HK40120363A - Anti-pd-1 antibody-attenuated il-2 immunoconjugates and uses thereof - Google Patents

Description

Anti-PD-1 antibody-attenuated IL-2 immunoconjugate and its uses

Cross-references to related applications

This application claims priority to U.S. Provisional Application No. 63/352,842, filed June 16, 2022; U.S. Provisional Application No. 63/481,630, filed January 26, 2023; and U.S. Provisional Application No. 63/502,746, filed May 17, 2023, the disclosure of each of which is hereby incorporated by reference in its entirety.

sequence list

This application contains a sequence list that has been submitted electronically in XML format and is hereby incorporated in its entirety by reference. The XML copy created on June 5, 2023, is named 102085.021703_SequenceListing.xml and is 696,000 bytes in size.

Technical Field

This article discloses modified human interleukin-2 (hIL-2) proteins, human antibody molecules or antigen-binding fragments thereof that are immune-specifically bound to human programmed cell death protein-1 (hPD-1), and immunoconjugates containing them.

Background Technology

Human IL-2 (hIL-2) is a type I tetraalpha helical bundle, a glycosylated cytokine produced by CD4+ and CD8+ T cells. Autocrine and paracrine IL-2 signaling occurs via conjugation of a high-affinity trimeric receptor complex containing IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (CD132), or a medium-affinity dimeric receptor complex containing IL-2Rβ (CD122) and IL-2Rγ (CD132). IL-2 exhibits dual, pleiotropic effects, stimulating both T cell proliferation to produce T cell effectors, T cell memory, and activated NK cells, and suppressive regulatory T cells to maintain immune homeostasis. Low-dose IL-2 primarily stimulates regulatory T cells, as well as some T effectors and NK cells, while high-dose IL-2 broadly stimulates cytotoxic T cells, T effectors, NK cells, and regulatory T cells. However, the use of IL-2 in the treatment of autoimmune diseases and as a cancer immunotherapy is limited by off-target effects and toxicity associated with IL-2 administration.

Summary of the Invention

This document discloses a modified human interleukin-2 (hIL-2) protein, which, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, comprises a substitution at amino acid position 20 and a substitution at amino acid position 38, wherein, relative to the unmodified hIL-2, the modified hIL-2 protein exhibits reduced potency against both high-affinity and intermediate-affinity hIL-2 receptors.

This article also discloses a modified human interleukin-2 (hIL-2) protein, which, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, includes a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution at amino acid position 20 and an R38E substitution at amino acid position 38.

This document also discloses a human antibody molecule or its antigen-binding fragment thereof, which specifically binds to human programmed cell death protein-1 (hPD-1) immunely, wherein the human antibody molecule or its antigen-binding fragment comprises:

a) Heavy chain complementarity-determining region 1 (CDR1) containing the amino acid sequence of SEQ ID NO:418, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:419, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:420, light chain CDR1 containing the amino acid sequence of SEQ ID NO:421, light chain CDR2 containing the amino acid sequence of SEQ ID NO:422, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:423;

b) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:388, light chain CDR1 containing the amino acid sequence of SEQ ID NO:389, light chain CDR2 containing the amino acid sequence of SEQ ID NO:390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:391;

c) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:398, light chain CDR1 containing the amino acid sequence of SEQ ID NO:399, light chain CDR2 containing the amino acid sequence of SEQ ID NO:400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:401; or

d) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:406, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:407, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:408, light chain CDR1 containing the amino acid sequence of SEQ ID NO:409, light chain CDR2 containing the amino acid sequence of SEQ ID NO:410, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:411.

This article also discloses immunoconjugates containing the following:

(a) A modified hIL-2 protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, comprising a substitution at amino acid position 20 and a substitution at amino acid position 38; and

(b) a human antibody molecule or an antigen-binding fragment thereof, said human antibody molecule or antigen-binding fragment thereof binding specifically to hPD-1, wherein said human antibody molecule or antigen-binding fragment thereof comprises:

(i) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:418, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:419, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:420, light chain CDR1 containing the amino acid sequence of SEQ ID NO:421, light chain CDR2 containing the amino acid sequence of SEQ ID NO:422, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:423;

(ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:388, light chain CDR1 containing the amino acid sequence of SEQ ID NO:389, light chain CDR2 containing the amino acid sequence of SEQ ID NO:390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:391;

(iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:398, light chain CDR1 containing the amino acid sequence of SEQ ID NO:399, light chain CDR2 containing the amino acid sequence of SEQ ID NO:400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:401; or

(iv) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:406, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:407, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:408, light chain CDR1 containing the amino acid sequence of SEQ ID NO:409, light chain CDR2 containing the amino acid sequence of SEQ ID NO:410, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:411.

Pharmaceutical compositions comprising any of the modified hIL-2 protein, human antibody molecules or their antigen-binding fragments or immunoconjugates disclosed herein are also disclosed.

This document also discloses polynucleotides comprising nucleic acid sequences encoding any modified hIL-2 protein, human antibody molecule or its antigen-binding fragment or immunoconjugate disclosed herein, as well as vectors comprising said polynucleotides and transformed cells comprising said vectors.

This document discloses a method for treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of any of the immunoconjugates or pharmaceutical compositions disclosed herein, thereby treating the disease or condition.

It also discloses the use of any of the immunoconjugates or pharmaceutical compositions disclosed herein in the preparation of medicaments for treating diseases, and the use of any of the immunoconjugates or pharmaceutical compositions disclosed herein in the treatment of diseases or conditions.

Attached Figure Description

The invention and the following detailed description will be further understood when read in conjunction with the accompanying drawings. Exemplary embodiments of the modified hIL-2 protein, anti-hPD-1 antibody, or antigen-binding fragment thereof, and immunoconjugate disclosed herein are illustrated in the drawings to illustrate the invention; however, the modified hIL-2 protein, anti-hPD-1 antibody, or antigen-binding fragment thereof, and immunoconjugate are not limited to the specific embodiments disclosed. In the drawings:

Figures 1A, 1B, 1C, 1D, 1E, 1F, 1G, and 1H illustrate exemplary antibody-hIL-2 immunoconjugates as described in Example 1 of this document. Unattenuated human IL-2 cytokines (gray rectangles) are fused directly (df) or via an L6 linker (L6) to the N-terminus or C-terminus of either the heavy chain or the two κ light chains of the antibody.

Figures 2A, 2B, 2C, 2D, 2E, 2F, 2G, and 2H illustrate exemplary antibody-hIL-2 immunoconjugates having an hCD25(1-164) extracellular domain designed to interfere with the binding of hIL-2 to human IL-2Rα. For the N-terminal variant, the human CD25/IL-2Rα extracellular domain (black triangle) is fused to unattenuated hIL-2 cytokines (gray rectangles) via an L20 linker (light gray line). The unattenuated hIL-2 cytokines are then fused directly to the antibody (df) or via an L6 linker. For the C-terminal variant, the hCD25/IL-2Rα extracellular domain is partially fused directly to the antibody (df) or via an L6 linker, followed by an L20 linker and unattenuated hIL-2 cytokines.

Figure 3 illustrates an exemplary 1H3-hIgG1-L6-hIL-2 immunoconjugate containing the CD25/IL-2Rα extracellular domain portion. The hCD25/IL-2Rα extracellular domain portion is fused to 1H3-hIgG1-L6-hIL-2 at the C-terminus of each heavy chain via an L6 linker, followed by an L20 linker and a substituted hIL-2 cytokine portion (attenuated hIL-2) as described in Example 2, which is intended to regulate binding to CD122/IL-2Rβ.

Figures 4A, 4B, 4C, and 4D show the experimental results of analyzing the binding of anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 to human PD-1 receptors on Jurkat cells in the presence of saturated concentrations of anti-hPD-1#1-mIgG2b-N297A and anti-hPD-1#2-mIgG2b-N297A (10 μM) prior to exposure to anti-hPD-1 antibodies.

Figure 5 illustrates exemplary anti-hPD-1-attenuated hIL-2 immunoconjugates with an L6 linker (L6) (left) or direct fusion (df) (right). An anti-hPD-1 antibody containing an hIgG4 or hIgG1 Fc domain (with or without L235E (LE) or L235A/G237A (LAGA) modification in the Fc domain) is fused to an attenuated hIL-2 cytokine at the C-terminus of the antibody heavy chain. Various substitutions are introduced into the hIL-2 cytokine to enhance attenuation potency.

Figures 6A and 6B show the results of the competitive assay, demonstrating that anti-hPD-1#1-mIgG2b-N297A (Figure 6A) or anti-hPD-1#2-mIgG2b-N297A (Figure 6B) binds to the anti-hPD-1 receptor on Jurkat cells in the presence of a saturated concentration of the anti-hPD-1-attenuated hIL-2 immunoconjugate (280 nM).

Figure 7 shows the results of the competitive assay, demonstrating that the anti-hPD-1-attenuation hIL-2 immunoconjugates 2H7-hIgG4-df-hIL-2(D20A/R38E), C51E6-5-L6-hIgG4-hIL-2(D20A/R38E) and A2-hIgG4-df-hIL-2(D20A/R38E) do not inhibit the binding of human PD-L1 to the human PD-1 receptor when using the PD-1/PD-L1 blocking bioassay.

Figure 8 illustrates the results of analyzing the effects of administration of the mediator, alternative anti-PD-1 antibodies (anti-mPD-1RMP1-14 mIgG2b-N297A and anti-mPD-1RMP1-30 mIgG2b-N297A), and alternative anti-PD-1-attenuated hIL-2 immunoconjugates (anti-mPD-1RMP1-14 mIgG2b-N297A-L6-hIL-2(F42K/Y45R/V69R) or anti-mPD-1RMP1-30 mIgG2b-N297A-L6-hIL-2(F42K/Y45R/V69R)) on the growth of established subcutaneous MC38 homologous tumors in C57BL/6 mice, as described in Example 18. The test agents were administered intraperitoneally at a dose of 5 mg/kg twice weekly for 4 weeks, starting on day 1. The dots in the graph represent the average tumor volume of 10 mice in each group.

Figures 9A, 9B, and 9C present the results of a study conducted to determine the efficacy of the alternative anti-hPD-1-attenuated hIL-2 immunoconjugate, anti-mPD-1RMP1-30 mIgG2b-N297A-hIL-2 (F42K/Y45R/V69R), in an MC38 mouse colon adenocarcinoma model. Figure 9A depicts the mean subcutaneous tumor volume (mm³) measured every 3–4 days over 8 days following the first administration of the test agent (administered three times at 5 mg/kg on days 1, 4, and ⁸ ). Tumor growth curves represent an average of 15 animals per group. Figure 9B summarizes the results of tumor immunophenotyping by flow cytometry on day 9, showing the proportion of different CD8 ⁺ T cell subsets relative to the mean absolute count of total CD8 ⁺ T cells. Figure 9C shows the immunophenotyping results on day 9, demonstrating a significant amplification of CD8 ⁺ T effector memory and a trend toward a decrease in regulatory T cells (cells/μL) in the tumor after exposure to the alternative immunoconjugate.

Figure 10 illustrates the experimental results of accelerated graft-versus-host disease in NOD-Prkdc ^em26Cd52 IL-2rg ^em26Cd22 /NjuCrl (NCG) mice exposed to an anti-hPD-1-attenuated hIL-2 immunoconjugate, as evidenced by the significant weight loss in the NCG-PBMC model.

Figures 11A and 11B show the results of dose-dependent expansion of blood CD8+ effector memory T cells (Figure 11A) and CD4 ⁺ effector memory T cells (Figure 11B) in NCG-PBMC model NOD-Prkdc ^em26Cd52 IL-2rg ^em26Cd22 /NjuCrl (NCG) mice treated with 2H7-hIgG1 ^- LAGA-df-hIL-2 (T3A/D20A/R38E/C125A).

Figure 12 shows the reduction in blood regulatory T cells/mL in NOD-Prkdc ^em26Cd52 IL-2rg ^em26Cd22 /NjuCrl (NCG) mice treated with 2H7-hIgG1-LAGA-df-hIL-2(T3A/D20A/R38E/C125A) in the NCG-PBMC model.

Figures 13A and 13B show that, in the presence of saturated concentrations of anti-hPD-1#1-mIgG2b-N297A and anti-hPD-1#2-mIgG2b-N297A (10 μM) prior to exposure, H7-632–hIgG1-LAGA-df-hIL-2 (T3A/D20A/R38E/C125A) (named “H7-767”) (Figure 13B) continued to bind to the human PD-1 receptor on Jurkat cells.

Figures 14A and 14B are diagrams illustrating the binding of recombinant human PD-1 captured by H7-767 fixed to the CM5 sensor chip, as evaluated by surface plasmon resonance (SPR), followed by (Figure 14A) the binding of H7-767, or (Figure 14B) the binding of PD-L1 or PD-L2.

Figure 15 demonstrates that, using the hPD-1/hPD-L1 blocking bioassay, H7-632-hIgG1-LAGA and H7-767 do not inhibit the binding of human PD-L1 to the human PD-1 receptor.

Figures 16A, 16B, 16C, and 16D illustrate the binding of the anti-hPD-1-attenuated hIL-2 immunoconjugates 2H7-hIgG4-df-hIL-2(D20A/R38E), C51E6-5-hIgG4-df-hIL-2(D20A/R38E), and A2-hIgG4-df-hIL-2(D20A/R38E) to human PD-1 receptors on Jurkat cells in the presence of saturated concentrations of anti-hPD-1#1-mIgG2b-N297A and anti-hPD-1#2-mIgG2b-N297A (10 μM) prior to exposure to the anti-hPD-1-attenuated hIL-2 immunoconjugates, as assessed by flow cytometry.

Figure 17 illustrates the binding of 2H7-hIgG4-df-hIL-2(D20A/R38E), C51E6-5-hIgG4-df-hIL-2(D20A/R38E), A2-hIgG4-df-hIL-2(D20A/R38E), and the unrelated antibody control 1H3-hIgG4-df-hIL-2(D20A/R38E) to recombinant HEK-293T cells expressing cynomolgus monkey PD-1, as assessed by flow cytometry.

Figures 18A, 18B, 18C, and 18D show the antagonistic activities of the anti-hPD-1-attenuated hIL-2 immunoconjugates 2H7-hIgG4-df-hIL-2 (D20A/R38E), C51E6-5-hIgG4-df-hIL-2 (D20A/R38E), and A2-hIgG4-df-hIL-2 (D20A/R38E) in the presence of anti-hPD-1#1 or anti-hPD-1#2. Figures 18A and 18B show the titration results of anti-hPD-1#1 or anti-hPD-1#2 in the presence of a fixed concentration of the anti-hPD-1-attenuated hIL-2 immunoconjugates. Figures 18C and 18D show the results of opposite experiments on the titration of anti-hPD-1-attenuated hIL-2 immunoconjugates with fixed concentrations of 100 nM anti-hPD-1#1 (Figure 18C) or 100 nM anti-hPD-1#2 (Figure 18D).

Figure 19 illustrates the effects of various test agents (including alternative anti-mouse PD-1/attenuated IL-2 immunoconjugates) on the growth of established subcutaneous MC38 homologous tumors in C57BL/6 mice. Each growth curve represents the mean tumor volume of ten mice in each treatment group.

Figure 20 illustrates the ability of MC38 tumor cells to grow in tumor-free mice compared to the mice in the MC38 primary tumor study shown in Figure 19, which had previously been treated with anti-mPD-1-hIL-2F42K/Y45R/V69R and demonstrated complete long-term regression of the established primary tumors. Animals from two cohorts (n=10 per cohort) had 5 × ^10⁵ MC38 tumor cells subcutaneously implanted in the left ventral region contralateral to the primary tumor site. Mice previously exposed to the alternative anti-mPD-1-hIL-2F42K/Y45R/V69R did not show tumor growth because they had developed sustained immunity, while the corresponding naïve mouse controls showed typical tumor growth in their flanks.

