutical composition according to any one of claims 8-11. 13. The method of claim 12, wherein the cancer is selected from the group consisting of renal cell carcinoma (RCC), melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), urothelial carcinoma (UC), Merkel cell carcinoma (MCC), ovarian cancer, gastric cancer, breast cancer, bladder cancer, bone cancer, brain cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, pancreatic cancer, prostate cancer, cutaneous squamous cell carcinoma (CSCC), and classical Hodgkin's lymphoma (cHL). 14. Use of an IL-2 conjugate according to any one of claims 1-7 or a pharmaceutical composition according to any one of claims 8-11 for treating cancer in a subject in need thereof. 15. The use according to claim 14, wherein the cancer is selected from the group consisting of renal cell carcinoma (RCC), melanoma, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), urothelial carcinoma (UC), Merkel cell carcinoma (MCC), ovarian cancer, gastric cancer, breast cancer, bladder cancer, bone cancer, brain cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, pancreatic cancer, prostate cancer, cutaneous squamous cell carcinoma (CSCC) and classical Hodgkin's lymphoma (cHL). 16. A method for producing an IL-2 conjugate, comprising: carrying out a reaction of an IL-2 polypeptide containing an unnatural amino acid of the formula wherein the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 3, position X-1 indicates the point of attachment to the preceding amino acid residue, position X+1 indicates the point of attachment to the next amino acid residue, and position X indicates the position of the amino acid that is replaced by the unnatural amino acid, wherein position X is K34, F41, K42, E61, P64, R37, T40, E67, Y44, or V68 relative to the amino acid positions of SEQ ID NO: 3, with mPEG-DBCO formulas or where n is an integer, such that mPEG-DBCO comprises mPEG having a molecular weight of about 5 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, or about 60 kDa, with the formation of IL-2 conjugate. 17. A method for producing an IL-2 conjugate, comprising: carrying out a reaction of an IL-2 polypeptide containing an unnatural amino acid of the formula wherein the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 3, wherein position X is P64 relative to the amino acid positions of SEQ ID NO: 3, position X-1 indicates the point of attachment to the preceding amino acid residue, position X+1 indicates the point of attachment to the next amino acid residue, with mPEG-DBCO formulas where n is an integer, such that mPEG-DBCO contains mPEG with a molecular weight of approximately 30 kDa, to form an IL-2 conjugate, wherein said IL-2 conjugate has formula (XII) or formula (XIII): Where wavy lines indicate covalent bonds to amino acid residues in SEQ ID NO: 3 that are not substituted.