Detailed Implementation

The disclosed modified hIL-2 protein, human antibody molecule or antigen-binding fragment thereof, and immunoconjugate can be more readily understood by referring to the following detailed description in conjunction with the accompanying drawings, which form a part of this disclosure. It should be understood that the disclosed modified hIL-2 protein, human antibody molecule or antigen-binding fragment thereof, and immunoconjugate are not limited to the specific modified hIL-2 protein, human antibody molecule or antigen-binding fragment thereof, and immunoconjugate described and/or shown herein, and the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to limit the claimed modified hIL-2 protein, human antibody molecule or antigen-binding fragment thereof, and immunoconjugate.

Unless otherwise specifically stated, any description of possible mechanisms of action or modes of action or reasons for improvement is intended to be illustrative only, and the disclosed modified hIL-2 protein, human antibody molecule or its antigen-binding fragment, and immunoconjugate are not subject to the correctness or incorrectness of any such recommended mechanisms of action or modes of action or reasons for improvement.

Throughout this document, descriptions relate to modified hIL-2 proteins, human antibody molecules or their antigen-binding fragments, and immunoconjugates, as well as methods using modified hIL-2 proteins, human antibody molecules or their antigen-binding fragments, and immunoconjugates. Where features or embodiments related to modified hIL-2 proteins, human antibody molecules or their antigen-binding fragments, and immunoconjugates are described or claimed in this disclosure, such features or embodiments are equally applicable to methods using modified hIL-2 proteins, human antibody molecules or their antigen-binding fragments, and immunoconjugates. Similarly, where features or embodiments related to methods using modified hIL-2 proteins, human antibody molecules or their antigen-binding fragments, and immunoconjugates are described or claimed in this disclosure, such features or embodiments are equally applicable to modified hIL-2 proteins, human antibody molecules or their antigen-binding fragments, and immunoconjugates.

Where numerical ranges are enumerated or established herein, a range includes its endpoints and all individual integers and fractions within the range, and also includes each of the various possible combinations of endpoints and internal integers and fractions of a subgroup forming a larger group of values within the range, to the same extent as each narrower range is explicitly enumerated. Where a numerical range is stated herein as being greater than the stated value, the range remains finite, and its upper limit is defined by a value operable within the context disclosed herein. Where a numerical range is stated herein as being less than the stated value, the lower limit of the range remains defined by a non-zero value. The ranges of modified hIL-2 proteins, human antibody molecules or their antigen-binding fragments, and immunoconjugates are not intended to be limited to the specific values enumerated when defining the range. All ranges are inclusive and composable.

Similarly, when a value is expressed as an approximation using the antecedent "about," it will be understood that the specific value forms another embodiment. Unless the context explicitly specifies otherwise, references to a specific numerical value include at least that specific value. The term "about" is used when referring to a range of numbers, a threshold, or a specific value to indicate that the listed value may differ from the stated value by up to 10%. Thus, the term "about" is used to cover variations of ±10% or less, ±5% or less, ±1% or less, ±0.5% or less, or ±0.1% or less from the specified value.

It should be understood that, for clarity, certain features of the modified hIL-2 protein, human antibody molecule or its antigen-binding fragment, and immunoconjugate disclosed herein, described in the context of individual embodiments, may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the modified hIL-2 protein, human antibody molecule or its antigen-binding fragment, and immunoconjugate disclosed herein, described in the context of a single embodiment, may also be provided individually or in any sub-combination.

As used herein, the singular forms “a”, “an”, and “the” include the plural.

Various terms are used throughout the specification and claims in connection with the described aspects. Unless otherwise stated, such terms have their ordinary meaning in the art. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein.

The term “comprising” is intended to include instances covered by the terms “substantially consisting of” and “consisting of”; similarly, the term “substantially consisting of” is intended to include instances covered by the term “consisting of”.

The term "antibody molecule" is used in a broad sense and includes both full-length immunoglobulin molecules and their antigen-binding fragments.

Immunoglobulins can be classified into five major classes based on the amino acid sequence of their heavy chain constant domain: IgA, IgD, IgE, IgG, and IgM. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. The antibody light chain of any vertebrate species can be designated as one of two distinct types (i.e., Kappa (κ) and Lambda (λ)) based on the amino acid sequence of its constant domain.

"Antigen-binding fragment" refers to the portion of an immunoglobulin molecule that retains the antigen-binding properties of the full-length parental antibody (i.e., "its antigen-binding fragment"). Exemplary antigen-binding fragments may have: heavy chain complementarity-determining regions (CDRs) 1, 2, and/or 3; light chain CDRs 1, 2, and/or 3; a heavy chain variable region (VH); a light chain variable region (VL); and combinations thereof. Antigen-binding fragments include: Fab fragments, which are monovalent fragments composed of VL, VH, constant light chain (CL), and constant heavy chain 1 (CH1) domains; F(ab) ₂ fragments, which are divalent fragments comprising two Fab fragments linked by disulfide bridges at the hinge region; Fd fragments composed of VH and CH1 domains; Fv fragments composed of VL and VH domains of a single arm of the antibody; and domain antibody (dAb) fragments composed of either a VH domain or a VL domain (Ward et al., Nature 341:544-546, 1989). VH and VL domains can be engineered and linked together via synthetic linkers to form various types of single-chain antibody designs, wherein when the VH and VL domains are expressed by separate single-chain antibody constructs, in these cases, the VH/VL domains pair intramolecularly or intermolecularly to form monovalent antigen-binding sites, such as single-chain Fv (scFv) or biantibodies, as described, for example, in International Patent Publications WO1998/44001, WO1988/01649, WO1994/13804, and WO1992/01047. These antibody fragments are obtained using techniques well known to those skilled in the art and are screened for practicality in the same manner as full-length antibodies.

The phrase "immunospecific binding" refers to the ability of a disclosed antibody molecule to preferentially bind to its target (hPD-1 in the case of anti-hPD-1 antibody molecules) rather than to other molecules in a sample containing a mixed molecular population. Immunospecific binding hPD-1 antibody molecules are substantially free of other antibodies with different antigen specificities (e.g., anti-hPD-1 antibodies are substantially free of antibodies that specifically bind to antigens other than hPD-1). However, immunospecific binding hPD-1 antibody molecules can exhibit cross-reactivity with other antigens (such as orthologs of hPD-1, including cynomolgus monkey (Macaca fascicularis) PD-1). The antibody molecules disclosed herein are capable of immunospecific binding to both naturally occurring hPD-1 and recombinant PD-1 produced in mammalian or prokaryotic cells.

The antibody variable region consists of four "framework" regions interrupted by three "antigen-binding sites". Antigen-binding sites are defined using various terms: (i) complementarity-determining regions (CDRs), three in the VH region (HCDR1, HCDR2, HCDR3) and three in the VL region (LCDR1, LCDR2, LCDR3), based on sequence variability (Wu and Kabat, *Journal of Experimental Medicine*, 132:211-50, 1970; Kabat et al., *Sequences of Proteins of Immunol*). (i) "Hypervariate region" ("HVR" or "HV"), three in VH (H1, H2, H3) and three in VL (L1, L2, L3), refers to the region of antibody variable region that is hypervariable in a structure as defined by Chothia and Lesk (Chothia and Lesk, Molecular Biology, 196:901-17, 1987). The AbM definition of CDR is also widely used; it is a compromise between the Kabat and Chothia numbering schemes, and is so named because it is used by Oxford Molecular's AbM antibody modeling software (Rees, A.R., Searle, S.M.J., Henry, A.H. and Pedersen, J.T. (1996) Sternberg M.J.E. (ed.), Protein Structure Prediction, Oxford University Press, 141–172). Other terms include "IMGT-CDR" (Lefranc et al., Developmental and Comparative Immunology 27:55-77, 2003) and "Specific Determining Residue Use" (SDRU) (Almagro, Molecular Recognition 17:132-43, 2004). The International Immunogenetics (IMGT) database (http://www.imgt.org) provides standardized numbers and definitions for antigen-binding sites. The correspondence between CDR, HV, and IMGT descriptions is described in Lefranc et al., Developmental and Comparative Immunology 27:55-77, 2003.

A "frame" or "frame sequence" is the remaining sequence of the variable region excluding the sequence defined as the antigen-binding site. Because antigen-binding sites can be defined using various terms as described above, the exact amino acid sequence of the framework depends on how the antigen-binding site is defined.

The terms "human antibody," "fully human antibody," and similar terms refer to antibodies with variable regions of both heavy and light chains, wherein both the framework and antigen-binding site are derived from human sequences. If the antibody contains a constant region, that constant region is also derived from a human sequence. Human antibodies contain variable regions of both heavy and/or light chains, and if the variable regions of the antibody are obtained from a system using human germline immunoglobulin or rearranged immunoglobulin genes, the variable regions are "derived" from human sequences. Such systems include phage-displayed human immunoglobulin gene libraries and transgenic nonhuman animals, such as mice or chickens, carrying human immunoglobulin loci as described herein. "Human antibodies" may contain amino acid differences compared to human germline or rearranged immunoglobulin sequences due to, for example, naturally occurring somatic mutations or intentional substitutions introduced into the variable domains (framework and antigen-binding sites) or constant domains. Typically, a “human antibody” is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical in amino acid sequence to the sequence encoded by human germline or rearranged immunoglobulin genes. In some cases, a “human antibody” may contain a common frame sequence derived from human frame sequence analysis, such as described by Knappik et al., *Journal of Molecular Biology* 296:57-86, 2000, or contain synthetic HCDR3 incorporated into a phage-displayed human immunoglobulin gene library, such as described by Shi et al., *Journal of Molecular Biology* 397:385-96, 2010, and International Patent Publication No. WO2009/085462. Antibodies whose antigen-binding sites originate from non-human species are not included in the definition of "human antibody".

Although human antibodies are derived from human immunoglobulin sequences, they can be generated using systems such as phage display that incorporates synthetic CDRs and/or synthetic frameworks, or they can be mutagenized in vitro to improve the antibody properties of variable or constant regions or both, thereby generating antibodies that are not naturally present in human antibody strains in vivo.

"Recombinant antibodies" include all antibodies prepared, expressed, generated, or isolated in a recombinant manner, such as: antibodies isolated from transgenic or transchromatic animals (e.g., mice) of the human immunoglobulin gene or hybridomas prepared therefrom (described further below); antibodies isolated from host cells converted to express antibodies; antibodies isolated from recombinant combined antibody libraries; and antibodies prepared, expressed, generated, or isolated by any other means involving splicing the human immunoglobulin gene sequence into other DNA sequences, or antibodies generated in vitro using Fab arm exchange.

"Monoclonal antibody" refers to a group of antibody molecules consisting of a single molecule. A monoclonal antibody composition exhibits single binding specificity and affinity for a specific epitope, or, in the case of a bispecific monoclonal antibody, dual binding specificity for two different epitopes. Therefore, a monoclonal antibody is a group of antibodies having a single amino acid composition in each heavy chain and each light chain, except for possible well-known alterations (such as the removal of a C-terminal lysine from the antibody heavy chain). Monoclonal antibodies may exhibit heterogeneous glycosylation within the antibody group. Monoclonal antibodies can be monospecific or multispecific, or monovalent, bivalent, or multivalent. The term monoclonal antibody includes bispecific antibodies.

An epitope is the part of an antigen that specifically binds to an antibody. Epitopes are typically composed of chemically active (e.g., polar, nonpolar, or hydrophobic) surface groups, such as amino acids or polysaccharide side chains, and can possess specific three-dimensional structural and charge properties. Epitopes can be composed of continuous and/or discontinuous amino acids forming conformational spatial units. For discontinuous epitopes, amino acids from different parts of the linear sequence of the antigen are very close in three-dimensional space through protein folding.

A “variant” is a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide through one or more modifications (e.g., substitution, insertion, or deletion). As used herein, the term “mutation” is intended to refer to one or more intentional substitutions of a polypeptide or polynucleotide.

"Treatment" and similar terms refer to therapeutic treatments and preventative or preventative measures, including reducing the severity and/or frequency of symptoms, eliminating symptoms and/or their underlying causes, reducing the frequency or likelihood of symptoms and/or their underlying causes, and improving or remedying harm caused directly or indirectly by the disease or condition. Treatment also includes prolonging survival compared to the expected survival of an untreated subject. Subjects to be treated include those who already have the disease or condition, those who are susceptible to the disease or condition, or those for whom the disease or condition is to be prevented.

As used herein, the terms “administer to a subject” and similar terms refer to a procedure of injecting the disclosed modified hIL-2 protein, immunoconjugate, or pharmaceutical composition into a subject so as to bring target cells, tissues, or fragments of the subject’s body into contact with the disclosed modified hIL-2 protein or an immunoconjugate containing therein.

The phrase “therapeutic effective amount” refers to the amount of modified hIL-2 protein, immunodrug conjugate, or pharmaceutical composition that effectively achieves a specific biological or therapeutic outcome (such as, but not limited to, the biological or therapeutic outcomes disclosed, described, or exemplified herein). Therapeutic effective amount can vary depending on factors such as an individual’s disease state, age, sex, and weight, as well as the ability of the modified hIL-2 protein, immunodrug conjugate, or pharmaceutical composition to elicit a desired response in a subject. Exemplary indicators of therapeutic effective amount include, for example, improvement in patient health, reduction of disease symptoms, cessation or slowing of disease symptom progression, and/or disappearance of disease symptoms.

As used herein, the term "subject" is intended to refer to any animal, particularly a mammal. Therefore, the methods described are applicable to both humans and non-human animals, but are most preferably used for humans. "Subject" and "patient" are used interchangeably herein.

Immunoconjugates and fusion proteins are used interchangeably in this article.

Modified human interleukin-2 (hIL-2) protein

This document discloses a modified hIL-2 protein, which, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, comprises a substitution at amino acid position 20 and a substitution at amino acid position 38, wherein, relative to the unmodified hIL-2, the modified hIL-2 protein exhibits reduced potency against both high-affinity and intermediate-affinity hIL-2 receptors. The disclosed modified hIL-2 protein is also referred to herein as “attenuated” hIL-2.

Suitable substitutions at amino acid position 20 include, for example, any one of the following substitutions: D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E.

Suitable substitutions at amino acid position 38 include, for example, any one of the following substitutions: R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D, or R38K.

In some embodiments, any one of the D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitutions may be combined with the R38E substitution.

The modified hIL-2 protein may contain the amino acid sequence of any one of SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620. The modified hIL-2 protein may contain the amino acid sequence of any one of SEQ ID NO: 134-150, 307, 344, 608, 611, 614, or 620. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 134. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 135. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 136. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 137. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 138. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 139. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 140. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 141. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 144. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 146. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 147. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 148. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 149. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 150. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 307. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 344. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 607. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 608. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 609. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 610. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 611. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 614. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 617. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 620. The modified hIL-2 protein with any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620 may further comprise a T3A substitution and/or a C125A substitution. In some embodiments, the modified hIL-2 protein with any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution. In some embodiments, the modified hIL-2 protein of any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a C125A substitution. In some embodiments, the modified hIL-2 protein of any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution and a C125A substitution.

Modified hIL-2 proteins can contain D20A substitution and R38E substitution.

As described herein, the term "reduced potency" and related terms such as "reduced potency" or "attenuation" of IL-2 activity refer to a reduction in the potency of modified hIL-2, as determined by an increase in the _EC50 value relative to the _EC50 value of unmodified hIL-2 in an IL-2-dependent assay. As described herein, modified hIL-2 will have reduced potency against high-affinity and intermediate-affinity IL-2 receptors. An IL-2-dependent assay for determining potency can be an engineered human erythroleukemia TF1 (TF1+IL-2Rβ) or human natural killer NK-92 cell proliferation assay as described herein. In one embodiment, the IL-2-dependent assay for determining potency is an engineered human erythroleukemia TF1 (TF1+IL-2Rβ) cell proliferation assay. In another embodiment, the IL-2-dependent assay for determining potency is a human natural killer NK-92 cell proliferation assay. Other IL-2-dependent assays for determining efficacy may include the TF1+IL-2Rβ or human natural killer NK-92pSTAT5 assays as described herein. Unmodified hIL-2 may be prokaryotically expressed hIL-2, such as (having the native human IL-2 amino acid sequence except for the C125S substitution used to remove unbound cysteine residues, and lacking normal human carbohydrate expression at residue T3), or unmodified hIL-2 may be hIL-2 having the amino acid sequence of SEQ ID NO:345 or hIL-2 having the amino acid sequence of SEQ ID NO:345 and a C125S substitution, expressed in mammalian cell lines such as CHO or HEK cell lines.

Compared to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, the modified hIL-2 protein may further include a substitution at amino acid position 3. Suitable substitutions include, for example, T3A. In some embodiments, the modified hIL-2 protein includes a T3A substitution, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:216.

Alternatively, the modified hIL-2 protein may further comprise a deletion at amino acid position 3, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345. In some embodiments, the modified hIL-2 protein comprises a deletion of amino acids 1-3, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:218.

Compared to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, the modified hIL-2 protein may further include a deletion or substitution at amino acid position 125. The substitution at amino acid position 125 may be C125A. In some embodiments, the modified hIL-2 protein includes a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein includes the amino acid sequence of SEQ ID NO:215. In some embodiments, the modified hIL-2 protein includes a T3A substitution, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein includes the amino acid sequence of SEQ ID NO:217. In some embodiments, the modified hIL-2 protein includes a deletion of amino acids 1-3, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein includes the amino acid sequence of SEQ ID NO:219.

Compared to unmodified hIL-2, modified hIL-2 protein can exhibit a reduction in potency to the high-affinity IL-2 receptor (hIL-2Rαβγ) of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold, for example, as quantified by comparing _EC50 values in the hIL-2-dependent cell proliferation assays described herein. In some embodiments, modified hIL-2 protein can exhibit a reduction in potency to the high-affinity IL-2 receptor (hIL-2Rαβγ) of unmodified hIL-2 of more than about 10,000-fold. For the modified hIL-2 protein described herein, a greater reduction in the potency of hIL-2 to the high-affinity hIL-2 receptor is possible and acceptable, but such reduction may not be quantifiable using the methods described herein due to limitations in cell proliferation assay conditions.

Furthermore, compared to unmodified hIL-2, modified hIL-2 protein can exhibit a reduction in potency of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold against the intermediate-affinity IL-2 receptor (hIL-2Rβγ), for example, as quantified by comparing _EC50 values in the hIL-2-dependent cell proliferation assay described herein. In some embodiments, modified hIL-2 protein can exhibit a reduction in potency of more than about 10,000-fold against the intermediate-affinity IL-2 receptor (hIL-2Rβγ) compared to unmodified hIL-2.

Compared to unmodified hIL-2, modified hIL-2 protein can exhibit up to approximately 10,000-fold reduced potency against high-affinity IL-2 receptors (hIL-2Rαβγ) and up to approximately 10,000-fold reduced potency against intermediate-affinity IL-2 receptors (hIL-2Rβγ), as quantified, for example, by comparing _EC50 values in the hIL-2-dependent cell proliferation assay described herein. Compared to unmodified hIL-2, modified hIL-2 protein can exhibit more than 10,000-fold reduced potency against high-affinity IL-2 receptors (hIL-2Rαβγ) and more than 10,000-fold reduced potency against intermediate-affinity IL-2 receptors (hIL-2Rβγ).

As demonstrated herein, the modified hIL-2 protein can fuse with an anti-PD-1 antibody or its antigen-binding fragment. The hIL-2 protein can fuse with the anti-PD-1 antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment. In some embodiments, the modified hIL-2 protein fuses directly with the anti-PD-1 antibody or its antigen-binding fragment via a peptide bond. The modified hIL-2 protein can, for example, fuse directly with the C-terminal amino acid residue of the anti-PD-1 antibody heavy chain via a peptide bond. In some embodiments, the modified hIL-2 protein fuses with the anti-PD-1 antibody or its antigen-binding fragment via a linker.

The fusion of modified hIL-2 protein with an antibody or its antigen-binding fragment can rescue the ability of the modified hIL-2 protein to bind to and activate the human intermediate-affinity IL-2 receptor on PD-1 expressing cells (such as T cells, and particularly tumor-infiltrating lymphocytes). In some embodiments, the hIL-2 protein fused with an antibody or its antigen-binding fragment exhibits potency against the intermediate-affinity IL-2 receptor on PD-1 expressing cells comparable to that of wild-type hIL-2 against the intermediate-affinity IL-2 receptor.

The fusion of modified hIL-2 protein with an antibody or its antigen-binding fragment can be used to selectively deliver IL-2 signaling to PD-1 target cells expressing the antibody or its antigen-binding fragment. Unbound by theory, it is believed that targeting specific cell populations with modified hIL-2 protein can significantly enhance the therapeutic effects of IL-2 (e.g., anti-tumor immunity) without off-target systemic toxicity.

This article also discloses a modified human interleukin-2 (hIL-2) protein, which, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, includes a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution at amino acid position 20 and an R38E substitution at amino acid position 38.

The modified hIL-2 protein may contain the amino acid sequence of any one of SEQ ID NO: 307, 607-611, 614, 617, or 620. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 307. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 607. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 608. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 609. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 610. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 611. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 614. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 617. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 620. The modified hIL-2 protein with any of the amino acid sequences SEQ ID NO: 307, 607-611, 614, 617, or 620 may further comprise a T3A substitution and/or a C125A substitution. In some embodiments, the modified hIL-2 protein with any of the amino acid sequences SEQ ID NO: 307, 607-611, 614, 617, or 620 further comprises a T3A substitution. In some embodiments, the modified hIL-2 protein with any of the amino acid sequences SEQ ID NO: 307, 607-611, 614, 617, or 620 further comprises a C125A substitution. In some embodiments, the modified hIL-2 protein of any of the amino acid sequences SEQ ID NO: 307, 607-611, 614, 617 or 620 further comprises a T3A substitution and a C125A substitution.

The modified hIL-2 protein may contain D20A and R38E substitutions. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO:149.

Compared to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, the modified hIL-2 protein may further include a substitution at amino acid position 3. Suitable substitutions include, for example, T3A. In some embodiments, the modified hIL-2 protein includes a T3A substitution, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:216.

Alternatively, the modified hIL-2 protein may further comprise a deletion at amino acid position 3, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345. In some embodiments, the modified hIL-2 protein comprises a deletion of amino acids 1-3, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:218.

Compared to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, the modified hIL-2 protein may further include a deletion or substitution at amino acid position 125. The substitution at amino acid position 125 may be C125A. In some embodiments, the modified hIL-2 protein includes a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein includes the amino acid sequence of SEQ ID NO:215. In some embodiments, the modified hIL-2 protein includes a T3A substitution, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein includes the amino acid sequence of SEQ ID NO:217. In some embodiments, the modified hIL-2 protein includes a deletion of amino acids 1-3, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein includes the amino acid sequence of SEQ ID NO:219.

Compared to unmodified hIL-2, modified hIL-2 protein can exhibit a reduction in potency to the high-affinity IL-2 receptor (hIL-2Rαβγ) of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold, for example, as quantified by comparing _EC50 values in the hIL-2-dependent cell proliferation assays described herein. In some embodiments, modified hIL-2 protein can exhibit a reduction in potency to the high-affinity IL-2 receptor (hIL-2Rαβγ) of unmodified hIL-2 of more than about 10,000-fold. For the modified hIL-2 protein described herein, a greater reduction in the potency of hIL-2 to the high-affinity hIL-2 receptor is possible and acceptable, but such reduction may not be quantifiable using the methods described herein due to limitations in cell proliferation assay conditions.

Furthermore, compared to unmodified hIL-2, modified hIL-2 protein can exhibit a reduction in potency of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold against the intermediate-affinity IL-2 receptor (hIL-2Rβγ), for example, as quantified by comparing _EC50 values in the hIL-2-dependent cell proliferation assay described herein. In some embodiments, modified hIL-2 protein can exhibit a reduction in potency of more than about 10,000-fold against the intermediate-affinity IL-2 receptor (hIL-2Rβγ) compared to unmodified hIL-2.

Compared to unmodified hIL-2, modified hIL-2 protein can exhibit up to approximately 10,000-fold reduced potency against high-affinity IL-2 receptors (hIL-2Rαβγ) and up to approximately 10,000-fold reduced potency against intermediate-affinity IL-2 receptors (hIL-2Rβγ), as quantified, for example, by comparing _EC50 values in the hIL-2-dependent cell proliferation assay described herein. Compared to unmodified hIL-2, modified hIL-2 protein can exhibit more than 10,000-fold reduced potency against high-affinity IL-2 receptors (hIL-2Rαβγ) and more than 10,000-fold reduced potency against intermediate-affinity IL-2 receptors (hIL-2Rβγ).

As demonstrated herein, the modified hIL-2 protein can fuse with an anti-PD-1 antibody or its antigen-binding fragment. The hIL-2 protein can fuse with the anti-PD-1 antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment. In some embodiments, the modified hIL-2 protein fuses directly with the anti-PD-1 antibody or its antigen-binding fragment via a peptide bond. The modified hIL-2 protein can, for example, fuse directly with the C-terminal amino acid residue of the anti-PD-1 antibody heavy chain via a peptide bond. In some embodiments, the modified hIL-2 protein fuses with the anti-PD-1 antibody or its antigen-binding fragment via a linker.

The fusion of modified hIL-2 protein with an antibody or its antigen-binding fragment can rescue the ability of the modified hIL-2 protein to bind to and activate the human intermediate-affinity IL-2 receptor on PD-1 expressing cells (such as T cells, and particularly tumor-infiltrating lymphocytes). In some embodiments, the hIL-2 protein fused with an antibody or its antigen-binding fragment exhibits potency against the intermediate-affinity IL-2 receptor on PD-1 expressing cells comparable to that of wild-type hIL-2 against the intermediate-affinity IL-2 receptor.

The fusion of modified hIL-2 protein with an antibody or its antigen-binding fragment can be used to selectively deliver IL-2 signaling to PD-1 target cells expressing the antibody or its antigen-binding fragment. Unbound by theory, it is believed that targeting specific cell populations with modified hIL-2 protein can significantly enhance the therapeutic effects of IL-2 (e.g., anti-tumor immunity) without off-target systemic toxicity.

Human anti-human programmed cell death protein-1 (hPD-1) antibody

This document discloses a human antibody molecule or its antigen-binding fragment thereof, which specifically binds to hPD-1 immune response, wherein the human antibody molecule or its antigen-binding fragment comprises:

a) Heavy chain complementarity-determining region 1 (CDR1) containing the amino acid sequence of SEQ ID NO:418, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:419, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:420, light chain CDR1 containing the amino acid sequence of SEQ ID NO:421, light chain CDR2 containing the amino acid sequence of SEQ ID NO:422, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:423;

b) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:388, light chain CDR1 containing the amino acid sequence of SEQ ID NO:389, light chain CDR2 containing the amino acid sequence of SEQ ID NO:390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:391;

c) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:398, light chain CDR1 containing the amino acid sequence of SEQ ID NO:399, light chain CDR2 containing the amino acid sequence of SEQ ID NO:400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:401; or

d) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:406, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:407, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:408, light chain CDR1 containing the amino acid sequence of SEQ ID NO:409, light chain CDR2 containing the amino acid sequence of SEQ ID NO:410, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:411.

In some embodiments, a human antibody molecule or its antigen-binding fragment comprises: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:418, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:419, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:420, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:421, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:422, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:423 (referred to herein as "H7-632").

In some embodiments, a human antibody molecule or its antigen-binding fragment comprises: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:386, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:387, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:388, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:389, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:390, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:391 (referred to herein as "2H7").

In some embodiments, a human antibody molecule or its antigen-binding fragment comprises: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:396, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:397, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:398, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:399, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:400, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:401 (referred to herein as "C51E6-5").

In some embodiments, a human antibody molecule or its antigen-binding fragment comprises: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:406, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:407, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:408, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:409, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:410, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:411 (referred to herein as “A2”).

The disclosed human antibody molecules or their antigen-binding fragments may exhibit one or more of the following activities:

● It binds to PD-1 without inhibiting the binding of PD-L1 to PD-1;

● Binds to PD-1 in the presence of standard care anti-PD-1 antibodies (e.g., and) used in clinical practice;

● It exhibits high selectivity for PD-1 and does not specifically bind to other related B7 family members; and

● Binds to PD-1 on activated human T cells ( _EC50 in flow cytometry binding assay is ~0.1-0.2 nM).

Human antibody molecules or their antigen-binding fragments may include: a heavy chain variable region containing the amino acid sequence of SEQ ID NO:416 and a light chain variable region containing the amino acid sequence of SEQ ID NO:417 (referred to herein as "H7-632").

Human antibody molecules or their antigen-binding fragments may include: a heavy chain variable region containing the amino acid sequence of SEQ ID NO:384 and a light chain variable region containing the amino acid sequence of SEQ ID NO:385 (referred to herein as "2H7").

Human antibody molecules or their antigen-binding fragments may include: a heavy chain variable region containing the amino acid sequence of SEQ ID NO:394 and a light chain variable region containing the amino acid sequence of SEQ ID NO:395 (referred to herein as "C51E6-5").

Human antibody molecules or their antigen-binding fragments may include: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:404 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:405 (referred to herein as "A2").

Human antibody molecules or their antigen-binding fragments may contain the constant region of the human IgG1 heavy chain.

Human antibody molecules or their antigen-binding fragments may have substitutions or deletions in constant regions to minimize Fc-mediated immune effector functions, such as FcγRIIIA-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), FcγRI and FcγRIIa-dependent antibody-dependent phagocytosis (ADCP), and C1q binding-mediated complement-dependent cytotoxicity (CDC). In some embodiments, the human antibody molecule contains an L235A substitution, wherein the amino acid numbers are based on EU numbers. In some embodiments, the human antibody molecule contains a G237A substitution, wherein the amino acid numbers are based on EU numbers. In some embodiments, the human antibody molecule contains both L235A and G237A substitutions, wherein the amino acid numbers are based on EU numbers.

Human antibody molecules or their antigen-binding fragments may comprise: a heavy chain containing the amino acid sequence of SEQ ID NO:414 and a light chain containing the amino acid sequence of SEQ ID NO:415 (referred to herein as "H7-632-hIgG1-LAGA").

Human antibody molecules or their antigen-binding fragments may comprise: a heavy chain containing the amino acid sequence of SEQ ID NO:424 and a light chain containing the amino acid sequence of SEQ ID NO:425 (referred to herein as "2H7-hIgG4").

Human antibody molecules or their antigen-binding fragments may comprise: a heavy chain containing the amino acid sequence of SEQ ID NO:426 and a light chain containing the amino acid sequence of SEQ ID NO:427 (referred to herein as "C51E6-5-hIgG4").

Human antibody molecules or their antigen-binding fragments may comprise: a heavy chain containing the amino acid sequence of SEQ ID NO:428 and a light chain containing the amino acid sequence of SEQ ID NO:429 (referred to herein as "A2-hIgG4").

Human antibody molecules or their antigen-binding fragments can be fused to a modified hIL-2 protein, wherein the modified hIL-2 protein comprises a substitution at amino acid position 20 and a substitution at amino acid position 38, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345. Human antibody molecules or their antigen-binding fragments can be fused to any of the modified hIL-2 proteins disclosed herein.

When not fused with antibody molecules or their antigen-binding fragments, modified hIL-2 proteins can exhibit reduced potency against both high-affinity and medium-affinity hIL-2 receptors compared to unmodified hIL-2.

Suitable substitutions at amino acid position 20 of the modified hIL-2 include, for example, any one of the following substitutions: D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E.

Suitable substitutions at amino acid position 38 of the modified hIL-2 protein include, for example, any one of the following substitutions: R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D, or R38K.

In some embodiments, any one of the D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitutions may be combined with the R38E substitution.

The modified hIL-2 protein may contain the amino acid sequence of any one of SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620. The modified hIL-2 protein may contain the amino acid sequence of any one of SEQ ID NO: 134-150, 307, 344, 608, 611, 614, or 620. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 134. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 135. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 136. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 137. In some embodiments, the modified hIL-2 protein contains the amino acid sequence of SEQ ID NO: 138. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 139. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 140. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 141. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 144. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 146. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 147. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 148. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 149. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 150. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 307. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 344. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 607. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 608. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 609. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 610. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 611. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 614. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 617. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 620. The modified hIL-2 protein with any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620 may further comprise a T3A substitution and/or a C125A substitution. In some embodiments, the modified hIL-2 protein with any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution. In some embodiments, the modified hIL-2 protein of any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a C125A substitution. In some embodiments, the modified hIL-2 protein of any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution and a C125A substitution.

Modified hIL-2 proteins can contain D20A substitution and R38E substitution.

Compared to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, the modified hIL-2 protein may further include a substitution at amino acid position 3. Suitable substitutions include, for example, T3A. In some embodiments, the modified hIL-2 protein includes a T3A substitution, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:216.

Alternatively, the modified hIL-2 protein may further comprise a deletion at amino acid position 3, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345. In some embodiments, the modified hIL-2 protein comprises a deletion of amino acids 1-3, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:218.

Compared to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, the modified hIL-2 protein may further include a deletion or substitution at amino acid position 125. The substitution at amino acid position 125 may be C125A. In some embodiments, the modified hIL-2 protein includes a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein includes the amino acid sequence of SEQ ID NO:215. In some embodiments, the modified hIL-2 protein includes a T3A substitution, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein includes the amino acid sequence of SEQ ID NO:217. In some embodiments, the modified hIL-2 protein includes a deletion of amino acids 1-3, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein includes the amino acid sequence of SEQ ID NO:219.

When not fused with a human antibody molecule or its antigen-binding fragment, the modified hIL-2 protein can exhibit a reduction in potency against the high-affinity IL-2 receptor (hIL-2Rαβγ) of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold, relative to unmodified hIL-2, for example, as quantified by comparing _EC50 values in an hIL-2-dependent cell proliferation assay as described herein. In some embodiments, when not fused with a human antibody molecule or its antigen-binding fragment, the modified hIL-2 protein can exhibit a reduction in potency against the high-affinity IL-2 receptor (hIL-2Rαβγ) of at least about 10,000-fold, relative to unmodified hIL-2. For the modified hIL-2 protein described herein, a greater reduction in the potency of hIL-2 to the high-affinity hIL-2 receptor is possible and acceptable, but such a reduction may not be quantifiable using the methods described herein due to limitations in cell proliferation assay conditions.

Furthermore, when not fused with a human antibody molecule or its antigen-binding fragment, the modified hIL-2 protein can exhibit a reduction in potency against the intermediate-affinity IL-2 receptor (hIL-2Rβγ) of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold relative to unmodified hIL-2, for example, as quantified by comparing _EC50 values in the hIL-2-dependent cell proliferation assay described herein. In some embodiments, when not fused with a human antibody molecule or its antigen-binding fragment, the modified hIL-2 protein can exhibit a reduction in potency against the intermediate-affinity IL-2 receptor (hIL-2Rβγ) of more than about 10,000-fold relative to unmodified hIL-2.

When not fused with a human antibody molecule or its antigen-binding fragment, modified hIL-2 protein can exhibit up to approximately 10,000-fold reduced potency against high-affinity IL-2 receptors (hIL-2Rαβγ) and up to approximately 10,000-fold reduced potency against intermediate-affinity IL-2 receptors (hIL-2Rβγ) compared to unmodified hIL-2, as quantified, for example, by comparing _EC50 values in the hIL-2-dependent cell proliferation assay described herein. When not fused with a human antibody molecule or its antigen-binding fragment, modified hIL-2 protein can exhibit more than 10,000-fold reduced potency against high-affinity IL-2 receptors (hIL-2Rαβγ) and more than 10,000-fold reduced potency against intermediate-affinity IL-2 receptors (hIL-2Rβγ) compared to unmodified hIL-2.

The fusion of modified hIL-2 protein with an antibody or its antigen-binding fragment can rescue the ability of the modified hIL-2 protein to bind to and activate the human intermediate-affinity IL-2 receptor on PD-1 expressing cells (such as T cells, and particularly tumor-infiltrating lymphocytes). In some embodiments, the hIL-2 protein fused with an antibody or its antigen-binding fragment exhibits potency against the intermediate-affinity IL-2 receptor on PD-1 expressing cells comparable to that of wild-type hIL-2 against the intermediate-affinity IL-2 receptor.

The modified hIL-2 protein can be fused to a human antibody molecule or its antigen-binding fragment at the N-terminus, C-terminus, N-terminus, C-terminus of the antibody light chain, N-terminus of the antibody heavy chain, or C-terminus of the antigen-binding fragment. In some embodiments, the hIL-2 protein is fused directly to the antibody or its antigen-binding fragment via a peptide bond. hIL-2 can, for example, be fused directly to the C-terminal amino acid residues of the antibody heavy chain via a peptide bond. In some embodiments, the hIL-2 protein is fused to the antibody or its antigen-binding fragment via a linker.

The fusion of human antibody molecules or their antigen-binding fragments with modified hIL-2 protein can be used to selectively deliver IL-2 signaling to PD-1-expressing cells. Beyond theoretical considerations, it is believed that targeting specific cell populations expressing PD-1 with modified hIL-2 protein can significantly enhance the therapeutic effects of IL-2 (e.g., anti-tumor immunity) while reducing or minimizing off-target systemic toxicity.

Immunoconjugates

This document discloses immunoconjugates comprising any modified hIL-2 protein disclosed herein and any human antibody molecule or antigen-binding fragment thereof disclosed herein. Immunoconjugates may comprise:

(a) A modified hIL-2 protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, comprising a substitution at amino acid position 20 and a substitution at amino acid position 38; and

(b) a human antibody molecule or an antigen-binding fragment thereof, said human antibody molecule or antigen-binding fragment thereof binding specifically to hPD-1, wherein said human antibody molecule or antigen-binding fragment thereof comprises:

(i) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:418, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:419, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:420, light chain CDR1 containing the amino acid sequence of SEQ ID NO:421, light chain CDR2 containing the amino acid sequence of SEQ ID NO:422, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:423;

(ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:388, light chain CDR1 containing the amino acid sequence of SEQ ID NO:389, light chain CDR2 containing the amino acid sequence of SEQ ID NO:390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:391;

(iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:398, light chain CDR1 containing the amino acid sequence of SEQ ID NO:399, light chain CDR2 containing the amino acid sequence of SEQ ID NO:400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:401; or

(iv) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:406, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:407, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:408, light chain CDR1 containing the amino acid sequence of SEQ ID NO:409, light chain CDR2 containing the amino acid sequence of SEQ ID NO:410, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:411.

Suitable substitutions at amino acid position 20 of the modified hIL-2 moiety of the immunoconjugate include any substitutions such as D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E.

Suitable substitutions at amino acid position 38 of the modified hIL-2 moiety of the immunoconjugate include, for example, any substitutions of R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D, or R38K.

In some embodiments, any one of the D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T, or D20E substitutions may be combined with the R38E substitution.

The modified hIL-2 protein portion of the immunoconjugate may comprise the amino acid sequence of any one of SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620. The modified hIL-2 protein portion of the immunoconjugate may comprise the amino acid sequence of any one of SEQ ID NO: 134-150, 307, 344, 608, 611, 614, or 620. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 134. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 136. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 137. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 138. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 139. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 140. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 141. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 144. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 146. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 147. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 148. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 149. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 150. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 307. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 344. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 607. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 608. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 609. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 610. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 611. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 614. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 617. In some embodiments, the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 620. The modified hIL-2 protein with any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620 may further comprise a T3A substitution and/or a C125A substitution. In some embodiments, the modified hIL-2 protein with any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution. In some embodiments, the modified hIL-2 protein of any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a C125A substitution. In some embodiments, the modified hIL-2 protein of any of the amino acid sequences SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617, or 620 further comprises a T3A substitution and a C125A substitution.

The modified hIL-2 protein moiety of the immunoconjugate may contain D20A substitution and R38E substitution.

Compared to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, the modified hIL-2 protein moiety of the immunoconjugate may further include a substitution at amino acid position 3. Suitable substitutions include, for example, T3A. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate includes a T3A substitution, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein moiety of the immunoconjugate comprises the amino acid sequence of SEQ ID NO:216.

Alternatively, the modified hIL-2 protein moiety of the immunoconjugate may further include a deletion at amino acid position 3, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate includes a deletion of amino acids 1-3, a D20A substitution, and an R38E substitution. In some aspects, the modified hIL-2 protein moiety of the immunoconjugate includes the amino acid sequence of SEQ ID NO:218.

Compared to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, the modified hIL-2 protein moiety of the immunoconjugate may further include a deletion or substitution at amino acid position 125. The substitution at amino acid position 125 may be C125A. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate includes a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate includes the amino acid sequence of SEQ ID NO:215. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate includes a T3A substitution, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate includes the amino acid sequence of SEQ ID NO:217. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate includes a deletion of amino acids 1-3, a D20A substitution, an R38E substitution, and a C125A substitution. In some embodiments, the modified hIL-2 protein portion of the immunoconjugate comprises the amino acid sequence of SEQ ID NO:219.

Compared to unmodified hIL-2, the modified hIL-2 protein moiety of the immunoconjugate can exhibit a reduction in potency against the high-affinity IL-2 receptor (hIL-2Rαβγ) of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold, for example, as quantified by comparing EC50 values in the hIL-2-dependent cell proliferation assays described herein. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate can exhibit a reduction in potency against the high-affinity IL-2 receptor (hIL-2Rαβγ) of more than about _10,000 -fold compared to unmodified hIL-2. For the modified hIL-2 protein described herein, a greater reduction in the potency of hIL-2 against the high-affinity hIL-2 receptor is possible and acceptable, but such reduction may not be quantifiable using the methods described herein due to limitations in cell proliferation assay conditions.

Furthermore, the modified hIL-2 protein moiety of the immunoconjugate can exhibit a reduction in potency against the intermediate-affinity IL-2 receptor (hIL-2Rβγ) of at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold, for example, as quantified by comparing _EC50 values in the hIL-2-dependent cell proliferation assay described herein. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate can exhibit a reduction in potency against the intermediate-affinity IL-2 receptor (hIL-2Rβγ) of more than about 10,000-fold, relative to the unmodified hIL-2.

Compared to unmodified hIL-2, the modified hIL-2 protein moiety of the immunoconjugate can exhibit up to approximately 10,000-fold reduced potency against high-affinity IL-2 receptors (hIL-2Rαβγ) and up to approximately 10,000-fold reduced potency against intermediate-affinity IL-2 receptors (hIL-2Rβγ), as quantified, for example, by comparing _EC50 values in the hIL-2-dependent cell proliferation assay described herein. Compared to unmodified hIL-2, the modified hIL-2 protein moiety of the immunoconjugate can exhibit up to approximately 10,000-fold or more reduced potency against high-affinity IL-2 receptors (hIL-2Rαβγ) and up to approximately 10,000-fold or more reduced potency against intermediate-affinity IL-2 receptors (hIL-2Rβγ).

The hIL-2 protein moiety of the immunoconjugate can be fused to an antibody or its antigen-binding fragment at the N-terminus, C-terminus, N-terminus, C-terminus of the antibody light chain, N-terminus of the antibody heavy chain, or C-terminus of the antigen-binding fragment. In some embodiments, the hIL-2 protein moiety of the immunoconjugate is fused directly to a human antibody molecule or its antigen-binding fragment via a peptide bond. The hIL-2 protein moiety of the immunoconjugate can also be fused directly to a C-terminal amino acid residue of the antibody heavy chain, for example, via a peptide bond. In some embodiments, the hIL-2 protein moiety of the immunoconjugate is fused to a human antibody molecule or its antigen-binding fragment via a linker.

The fusion of modified hIL-2 protein with a human antibody molecule or its antigen-binding fragment can rescue the ability of the modified hIL-2 protein to activate intermediate-affinity IL-2 receptors. In some embodiments, the immunoconjugate can activate intermediate-affinity IL-2 receptors to a degree comparable to wild-type hIL-2 activation of intermediate-affinity IL-2 receptors.

In some embodiments, the human antibody molecule of the immunoconjugate or its antigen-binding fragment portion comprises: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:418, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:419, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:420, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:421, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:422, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:423.

In some embodiments, the human antibody molecule of the immunoconjugate or its antigen-binding fragment portion comprises: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:386, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:387, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:388, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:389, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:390, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:391.

In some embodiments, the human antibody molecule of the immunoconjugate or its antigen-binding fragment portion comprises: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:396, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:397, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:398, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:399, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:400, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:401.

In some embodiments, the human antibody molecule of the immunoconjugate or its antigen-binding fragment portion comprises: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:406, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO:407, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:408, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:409, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:410, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:411.

The human antibody molecule or its antigen-binding fragment portion of the immunoconjugate may include: a heavy chain variable region containing the amino acid sequence of SEQ ID NO:416 and a light chain variable region containing the amino acid sequence of SEQ ID NO:417.

The human antibody molecule of the immunoconjugate or its antigen-binding fragment may include: a heavy chain variable region containing the amino acid sequence of SEQ ID NO:384 and a light chain variable region containing the amino acid sequence of SEQ ID NO:385.

The human antibody molecule or its antigen-binding fragment portion of the immunoconjugate may include: a heavy chain variable region containing the amino acid sequence of SEQ ID NO:394 and a light chain variable region containing the amino acid sequence of SEQ ID NO:395.

The human antibody molecule or its antigen-binding fragment portion of the immunoconjugate may include: a heavy chain variable region containing the amino acid sequence of SEQ ID NO:404 and a light chain variable region containing the amino acid sequence of SEQ ID NO:405.

The human antibody molecule or its antigen-binding fragment portion of the immunoconjugate may contain the constant region of the IgG1 heavy chain.

The human antibody molecule of the immunoconjugate or its antigen-binding fragment may have substitutions or deletions in a constant region to minimize Fc-mediated immune effector functions, such as FcγRIIIA-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), FcγRI and FcγRIIa-dependent antibody-dependent phagocytosis (ADCP), and C1q binding-mediated complement-dependent cytotoxicity (CDC). In some embodiments, the human antibody molecule portion of the immunoconjugate contains an L235A substitution, wherein the amino acid numbering is according to EU numbers. In some embodiments, the human antibody molecule portion of the immunoconjugate contains a G237A substitution, wherein the amino acid numbering is according to EU numbers. In some embodiments, the human antibody molecule portion of the immunoconjugate contains both L235A and G237A substitutions, wherein the amino acid numbering is according to EU numbers.

The human antibody molecule or its antigen-binding fragment portion of the immunoconjugate may comprise: a heavy chain containing the amino acid sequence of SEQ ID NO:414 and a light chain containing the amino acid sequence of SEQ ID NO:415.

The human antibody molecule or its antigen-binding fragment portion of the immunoconjugate may comprise: a heavy chain containing the amino acid sequence of SEQ ID NO:424 and a light chain containing the amino acid sequence of SEQ ID NO:425.

The human antibody molecule or its antigen-binding fragment portion of the immunoconjugate may comprise: a heavy chain containing the amino acid sequence of SEQ ID NO:426 and a light chain containing the amino acid sequence of SEQ ID NO:427.

The human antibody molecule or its antigen-binding fragment portion of the immunoconjugate may comprise: a heavy chain containing the amino acid sequence of SEQ ID NO:428 and a light chain containing the amino acid sequence of SEQ ID NO:429.

Immunoconjugates may have one or more of the following properties:

● It binds to PD-1, but does not inhibit the binding of PD-L1 to PD-1;

● Binds to PD-1 in the presence of standard care anti-PD-1 antibodies (e.g., and) used in clinical practice;

● It exhibits high selectivity for PD-1 and does not specifically bind to other related B7 family members;

● Binds to PD-1 on activated human T cells ( _EC50 in flow cytometry binding assay is ~0.1-0.2 nM);

● The potency of modified hIL-2 against the high-affinity IL-2 receptor (hIL-2Rαβγ) is reduced by at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold, relative to unmodified hIL-2, for example, as quantified by comparing _EC50 values in the hIL-2-dependent cell proliferation assay described herein. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate may exhibit a potency reduction of more than about 10,000-fold against the high-affinity IL-2 receptor (hIL-2Rαβγ) relative to unmodified hIL-2. For the modified hIL-2 protein described herein, a greater reduction in the potency of hIL-2 against the high-affinity hIL-2 receptor is possible and acceptable, but such reduction may not be quantifiable using the methods described herein due to limitations in cell proliferation assay conditions.

● The potency against the intermediate-affinity IL-2 receptor (hIL-2Rβγ) is reduced by at least about 200-fold, at least about 500-fold, at least about 1,000-fold, at least about 2,000-fold, at least about 5,000-fold, at least about 6,500-fold, or at least about 10,000-fold relative to unmodified hIL-2, for example, as quantified by comparing _EC50 values in an hIL-2-dependent cell proliferation assay as described herein. In some embodiments, the modified hIL-2 protein moiety of the immunoconjugate may exhibit a potency reduction of more than about 10,000-fold against the intermediate-affinity IL-2 receptor (hIL-2Rβγ) relative to unmodified hIL-2;

● Rescue and expansion of PD-1-expressing human memory T cell subsets in a GvHD animal model; and

● After a single dose of 1 and 10 mg/kg to cynomolgus monkeys, there were minimal or no effects on body weight, blood chemistry, or hematological parameters.

In some embodiments, the immunoconjugate comprises a modified hIL-2 protein comprising T3A, R38E, D20A, and C125A substitutions, fused to the C-terminus of a heavy chain of a human anti-hPD-1 antibody, the antibody comprising a human IgG1 framework having L235A and G237A substitutions. In some embodiments, the immunoconjugate comprises a fusion of a light chain containing the amino acid sequence of SEQ ID NO: 415 and a heavy chain containing the amino acid sequence of SEQ ID NO: 532 of the hIL-2 protein.

The disclosed immunoconjugate selectively delivers IL-2 signaling to PD-1-expressing T cells. The human antibody molecule or its antigen-binding fragment of the immunoconjugate is used solely for the delivery of modified hIL-2 to PD-1-expressing cells and does not block PD-1 receptor function, as with classic anti-PD-1 inhibitor antibodies such as [examples of antibodies against PD-1]. The primary mechanism of action of the disclosed immunoconjugate is through T-cell-selective activity of IL-2. The human PD-1 receptor is primarily expressed on a small subset of T cells with potent tumor reactivity. Without being bound by theory, it is believed that targeting the modified hIL-2 protein portion of the immunoconjugate to this T-cell population can significantly enhance anti-tumor immunity while reducing or minimizing off-target systemic IL-2-mediated toxicity mediated by PD-1-deficient cell populations.

Drug compositions, polynucleotides, carriers and cells

This document discloses pharmaceutical compositions comprising any of the modified hIL-2 proteins disclosed herein, any human antibody molecules or antigen-binding fragments thereof disclosed herein, or any immunoconjugates disclosed herein. In some embodiments, the pharmaceutical composition comprises any of the modified hIL-2 proteins disclosed herein. In some embodiments, the pharmaceutical composition comprises any human antibody molecules or antigen-binding fragments thereof disclosed herein. In some embodiments, the pharmaceutical composition comprises any of the immunoconjugates disclosed herein.

This document discloses polynucleotides comprising nucleic acid sequences encoding any of the modified hIL-2 proteins disclosed herein, any human antibody molecules or antigen-binding fragments thereof disclosed herein, or any immunoconjugates disclosed herein. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding any of the modified hIL-2 proteins disclosed herein. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding any of the human antibody molecules or antigen-binding fragments thereof disclosed herein. In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding any of the immunoconjugates disclosed herein.

This document discloses vectors comprising polynucleotides, said polynucleotides comprising nucleic acid sequences encoding any of the modified hIL-2 proteins disclosed herein, any human antibody molecules or antigen-binding fragments thereof disclosed herein, or any immunoconjugates disclosed herein. In some embodiments, the vector comprises a polynucleotide comprising a nucleic acid sequence encoding any of the modified hIL-2 proteins disclosed herein. In some embodiments, the vector comprises a polynucleotide comprising a nucleic acid sequence encoding any of the human antibody molecules or antigen-binding fragments thereof disclosed herein. In some embodiments, the vector comprises a polynucleotide comprising a nucleic acid sequence encoding any of the immunoconjugates disclosed herein.

This article also discloses transformed cells containing any of the vectors disclosed herein.

Treatment methods and uses

This document discloses a method for treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of any of the immunoconjugates or pharmaceutical compositions disclosed herein, thereby treating the disease.

The use of any of the immunodrug conjugates or pharmaceutical compositions disclosed herein in the preparation of medicaments for treating diseases is also disclosed. The use of any of the immunodrug conjugates or pharmaceutical compositions described herein for treating diseases or conditions is also disclosed.

The disclosed immunoconjugates and pharmaceutical compositions can be used to treat diseases or conditions that are beneficial to the stimulation of a subject's immune system. In some embodiments, the subject has an inadequate or absent immune response, and the disclosed immunoconjugates and pharmaceutical compositions stimulate the subject's immune response. The antibody portion of the immunoconjugate can be used to direct the modified hIL-2 protein to the subject's immune cells, for example, by binding to an antigen expressed on the surface of immune cells. In the case of the disclosed modified hIL-2 protein-human anti-hPD-1 antibody immunoconjugate, for example, the anti-PD-1 antibody portion of the immunoconjugate (or its antigen-binding fragment) can bind to PD-1 expressed on T cells, thereby delivering the modified hIL-2 protein to the T cells. Targeting the modified IL-2 protein to specific cells can significantly enhance the therapeutic effect of the IL-2 protein without off-target systemic toxicity mediated by cell populations lacking antigen expression. The disclosed methods and uses can be used to treat, for example, cancer, autoimmune diseases and inflammatory diseases, as well as chronic infections and infectious diseases. Exemplary cancers include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, non-small cell lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, leukemia, skin cancer, melanoma, squamous cell carcinoma, bone cancer, and kidney cancer. Exemplary autoimmune and inflammatory diseases include systemic lupus erythematosus (SLE), type 1 diabetes, rheumatoid arthritis, ankylosing spondylitis, psoriasis, Behcet's disease, granulomatous polyangiitis, Takayasu's disease, Crohn's disease, ulcerative colitis, autoimmune hepatitis, sclerosing cholangitis, Sjoren's syndrome, alopecia areata, and inflammatory myopathy. Exemplary infectious diseases include HIV and hepatitis B.

In some embodiments, the disease is cancer. The methods and uses may comprise administering to a subject a therapeutically effective amount of any of the modified hIL-2 protein-antibody conjugates disclosed herein, thereby treating the cancer. In some aspects, the cancer is melanoma. In some aspects, the cancer is non-small cell lung cancer.

Example

The following examples are provided to further describe some embodiments of the embodiments disclosed herein. These examples are intended to illustrate, and not limit, the disclosed embodiments.

General Method

Option A. Flow cytometry screening of human PD-1 binding to anti-hPD-1 antibody or anti-hPD-1 antibody-attenuated hIL-2 fusion product.

To test binding to hPD-1, the antibody and antibody-attenuated hIL-2 fusion protein were characterized in complete titration curves. Jurkat cell lines were transfected with a mammalian vector encoding amino acid 1-185 (SEQ ID NO: 346) of human PD-1 to stably express the extracellular domain and part of the transmembrane domain of human PD-1, and this transfected cell line was used to determine the binding of the anti-hPD-1 antibody. Jurkat+hPD-1 cells were washed and added at 100,000 cells per well to FACS buffer (PBS, 0.2% heat-inactivated fetal bovine serum) in 96-well plates. Cells were blocked for 10 min at 4°C with a 1:50 dilution of human FCR blocking agent (Miltenyi) and washed with FACS buffer.

The antibody or antibody-attenuated hIL-2 immunoconjugate (fusion protein) was serially diluted six-fold in FACS buffer to obtain an 8-point curve, and added to Jurkat cells expressing human PD-1 at a volume of 100 μL on ice for 1 hour. Cells were washed and resuspended in FACS buffer containing a 1:40 dilution of allophycocyanin-conjugated anti-human IgG Fc monoclonal antibody. Cells were washed again and resuspended in FACS buffer containing a 1:1000 dilution of Sytox Green (Thermo Fisher Scientific), and flow cytometry was performed on a BD FACS Canto II, BD Celesta, or BD Fortessa (BD Biosciences). Geometric mean fluorescence intensity (gMFI) was calculated using FlowJo software version 10. The half-maximum effective concentration ( _EC50 ) was calculated using GraphPad Prism7 software based on the gMFI of the allophycocyanin signal across titrated concentrations.

Option B. Competitive screening by flow cytometry of anti-hPD-1 antibody or anti-hPD-1 antibody-attenuated hIL-2 fusion with human PD-1 binding.

The ability of the antibody and antibody-attenuated hIL-2 fusion protein to bind human PD-1 was tested in the presence of saturated concentrations of anti-hPD-1#1-mIgG2b-N297A (containing the heavy and light chain variable region sequences of nivolumab (clone 5C4), formatted on a mouse IgG2b-N297A background as described in US Patent Publication No. US2009/0217401A1) (SEQ ID NO: 348 and 349) or anti-hPD-1#2-mIgG2b-N297A (containing the heavy and light chain variable region sequences of pembrolizumab (clone 109A-H/K09A-L-11), formatted on a mouse IgG2b-N297A background as described in International Publication No. WO2008/156712A1) (SEQ ID NO: 350 and 351).

The antibody or antibody-attenuated hIL-2 fusion protein was serially diluted six-fold to obtain an 8-point titration profile, the antibody or fusion protein having or not having a saturated amount of 10 μM anti-hPD-1#1-mIgG2b-N297A or anti-hPD-1#2-mIgG2b-N297A. Briefly, Jurkat cells stably expressing hPD-1 were washed (as described in Protocol A above) and resuspended in FACS buffer containing a 1:50 dilution of human FcR blocking agent. The cells were incubated at 4°C for 10 min and washed. The cells were then resuspended in a 100 μL volume with anti-hPD-1#1-mIgG2b-N297A or anti-hPD-1#2-mIgG2b-N287A diluted to 10 μM in FACS buffer and incubated at 4°C for one hour. Cells were washed and incubated at 4°C for one hour with either the test antibody or the antibody-attenuated hIL-2 fusion protein serially diluted six-fold in 100 μL volumes to obtain an 8-point curve. To detect binding of the test anti-hPD-1 antibody or anti-hPD-1-attenuated hIL-2 fusion protein, cells were washed again and incubated on ice for 45 minutes with a 1:40 dilution of allophycocyanin-conjugated anti-human IgG Fc monoclonal antibody. Cells were then washed and resuspended in FACS buffer containing a 1:1000 dilution of Sytox Green (Thermo Fisher Scientific). For comparison, Jurkat cells stably expressing human PD-1 were incubated only with titrated test antibodies or antibody-degraded hIL-2 fusion proteins (without anti-hPD-1#1-mIgG2b-N297A or anti-hPD-1#2-mIgG2b-N297A) and subsequently with a 1:40 dilution of allophycocyanin-conjugated anti-human IgG Fc secondary. As controls, the variable regions of anti-hPD-1#1 and anti-hPD-1#2 were cloned into the hIgG4 framework and evaluated with and without the addition of anti-hPD-1#1-mIgG2b-N297A or anti-hPD-1#2-mIgG2b-N297A. Flow cytometry was performed on BD Canto II, BD Celesta, or BD Fortessa (BD Biosciences), and gMFI was calculated using FlowJo software version 10. _EC50 values were calculated using GraphPad Prism 7 software based on gMFI of allophycocyanin signals across titration concentrations.

Option C. Cell-based screening based on characterization of non-antagonist anti-hPD-1 antibody or anti-hPD-1 antibody-attenuated hIL-2 fusion.

Characterizing the ability of human PD-1 antibody and anti-hPD-1-attenuated hIL-2 fusion protein to block the binding of hPD-1 to the ligand hPD-L1 (SEQ ID NO: 584). The anti-hPD-1 antibody and anti-hPD-1-attenuated hIL-2 fusion protein were characterized as antagonists or non-antagonists using an in vitro cell-based human PD-1/PD-L1 blocking bioassay (Promega, catalog number J1255). This co-culture assay utilized two cell lines: FCγR11b artificial antigen-presenting cells/Chinese ovarian hamster K1 (aAPC/CHO-K1) and Jurkat effector cells. aAPC/CHO K1 cells stably expressed both the human PD-L1 ligand and cell surface protein to activate the homologous T cell receptor (TCR), while Jurkat effector cells expressed hPD-1 and a luciferase reporter gene under the control of the activated nuclear factor response element (NFAT-RE) of T cells. When these cells were co-cultured in the presence of a non-antagonistic antibody, the hPD-1/hPD-L1 interaction inhibited TCR signaling, and no luminescence was detected. In the presence of an antibody antagonizing the interaction between hPD-1 and hPD-L1 (SEQ ID NO:584), the inhibitory signal was disrupted, and luminescence was detected.

Thawing and assaying were performed according to the manufacturer's instructions. In short, aAPC/CHO-K1 cells were first thawed and seeded at 30,000 cells per well in a 5% _CO2 incubator at 37°C for 18 hours on flat-bottomed 96-well plates. After cell adhesion, the culture medium was removed, and 200 nM or 1000 nM of the test antibody or antibody-degraded hIL-2 fusion protein was diluted in 40 μL of assay buffer (RPMI 1640 medium + 1% FBS) and added to the aAPC/CHO-K1 cells. A human IgG4 isotype control monoclonal antibody targeting keyfora hemocyanin (KLH) clone C3 (SEQ ID NO: 585 and 586) was used as a negative control. Jurkat effector cells expressing hPD-1 were added to a 40 μL volume at 24,000 cells per well. The final concentration of the fixed antibody tested was 100 nM or 500 nM. In some instances, a series of concentrations of hIL-2 fusion protein against hPD-11 or hPD-1-attenuation were tested in this co-culture assay, with the highest concentration in the five-fold titration series being 500 nM (Figure 7).

Co-culturing assays were performed at 37°C in a 5% _CO2 incubator for an additional 18–20 hours. To read the luminescence signal, the plate was allowed to reach room temperature, and 80 μL of Bio-Glo ^™ reagent was added to each well. The plate was incubated in the dark at room temperature for 15 minutes, and the luminescence was read on a Victor X-ray spectrophotometer (PerkinElmer). The average of every three relative luminescence units (RLU) was calculated, and the results were plotted using GraphPad Prism 7 software.

Protocol D. In vitro phosphorylated STAT5 assay to test hIL-2 variant attenuation

Phosphorylated STAT5 was used to determine the attenuation level of hIL-2 receptor activation activity of the antibody-attenuated hIL-2 fusion protein. Variants were tested in both hIL-2-responsive human natural killer (NK-92) cells and engineered human erythroleukemia (TF1) cells. The NK-92 cell line naturally expresses a high-affinity hIL-2 receptor (IL-2Rαβγ) at physiological levels, while the TF1 cell line, which naturally expresses IL-2Rγ (SEQ ID NO:352), was engineered to stably express human CD122 (IL-2Rβ) (SEQ ID NO:353) to express the intermediate-affinity hIL-2 receptor complex (IL-2Rβγ). This stable TF1+IL-2Rβ cell line does not express IL-2Rα (SEQ ID NO:354). Both the NK-92 and TF1+IL-2Rβ cell lines were used to assess the attenuation level of IL-2 potency in these cell-based potency assays, such as fixed-concentration screening and complete titration curves.

For fixed concentration screening, 100,000 NK-92 cells or TF1+IL-2Rβ cells were plated into 96 wells of 50 μL of fresh growth medium lacking human IL-2 cytokines and incubated overnight at 37°C in a _CO2 incubator. After 15–16 hours, human IL-2-starved cells were treated with 25.7 nM recombinant hIL-2 (denoted as rhIL-2) (SEQ ID NO:345) or a test antibody-degraded hIL-2 fusion protein for NK-92 cell assays, or with 33.3 nM hIL-2 or a test hIL-2 variant for TF1+IL-2Rβ cell assays. Cells were incubated at 37°C in 5% _CO2 for 10 minutes. Cells were fixed with Cytofix buffer (BD Biosciences) at 37°C for 10 minutes and then permeabilized on ice with Perm buffer III (BD Biosciences) for 30 minutes. hIL-2-dependent Stat5 phosphorylation was detected after staining fixed and permeabilized cells with Alexa Fluor-647-conjugated anti-Stat5 antibody (BD Biosciences) at 0.5 μL/sample for 45 min in the dark at room temperature. Cells were washed and the reagent was diluted in BD Pharmaceuticals buffer (BD Biosciences). Stained cells were acquired on a FACS-Celesta hematology counter (BD Biosciences) and analyzed using FlowJo software version 10.7.2. Assays were performed in populations, but each plate was normalized using rhIL-2. The degree of decay of the selected antibody-decaying hIL-2 fusion protein was assessed in 8-point, 6-fold sequential titration curves ranging from 1200 nM to 7 pM on both the NK-92 and TF1+IL-2Rβ cell lines. The pStat5 curve procedure was performed in the same manner as described above. _EC50 values were calculated based on the geometric mean fluorescence intensity (gMFI) across titration concentrations using GraphPad Prism 7 software. The fold change in rhIL-2 activity was calculated by dividing the _EC50 value of the variant by the _EC50 of hIL-2.

Protocol E. In vitro cell proliferation assay to test the attenuation of antibody-attenuated hIL-2 fusion protein.

The attenuated hIL-2 activity of the antibody-attenuated hIL-2 fusion protein was also tested in an hIL-2-dependent cell proliferation assay. 10,000 NK-92 cells (expressing high-affinity receptor hIL-2Rαβγ) or TF1+IL-2Rβ cells (expressing intermediate-affinity receptor hIL-2Rβγ) suspended in 50 μL of fresh growth medium without hIL-2 cytokines were plated in each well of a 96-well U-bottom cell culture plate. An eight-point, 6-fold serial titration of the antibody-attenuated hIL-2 fusion protein at a maximum concentration of 996 nM was diluted in fresh medium and applied to the cells in the wells. Cells were incubated at 37°C in a 5% _CO2 incubator for 3 days for TF1+IL-2Rβ cells or 4 days for NK-92 cells. To measure proliferation, Cell-Titer-Glo (Promega) was added to the wells, incubated at room temperature for 10 minutes, and the luminescence of each well was read for 0.1 seconds using a VictorX multi-label plate reader (PerkinElmer). _EC50 values were calculated based on relative luminescence units (RLU) across titration concentrations using GraphPad Prism 7 software. The fold change in rhIL-2 activity was calculated by dividing the _EC50 value of the variant by the _EC50 value of hIL-2. Assays were performed in a cohort, but each plate was normalized using the rhIL-2 _EC50 value.

Example 1: Optimization of antibody-attenuated hIL-2 fusion protein variants and determination of their hIL-2 activity on intermediate-affinity and high-affinity hIL-2 receptor complexes.

To determine the optimal structure of the antibody-attenuated hIL-2 fusion protein, unattenuated hIL-2 was fused with an anti-DNase I antibody (clone 1H3) named 1H3-hIgG1 (SEQ ID NO:379, SEQ ID NO:374) in the variable region of the antibody in various ways as shown in Figure 1. Variants include the fusion of hIL-2 at the N-terminus of the human anti-DNase I antibody (clone 1H3) immunoglobulin hIgG1 heavy chain or human κ light chain via a direct fusion (df) of hIL-2 N-terminal light chain df (SEQ ID NO:379, SEQ ID NO:356) or hIL-2 N-terminal heavy chain df (SEQ ID NO:358, SEQ ID NO:374) or a hexaamino acid linker (L6) (SEQ ID NO:355) of hIL-2 N-terminal light chain L6 fusion (SEQ ID NO:379, SEQ ID NO:357) and hIL-2 N-terminal heavy chain L6 fusion (SEQ ID NO:359, SEQ ID NO:374). Variations were also generated in which the hIL-2 moiety fused with the C-terminus of both the heavy and light chains via df or L6, and are represented as hIL-2 C-terminal heavy chain df (SEQ ID NO:360, SEQ ID NO:374), hIL-2 C-terminal heavy chain L6 fusion (SEQ ID NO:361, SEQ ID NO:374), hIL-2 C-terminal light chain df (SEQ ID NO:379, SEQ ID NO:362), and hIL-2 C-terminal light chain L6 fusion (SEQ ID NO:379, SEQ ID NO:363). Further variations were generated in which the CD25/IL-2Rα extracellular domain (amino acids 1-164) (SEQ ID NO:126) fused with the N-terminus or C-terminus of the heavy or κ light chain to interfere with the binding of IL-2 to CD25 of the IL-2 receptor (Figure 2). In these constructs, the extracellular domain of human CD25 (amino acids 1-164) (SEQ ID NO: 126) is fused to human IL-2 via a 20-amino acid linker (L20) (SEQ ID NO: 364), and then directly fused to the N-terminus or fused to the 1H3-hIgG1 heavy or light chain via an L6 linker (SEQ ID NO: 355): hCD25-L20-hIL-2 N-terminal heavy chain df (SEQ ID NO: 365, SEQ ID NO: 355). SEQ ID NO:374), hCD25-L20-hIL-2N terminal heavy chain L6 fusion (SEQ ID NO:366, SEQ ID NO:374), hCD25-L20-hIL-2N terminal light chain df (SEQ ID NO:379, SEQ ID NO:367), hCD25-L20-hIL-2N terminal light chain L6 fusion (SEQ ID NO:379, SEQ ID NO:368). Finally, a final set of variants were generated in which the extracellular domain portion of CD25/IL-2Rα (SEQ ID NO:126) was fused to the C-terminus of the heavy chain and κ light chain: hCD25-L20-hIL-2 C-terminal heavy chain df (SEQ ID NO:369, SEQ ID NO:374), hCD25-L20-hIL-2 C-terminal heavy chain L6 fusion (SEQ ID NO:370, SEQ ID NO:374), hCD25-L20-hIL-2 C-terminal light chain df (SEQ ID NO:379, SEQ ID NO:371), and hCD25-L20-hIL-2 C-terminal light chain L6 fusion (SEQ ID NO:379, SEQ ID NO:372). These antibody-hIL-2 fusion proteins were generated, expressed, and purified using standard techniques. The aforementioned 16 N-terminal or C-terminal and adapter variants were evaluated in an in vitro cell-based phosphorylated STAT5 assay using an 8-point, 6-fold serial titration as described in Protocol D.

Table 1 summarizes the _EC50 calculated using geometric mean fluorescence intensity (gMFI) on an 8-point, 6-fold sequential titration curve using FlowJo version 10 software. The fold change for each variant relative to rhIL-2 was also calculated as a measure of the level of decay compared to the activity of the rhIL-2 positive control. Some _EC50 values could not be calculated using GraphPad Prism 7 software and were marked as not calculated (NC); however, these variants showed no decay based on the dose-titer curves.

Compared to rhIL-2 expressing either high-affinity hIL-2 receptor (NK-92) or intermediate-affinity hIL-2 receptor (TF1+IL-2Rβ) on cell lines, fusion of the hIL-2 moiety with the N-terminus or C-terminus of the immunoglobulin heavy chain did not result in decreased IL-2 activity. Direct fusion (df) of hIL-2 with the antibody component of the fusion protein did not lead to changes in IL-2 activity compared to fusion using a six-amino acid linker (L6) between IL-2 and the antibody component. Similarly, fusion of the IL-2 component with either the heavy or light chain of the antibody component did not lead to changes in IL-2 activity compared to rhIL-2. All N-terminal or C-terminal and linker fusion protein variants in which the hCD25/hIL-2Rα moiety is fused to hIL-2 are expected to exhibit reduced binding of the fusion protein to the CD25 of the hIL-2 receptor on cells. In experiments, these constructs exhibited strongly attenuated hIL-2 activity (at least 45-fold attenuation) against the high-affinity IL-2 receptor (NK-92) and 18-fold attenuation against the intermediate-affinity hIL-2 receptor (TF1+IL-2Rβ).

Example 2: Generation of antibody-attenuated hIL-2 fusion protein variants and determination of their binding kinetics with recombinant human CD25 and/or human CD122.

Since hIL-2 activity is not reduced in various N-terminal or C-terminal immunoglobulin heavy chain fusion proteins, the hIL-2 C-terminal heavy chain L6 fusion protein named "1H3-hIgG1-L6-hIL-2" (SEQ ID NO: 361, 374) was used as the basic construct for antibody-attenuated hIL-2 fusion protein variants with substitutions in the hIL-2 moiety. To investigate the role of these residues in recognizing human CD25/IL-2Rα and/or human CD122/IL-2Rβ or CD132/IL-2Rγ (human IL-2R subunits), single, two, and/or multiple amino acid substitutions were introduced into selected residues of human IL-2. More than three hundred antibody-attenuated hIL-2 fusion protein variants with substitutions in the hIL-2 moiety were generated and evaluated in six rounds. These variants were first screened on IL-2-dependent cell lines (NK-92 and TF1+IL-2Rβ) using a fixed-concentration, flow-based phosphorylated STAT5 (pSTAT5) assay in dose-titer curves. Phosphorylated STAT5 is a downstream signal of IL-2 activity and was used as a snapshot measure of IL-2 potency. IL-2-dependent cell proliferation assays were also performed to measure IL-2 activity over a 3–4 day time period. Selection criteria for attenuated hIL-2 included: (1) reduced IL-2 potency in both the NK-92 and TF1+IL-2Rβ cell lines, with agonist activity greater than 50% in both cell lines; and (2) moderate to high yield.

Human anti-DNase I antibody-hIL-2 fusion protein is produced by fusing human IL-2 or human IL-2 variants (SEQ ID NO: 1-344, 377, 378 and 575) with the C-terminus of the heavy chain of a human anti-DNase I antibody (clone 1H3, having a human IgG1 isotype) via an L6 linker. The fusion protein is then combined with the hIgG1 light chain (1H3-hkappa LC; SEQ ID NO: 374) to produce the 1H3-hIgG1-L6-hIL-2 fusion protein (provided in Table 28). Mouse anti-yellow fever virus antibody-hIL-2 fusion proteins are also produced by fusing a human IL-2 variant with the C-terminus of a mouse anti-yellow fever virus antibody (clone 2D12, having a mouse IgG1 isotype) heavy chain via an L6 linker, replacing the reduced immune effector function with D265A. This fusion protein is then combined with a 2D12-mIgG1 light chain (2D12-mKappa LC; SEQ ID NO: 376) to produce a 2D12-mIgG1-D265A-L6-hIL-2 fusion protein (provided in Table 28). Some of these mouse anti-yellow fever virus antibody-hIL-2 fusion proteins are formatted into the human IgG1 constant region and produced in the same manner as described above (i.e., combined with a 2D12-hKappa light chain (2D12-hKappa LC; SEQ ID NO: 573)). IL-2 amino acid substitutions are repeated six times, designated as groups 1 through 6. Standard techniques were used to generate and express fusion proteins of 1H3-hIgG1-L6-hIL-2, 2D12-mIgG1-D265A-L6-hIL-2, and 2D12-hIgG1-L6-hIL-2, and to purify protein A.

Group 1 contains only the initial series of 2D12-mIgG1-D265A-L6-hIL-2 or 2D12-hIgG1-L6-hIL-2 fusion proteins, which contain human IL-2 and are expected to involve binding only to one of the IL-2 receptor subunits CD25/IL-2Rα, CD122/IL-2Rβ, or CD132/IL-2Rγ, or a combination of substitutions. The fusion proteins in this group include the following IL-2 substitutions expected to regulate binding to CD25/IL-2Rα: F42K (SEQ ID NO:1), V69A (SEQ ID NO:2), V69E (SEQ ID NO:3), V69F (SEQ ID NO:4), V69G (SEQ ID NO:5), V69H (SEQ ID NO:6), V69I (SEQ ID NO:7), V69K (SEQ ID NO:8), V69L (SEQ ID NO:9), V 69M(SEQ ID NO:10), V69Q(SEQ ID NO:11), V69S(SEQ ID NO:12), V69T(SEQ ID NO:13), V69W(SEQ ID NO:14), V69Y(SEQ IDNO:15), V69R(SEQ ID NO:581), (F42K/F44K)(SEQ ID NO:16), (F44K/Y45R)(SEQ ID NO:17), (F42K/V69R)(SEQ ID NO:18), (Y45R/V69R) (SEQ ID NO:19), (F42K/F44K/Y45R) (SEQ ID NO:20), (F42A/Y45A/L72G) (SEQ ID NO:574), (R38A/ F42K/Y45R)(SEQ ID NO:21), (R38E/F42K/Y45R)(SEQ ID NO:22), (K43E/F42K/Y45R)(SEQ ID NO:23), (K43T/F42K/Y45 R)(SEQ ID NO:24), (F42K/Y45R/E62A)(SEQ ID NO:25), (P65R/F42K/Y45R)(SEQ ID NO:26), (P65S/F42K/Y45R)(SEQ ID NO:27), (V69A/F42K/Y45R)(SEQ ID NO:28), (V69D/F42K/Y45R)(SEQ ID NO:29) or (V69R/F42K/Y45R)(SEQ ID NO:30). The substitutions in this group include the following substitutions that are expected to regulate binding to CD122/IL-2Rβ: D20A (SEQ ID NO:31), D20N (SEQ ID NO:32), D20K (SEQ ID NO:33), N88A (SEQ ID NO:34), N88G (SEQ ID NO:35), N88H (SEQ ID NO:36), N88K (SEQ ID NO:37), (D20A/D84A)(SEQ ID NO:36), (D20A/D84A)(SEQ ID NO:37), (D20A/D84A)(SEQ ID NO:38 ... Group 1 also includes the following IL-2 substitutions that are expected to regulate the binding of IL-2 to CD132/IL-2-Rγ: Q126L (SEQ ID NO: 377) or Q126E (SEQ ID NO: 378). The IL-2 substitutions studied in Group 1 are not expected to regulate binding to more than one IL-2 receptor subunit. (D20A/E15A)(SEQ ID NO: 39), (D20A/E95A)(SEQ ID NO: 40), (D20A/N88A)(SEQ ID NO: 41), (D20A/S87A)(SEQ ID NO: 42), (D84A/N88A)(SEQ ID NO: 43), (E15A/N88A)(SEQ ID NO: 44), or (S87A/N88A)(SEQ ID NO: 45).

Group 2 contains a series of 1H3-hIgG1-L6-hIL-2 fusion proteins, which contain one or more substitutions in human IL-2 that are expected to be involved only in CD25/IL-2Rα binding. The fusion proteins in this group include the following IL-2 substitutions expected to regulate binding to CD25/IL-2Rα: R38A (SEQ ID NO:46), R38D (SEQ ID NO:47), R38E (SEQ ID NO:48), R38Q (SEQ ID NO:49), F42R (SEQ ID NO:50), F42A (SEQ ID NO:51), F42D (SEQ ID NO:52), and F42H (SEQ ID NO:53). K43A(SEQ ID NO:54), K43E(SEQ ID NO:55), K43Q(SEQ ID NO:56), Y45A(SEQ ID NO:57), Y45K(SEQ ID NO:58), Y45S(SEQ ID NO:59), Y45R(SEQ ID NO:60), E61A(SEQ ID NO:61), E61R(SEQ ID NO:62), E61K(SEQ ID NO:63), E 62A(SEQ ID NO:64), E62R(SEQ ID NO:65), E62K(SEQ ID NO:66), E62Y(SEQ ID NO:67), E68Y(SEQ ID NO:68), E 68A(SEQ ID NO:69), E68K(SEQ ID NO:70), E68R(SEQ ID NO:71), E68L(SEQ ID NO:72), L72Y(SEQ ID NO:73), L7 2R (SEQ ID NO:74), L72A (SEQ ID NO:75), L72D (SEQ ID NO:76), L72H (SEQ ID NO:77), L72F (SEQ ID NO:78), (R38D/E61R) (SEQ ID NO:79), (R38D/E61R/K43E) (SEQ ID NO:80), or (T3A/F42A/Y45A/L72G/C125A) (SEQ ID NO:81). Substituting T3A into the IL-2 amino acid sequence removes the predicted O-linked glycosylation site on human IL-2 (see, for example, International Publication No. WO2012/107417), and substituting C125A into the IL-2 amino acid sequence removes unpaired cysteine residues (see, for example, International Publication No. WO2018/184964). The IL-2 substitutions studied in Group 2 are not expected to regulate the binding of IL-2 to CD132/IL-2-Rγ, nor are they expected to regulate binding to more than one IL-2 receptor subunit.

Group 3 contains a series of 1H3-hIgG1-L6-hIL-2 fusion proteins, which contain one or more substitutions in human IL-2 expected to be involved only in CD122/IL-2Rβ binding. The fusion proteins in this group include the following IL-2 substitutions expected to regulate binding to CD122/IL-2Rβ: E15A (SEQ ID NO:82), E15R (SEQ ID NO:83), E15K (SEQ ID NO:84), H16A (SEQ ID NO:85), H16Y (SEQ ID NO:86), H16E (SEQ ID NO:87), L19A (SEQ ID NO:88), D20I (SEQ ID NO:89), D20S (SEQ ID NO:90), D20H (SEQ ID NO:91), D20T (SEQ ID NO:88), D20I (SEQ ID NO:89), D20S (SEQ ID NO:90), D20H (SEQ ID NO:91), D20T (SEQ ID NO:89 ...I (SEQ ID NO:89), D20I (SEQ ID NO:89), D20I (SEQ ID NO:90), D20I (SEQ ID NO:91), D20I (SEQ ID NO:89), D20I (SEQ ID NO:89), D20I (SEQ ID NO:89), D20I (SEQ ID NO:89), D20I (SEQ ID NO:89), D20I (SEQ ID NO:89), D20I (SEQ ID NO:89), ID NO:92), D20W(SEQ ID NO:93), D20Y(SEQ ID NO:94), D20R(SEQ ID NO:95), D20F(SEQ ID NO:96), R81A(SEQ ID NO:97), D84A(SEQ ID N O:98), D84R(SEQ ID NO:99), D84K(SEQ ID NO:100), S87A(SEQ ID NO:101), N88Y(SEQ ID NO:102), N88D(SEQ ID NO:103), N88R(SEQ ID NO: 104), N88E(SEQ ID NO:105), N88F(SEQ ID NO:106), N88I(SEQ ID NO:107), I92A(SEQ ID NO:108), I92Y(SEQ ID NO:109), I92S(SEQ ID NO :110), I92F(SEQ ID NO:111), I92R(SEQ ID NO:112), I92D(SEQ ID NO:113), I92E(SEQ ID NO:114), E95A(SEQ ID NO:115), E95R(SEQ ID NO: 116), E95K(SEQ ID NO:117), (D20Y/H16E)(SEQ ID NO:118), (D20Y/H16A)(SEQ ID NO:119), (D20Y/H16Y)(SEQ ID NO:120), (D20Y/I92A)(S EQ ID NO:121), (D20Y/I92S)(SEQ ID NO:122), (D20Y/I92R)(SEQ ID NO:123), (D20Y/E95R)(SEQ ID NO:124) or (D20Y/E95A)(SEQ ID NO:125).

Group 4 contains a series of fusion proteins containing 1H3-hIgG1-L6-hIL-2HC fused to the extracellular domain portion of CD25/IL-2Rα (SEQ ID NO:126), a 20-amino acid linker (L20) (SEQ ID NO:364), and a human IL-2 variant containing one or more residues substituted that are expected to be involved in binding to CD122/IL-2Rβ. The fusion proteins in this group include the following IL-2 substitutions expected to regulate binding to CD122/IL-2Rβ: E15A (SEQ ID NO:82), D20I (SEQ ID NO:89), D20S (SEQ ID NO:90), D20H (SEQ ID NO:91), D20W (SEQ ID NO:93), D20Y (SEQ ID NO:94), D20R (SEQ ID NO:95), D20F (SEQ ID NO:96), D84K (SEQ ID NO:97), D20R (SEQ ID NO:98), D20F (SEQ ID NO:98), D20R (SEQ ID NO:99), D20R (SEQ ID NO:99), D20F (SEQ ID NO:99), D20R ... The following are listed: S87A (SEQ ID NO:101), N88Y (SEQ ID NO:102), N88D (SEQ ID NO:103), N88R (SEQ ID NO:104), N88E (SEQ ID NO:105), N88F (SEQ ID NO:106), N88I (SEQ ID NO:107), I92A (SEQ ID NO:108), E95A (SEQ ID NO:115), or E95K (SEQ ID NO:117). The antibody-degraded hIL-2 fusion protein in this group is represented as 1H3-hIgG1-L6-hCD25(1-164)-L20-hIL-2.

Group 5 contains a series of 1H3-hIgG1-L6-hIL-2 variants, which include IL-2 with substitution combinations expected to involve IL-2 binding to CD25/IL-2Rα and CD122/IL-2Rβ or CD132/IL-2Rγ. Additionally, some variants lack the first three amino acids at the N-terminus of the hIL-2 moiety (Δ1-3APT). The fusion proteins in group 5 include the following IL-2 substitutions expected to regulate the binding of IL-2 to CD25/IL-2Rα and CD122/IL-2Rβ: (F42D/D20A) (SEQ ID NO:127), (F42R/D20A) (SEQ ID NO:128), (F42K/D20A) (SEQ ID NO:129), (F42A/D20A) (SEQ ID NO:130), (F42H/D20A) (SEQ ID NO:131), and (Y45R/D20A) (SEQ ID NO:132). , (Y45K/D20A)(SEQ ID NO:133), (R38N/D20A)(SEQ ID NO:134), (R38G/D20A)(SEQ ID NO:135), (R38H/D20A)(SEQ IDNO:136), ( R38I/D20A)(SEQ ID NO:137), (R38L/D20A)(SEQ ID NO:138), (R38M/D20A)(SEQ ID NO:139), (R38F/D20A)(SEQ ID NO:140), (R3 8P/D20A)(SEQ ID NO:141), (R38S/D20A)(SEQ ID NO:142), (R38T/D20A)(SEQ ID NO:143), (R38W/D20A)(SEQ ID NO:144), (R38 Y/D20A)(SEQ ID NO:145), (R38V/D20A)(SEQ ID NO:146), (R38A/D20A)(SEQ ID NO:147), (R38Q/D20A)(SEQ ID NO:148), (D20A /R38E)(SEQ ID NO:149), (R38D/D20A)(SEQ ID NO:150), (K43E/D20A)(SEQ ID NO:151), (E61A/D20A)(SEQ ID NO:152), (E62A/ D20A)(SEQ ID NO:153), (E62Y/D20A)(SEQ ID NO:154), (L72D/D20A)(SEQ ID NO:155), (L72H/D20A)(SEQ ID NO:156), (L72R/D20A )(SEQ ID NO:157), (F42D/I92D)(SEQ ID NO:158), (F42R/I92D)(SEQ ID NO:159), (F42H/I92D)(SEQ ID NO:160), (F42A/I92D) (SEQ ID NO:161), (H16A/F42A)(SEQ ID NO:575), (K43E/I92D)(SEQ ID NO:162), (Y45R/I92D)(SEQ ID NO:163), (Y45K/I92D)( SEQ ID NO:164), (E62A/I92D) (SEQ ID NO:165), (E62Y/I92D) (SEQ ID NO:166), (L72D/I92D) (SEQ ID NO:167), (L72H/I92D) (S EQ ID NO:168), (L72R/I92D) (SEQ ID NO:169), (R38D/I92D) (SEQ ID NO:170), (R38E/I92D) (SEQ ID NO:171), (R38Q/I92D) (SEQI D NO:172), (R38A/I92D) (SEQ ID NO:173), (R38E/N88R) (SEQ ID NO:174), (R38E/D84R) (SEQ ID NO:175), (R38E/D84K) (SEQ ID NO:176), (F42A/Y45R/D20A)(SEQ ID NO:177), (F42H/Y45R/D20A)(SEQ ID NO:178), (R38D/E61R/D20A)(SEQ ID NO:179), (R38 E/E61R/D20A)(SEQ ID NO:180), (R38Q/E61R/D20A)(SEQ ID NO:181), (R38A/E61R/D20A)(SEQ ID NO:182), (R38A/D20A/E95A)(S EQ ID NO:183), (D20A/E95A/R38D)(SEQ ID NO:184), (D20A/E95A/R38E)(SEQ ID NO:185), (D20A/E95A/R38Q)(SEQ ID NO:186),

(D20A/E95A/F42R)(SEQ ID NO:187), (D20A/E95A/F42A)(SEQ ID NO:188), (D2 0A/E95A/F42D)(SEQ ID NO:189), (D20A/E95A/F42H)(SEQ ID NO:190), (D20A/ E95A/F42K)(SEQ ID NO:191), (D20A/E95A/K43A)(SEQ ID NO:192), (D20A/E95 A/K43E)(SEQ ID NO:193),(D20A/E95A/K43Q)(SEQ ID NO:194),(D20A/E95A/Y45 A)(SEQ ID NO:195)、(D20A/E95A/Y45K)(SEQ ID NO:196)、(D20A/E95A/Y45S)( SEQ ID NO:197), (D20A/E95A/Y45R) (SEQ ID NO:198), (D20A/E95A/E61A) (SEQ ID NO:199), (D20A/E95A/E62A) (SEQ ID NO:200), (D20A/E95A/E62R) (SEQ ID N O:201), (D20A/E95A/E62K)(SEQ ID NO:202), (D20A/E95A/E62Y)(SEQ ID NO:20 3), (D20A/E95A/E68Y)(SEQ ID NO:204), (D20A/E95A/E68A)(SEQ ID NO:205), (D20A/E95A/E68L)(SEQ ID NO:206), (D20A/E95A/L72Y)(SEQ IDNO:207), (D20A /E95A/L72R)(SEQ ID NO:208), (D20A/E95A/L72A)(SEQ ID NO:209), (D20A/E9 5A/L72D)(SEQ ID NO:210), (D20A/E95A/L72H)(SEQ ID NO:211), (D20A/E95A/L 72F)(SEQ ID NO:212), (F42K/Y45R/D20A/S87A)(SEQ ID NO:213), (F42K/Y45R /D20A/E95A)(SEQ ID NO:214), (D20A/R38E/C125A)(SEQ ID NO:215), (T3A/D20 A/R38E)(SEQ ID NO:216), (T3A/D20A/R38E/C125A)(SEQ ID NO:217), (Δ1-3APT /D20A/R38E)(SEQ ID NO:218) or (Δ1-3APT/D20A/R38E/C125A)(SEQ ID NO:219). The fusion proteins in group 5 include the following IL-2 substitutions expected to regulate the binding of IL-2 to CD25/IL-2Rα and CD132/IL-2R: (R38E/Q22A)(SEQ ID NO:220), (R38E/T123A)(SEQ ID NO:221), (R38E/I129A)(SEQ ID NO:222), (R38E/S130A)(SEQ ID NO:223), (R38E/Q1 (26A)(SEQ ID NO:224), (R38E/Q126D)(SEQ ID NO:225), (R38E/Q126V)(SEQ ID NO:226), (R38E/Q22A/S130A)(SEQ ID NO:227), (F42K/Y45R/Q126D)(SEQ ID NO:228), or (D20A/E95A/Q126D)(SEQ ID NO:229). SEQ ID NOs 127-229 and 575 list the group 5 antibody-attenuated hIL-2 fusion proteins numbered according to mutations in the hIL-2 sequence of the IL-2 sequence.

Group 6 contains a series of 1H3-hIgG1-L6-hIL-2 fusion proteins, which comprise human IL-2 substitution combinations expected to involve IL-2 binding to CD25/IL-2Rα and CD122/IL-2Rβ but not to CD132/IL-2Rγ. The fusion proteins in Group 6 include the following substitution combinations in IL-2 expected to regulate IL-2 binding to CD25/IL-2Rα and CD122/IL-2Rβ: (D20A/E61R)(SEQ ID NO:230), (D20A/E61N)(SEQ ID NO:231), (D20A/E61D)(SEQ ID NO:232), (D20A/E61Q)(SEQ ID NO:233), (D20A/E61G)(SEQ ID NO:234), (… D20A/E61H)(SEQ ID NO:235), (D20A/E61I)(SEQ ID NO:236), (D20A/E61L)(SEQ ID NO:237), (D20A/E61K)(SEQ ID NO:238), (D20A/E61M)(SEQ ID NO:239), (D20A/E61F)(SEQ ID NO:240), (D20A/E61P)(SEQ ID NO:241), (D20A/E61S )(SEQ ID NO:242), (D20A/E61T)(SEQ ID NO:243), (D20A/E61W)(SEQ ID NO:244), (D20A/E61Y)(SEQ ID NO:245), ( D20A/E61V)(SEQ ID NO:246), (D20A/F42N)(SEQ ID NO:247), (D20A/F42Q)(SEQ ID NO:248), (D20A/F42E)(SEQ ID NO:249), (D20A F42G) (SEQ ID NO:250), (D20A/F42I) (SEQ ID NO:251), (D20A/F42L) (SEQ ID NO:252), (D20A/F42 M)(SEQ ID NO:253), (D20A/F42P)(SEQ ID NO:254), (D20A/F42S)(SEQ ID NO:255), (D20A/F42T)(SEQ ID NO:256), (D20A/F42W)(SEQ ID NO:257), (D20A/F42Y)(SEQ ID NO:258), (D20A/F42V)(SEQ ID NO:259), (D20A/Y45A)(SEQID NO:260), (D20A/Y45N)(SEQ ID NO:261), (D20A/Y45D)(SEQ ID NO:262), (D20A/Y45Q)(SEQ ID NO:263), (D20A/Y45E )(SEQ ID NO:264), (D20A/Y45G)(SEQ ID NO:265), (D20A/Y45H)(SEQ ID NO:266), (D20A/Y45I)(SEQ ID NO:267), (D20A/Y45L)(SEQ ID NO:268), (D20A/Y45M)(SEQ ID NO:269), (D20A/Y45F)(SEQ ID NO:270), (D20A/Y45P)(SEQ ID NO:271), (D20A/Y45S)(SEQ ID NO:272), (D20A/Y45T)(SEQ ID NO:273), (D20A/Y45W)(SEQ ID NO:274), (D20A/Y45 V)(SEQ ID NO:275), (I92D/F42N)(SEQ ID NO:276), (I92D/F42Q)(SEQ ID NO:277), (I92D/F42E)(SEQ ID NO:278), ( I92D/F42G)(SEQ ID NO:279), (I92D/F42I)(SEQ ID NO:280), (I92D/F42L)(SEQ ID NO:281), (I92D/F42K)(SEQ ID NO:282), (I92D/F42M)(SEQ ID NO:283), (I92D/F42P)(SEQ ID NO:284), (I92D/F42S)(SEQ ID NO:285), (I92D/F42 T)(SEQ ID NO:286), (I92D/F42W)(SEQ ID NO:287), (I92D/F42Y)(SEQ ID NO:288), (I92D/F42V)(SEQ ID NO:289) , (I92D/Y45A)(SEQ ID NO:290), (I92D/Y45N)(SEQ ID NO:291), (I92D/Y45D)(SEQ ID NO:292), (I92D/Y45Q)(SEQ ID NO:293), (I92D/Y45E)(SEQ ID NO:294), (I92D/Y45G)(SEQ ID NO:295), (I92D/Y45H)(SEQ ID NO:296), (I92D/Y45 I)(SEQ ID NO:297), (I92D/Y45L)(SEQ ID NO:298), (I92D/Y45M)(SEQ ID NO:299), (I92D/Y45F)(SEQ ID NO:300), (I92D/Y45P)(SEQ ID NO:301), (I92D/Y45S)(SEQ ID NO:302), (I92D/Y45T)(SEQ ID NO:303), (I92D/Y45W)(SEQ I D NO:304), (I92D/Y45V)(SEQ ID NO:305), (R38E/D20H)(SEQ ID NO:306), (R38E/D20S)(SEQ ID NO:307), (F42A/N8 8R)(SEQ ID NO:308), (F42A/N88D)(SEQ ID NO:309), (R38E/D84A)(SEQ ID NO:310), (R38E/D84N)(SEQ ID NO:311), (R38E/D84Q)(SEQ ID NO:312), (R38E/D84E)(SEQ ID NO:313), (R38E/D84G)(SEQ ID NO:314), (R38E/D84H)(SEQ ID NO:315), (R38E/D84I)(SEQ ID NO:316), (R38E/D84L)(SEQ ID NO:317), (R38E/D84M)(SEQ ID NO:318), (R38E/D8 4F)(SEQ ID NO:319), (R38E/D84P)(SEQ ID NO:320), (R38E/D84S)(SEQ ID NO:321), (R38E/D84T)(SEQ ID NO:322) , (R38E/D84W)(SEQ ID NO:323), (R38E/D84Y)(SEQ ID NO:324), (R38E/D84V)(SEQ IDNO:325), (R38E/I92A)(SEQ I D NO:326), (R38E/I92R)(SEQ ID NO:327), (R38E/I92N)(SEQ ID NO:328), (R38E/I92Q)(SEQ ID NO:329), (R38E/I92 E)(SEQ ID NO:330), (R38E/I92G)(SEQ ID NO:331), (R38E/I92H)(SEQ ID NO:332), (R38E/I92L)(SEQ ID NO:333) , (R38E/I92K)(SEQ ID NO:334), (R38E/I92M)(SEQ ID NO:335), (R38E/I92F)(SEQ ID NO:336), (R38E/I92P)(SEQ I (R38E/I92S)(SEQ ID NO:338), (R38E/I92T)(SEQ ID NO:339), (R38E/I92W)(SEQ ID NO:340), (R38E/I92Y)(SEQ ID NO:341), (R38E/I92V)(SEQ ID NO:342), (R38E/H16E)(SEQ ID NO:343), or (R38K/D20A)(SEQ ID NO:344). SEQ ID NOs 230-344 list the group 6 antibody-attenuated hIL-2 fusion proteins numbered according to mutations in the hIL-2 sequence of the IL-2 sequence.

Binding kinetics of several purified 1H3-hIgG1-L6-hIL-2 variants of individual recombinant human CD25 and human CD122 were determined using biolayer interferometry (BLI). Briefly, binding experiments were performed at 25 °C using an Octet Red96 instrument (Pall ForteBio). C-terminal multihistidine-tagged extracellular domains of human CD25 and human CD122 were captured onto an anti-His2 sensor (Pall ForteBio). Starting at a maximum concentration of 300 nM, the receptor-loaded sensor was immersed in seven consecutive 3-fold dilutions for each 1H3-hIgG-L6-hIL-2 variant. The 1H3-hIgG1-L6-hIL-2 fusion protein was diluted in an assay buffer consisting of phosphate-buffered saline (PBMS) supplemented with 0.1% BSA and 0.02% Tween-20 (pH 7.2). The loaded sensor was regenerated using 10 mM glycine buffer (pH 1.7). Kinetic constants were calculated using a monovalent binding model.

Table 2 records the association constant (k _{_on} ), dissociation constant (k_{_off} ), and equilibrium constant (K_{_D} ) of 74 immunoglobulin-hIL-2 fusion protein variants that bind to recombinant human CD25 or recombinant human CD122.

Table 2. Binding kinetics of 1H3-hIgG-L6-hIL-2 fusion protein obtained by Octet BLI with recombinant human CD25 or CD122

Table 3 records the association constant (k _on ), dissociation constant (k _off ), and equilibrium constant (K _D ) of 74 1H3-hIgG1-L6-hIL-2 fusion proteins that bind to recombinant human CD122.

Example 3: Screening for attenuation of high-affinity and intermediate-affinity hIL-2 receptors using fixed concentrations of cell-based potency pSTAT5

As described in Protocol D, the attenuation of the antibody-attenuated hIL-2 fusion protein described in Example 2 was tested using NK-92 (expressing high-affinity hIL-2 receptor) and TF1+IL-2Rβ (expressing medium-affinity hIL-2 receptor) cell lines in a fixed-concentration pSTAT5 screening. Tables 4-8 list the fold changes in geometric mean fluorescence intensity (gMFI) of the antibody-attenuated hIL-2 fusion protein from free cytokine wild-type rhIL-2, i.e., measurements of reduced IL-2 activity. For fixed-concentration screening, the fold change was calculated by dividing the gMFI of rhIL-2 by the gMFI of the variant. For experiments with complete titration curves, the fold change relative to rhIL-2 was calculated by dividing the _EC50 value of rhIL-2 by the _EC50 of the variant. The fold change was rounded to the nearest integer. Compared to gMFI produced by rhIL-2, decreased gMFI in both the NK-92 and TF1+IL-2Rβ cell lines indicates attenuation of IL-2 activity in both high-affinity and intermediate-affinity receptors. The Group 1 variant described in Example 2 was not tested in a pSTAT5 efficacy screening based on fixed cell concentrations.

The IL-2 agonist activity of each variant tested was also evaluated and characterized as a complete or partial IL-2 agonist, or as having no IL-2 activity (inactive). A dose-titer curve of the 1H3-hIgG1-L6-hIL-2 fusion protein reaching the maximum gMFI level exhibited by the rhIL-2 positive control was considered an antibody-attenuated hIL-2 fusion protein with complete agonist activity. The percentage of partial agonist activity to complete activity was calculated using the maximum gMFI of rhIL-2 as 100%. Antibody-attenuated hIL-2 fusion proteins with less than 10% of the maximum gMFI of rhIL-2 at the highest concentration of 1200 nM were considered to have no agonist activity (inactive). Certain _EC50 values and attenuation levels could not be accurately calculated using GraphPad Prism 7 software because the activity did not reach its maximum value, and therefore these values are estimates.

pSTAT5 fixed concentration results demonstrated that although some single residue substitutions attenuated IL-2 activity on the high-affinity cell line (NK-92), a combination of substitutions that modulate the binding of both α and β chains or both α and γ chains was required to significantly attenuate IL-2 activity on the high-affinity IL-2 receptor (more than 20-fold attenuation of recombinant hIL-2).

Table 4. Fold change relative to rhIL-2 in screening with fixed concentrations of pSTAT5 on the 1H3-hIgG1-L6-hIL-2 fusion protein from Group 2

NT-1 was first tested in a complete titration with pSTAT5, and no data were available for a fixed concentration determination.

Table 5. Fold change relative to rhIL-2 in screening with fixed concentrations of pSTAT5 on the 1H3-hIgG1-L6-hIL-2 fusion protein from group 3.

NT-1 was first tested in a complete titration with pSTAT5, and no data were available for a fixed concentration determination.

Table 6. Fold change relative to rhIL-2 in screening with fixed concentrations of pSTAT5 on the 1H3-hIgG1-L6-hIL-2 fusion protein from group 4

Table 7. Fold change relative to rhIL-2 in fixed-concentration pSTAT5 screening of 1H3-hIgG1-L6-hIL-2 fusion protein from group 5

Table 8. Fold change relative to rhIL-2 in screening with fixed concentrations of pSTAT5 on the 1H3-hIgG1-L6-hIL-2 fusion protein from group 6

Example 4: Screening for attenuation of IL-2 fusion proteins in each of the high-affinity and intermediate-affinity hIL-2 receptors using cell-based efficacy pSTAT5 dose titration.

The attenuation of the selected antibody-attenuated hIL-2 fusion protein (1H3-hIgG1-L6-hIL-2 fusion protein from groups 2-6) described in Example 2 was tested in pSTAT5 titration curves using NK-92 and TF1+IL-2Rβ cell lines as described in Protocol D.

Four-parameter logistic curves were generated using gMFI of the Alexa Fluor 647pSTAT5 positive signal, and then _EC50 values were calculated using GraphPad Prism 7 software. These values were compared with a recombinant hIL-2 (rhIL-2) control as measurements of attenuation. Tables 9-13 summarize the fold changes in rhIL-2 activity calculated using gMFI of the Alexa Fluor 647 signal.

An increase in the fold change in rhIL-2 indicates the degree of decay in hIL-2 activity. The agonistic activity of each antibody-decaying hIL-2 fusion protein tested in pSTAT5 titration profiles was also evaluated and characterized as complete, partial, or inactive (inactive). Antibody-decaying hIL-2 fusion proteins that reached maximum gMFI levels, like rhIL-2, were considered variants with complete agonist activity. Partial agonist activity was calculated as described in Example 3. Inactive antibody-decaying hIL-2 fusion proteins were classified as having less than 10% activity compared to rhIL-2. Certain fold changes relative to rhIL-2 could not be accurately calculated using GraphPad Prism 7 software (indicated as not calculated or “NC”) because a complete four-parameter logistic curve was not generated, and therefore these values are estimates (labeled as ^a in Tables 9-13). However, on the graphs, these variants showed decay of more than 10,000-fold relative to rhIL-2 (data not shown). This is represented in Table 9-13 as ">10,000 on the graph; NC".

The complete titration of pSTAT5 curves demonstrated results similar to those presented in Example 3, where substitutions regulating binding to both the α and β chains significantly attenuated IL-2 activity on the high-affinity IL-2 receptor compared to single substitutions binding only the α or β chain. Furthermore, the complete titration of pSTAT5 assays was able to distinguish variants with substitutions leading to inactivation from variants exhibiting high attenuation. Finally, the comparison of dose-titer curves demonstrated a more precise attenuation level than fixed-concentration assays.

Table 9. Fold change relative to rhIL-2 and agonistic activity on the 1H3-hIgG1-L6-hIL-2 fusion protein from group 2

NT = Untested

^a = the change factor is only an estimate because a complete four-parameter logic curve has not been reached.

Table 10. Fold change relative to rhIL-2 and agonistic activity on the 1H3-hIgG1-L6-hIL-2 fusion protein variant from group 3

NT = Untested; NC = Not computed using GraphPad Prism 7

^a = the change factor is only an estimate because a complete four-parameter logic curve has not been reached.

Example 5: Testing the attenuation of high-affinity and intermediate-affinity hIL-2 receptors using cell-based proliferation assays

As described in Protocol E, the attenuation of IL-2 activity of antibody-attenuated hIL-2 fusion proteins (2D12-mIgG1-D265A-L6-hIL-2, 2D12-hIgG1-L6-hIL-2, and 1H3-hIgG1-L6-hIL-2 fusion proteins) from groups 1-6 of Example 2 was tested in proliferation assays of both the NK-92 and TF1+IL-2Rβ cell lines. The results are provided in Tables 14-19.

In this cell-based proliferation assay, selected 1H3-hIgG1-L6-hIL-2 fusion proteins from Example 4, exhibiting significant attenuation in the pSTAT5 titration curve, were tested. pSTAT5 is a downstream reading of IL-2 activity, and the assay requires only 10 minutes of stimulation, potentially providing a snapshot of IL-2-dependent activity. For the proliferation assay, cells were incubated for 3–4 days with 2D12-mIgG1-D265A-L6-hIL-2, 2D12-hIgG1-L6-hIL-2, 1H3-hIgG1-L6-hIL-2 fusion proteins, or a recombinant hIL-2 control, to provide a more physiologically relevant reading of in vivo IL-2-dependent activity. The IL-2-dependent activity of other 2D12-mIgG1-D265A-L6-hIL-2 and 2D12-hIgG1-L6-hIL-2 fusion proteins generated but not tested in the pSTAT5 assay was also determined using this proliferation assay.

Similar to cell-based pSTAT5 dosing titration experiments, the calculated _EC50 was determined based on relative luminescent units (RLU) rather than gMFI, and once the _EC50 was calculated, the results analysis was performed as in Example 4. As with the results identified in Example 4, proliferation curves demonstrated that some substitutions regulating binding to both the α and β chains significantly attenuated IL-2 activity on high-affinity receptors compared to single substitutions binding only the α or β chain. The proliferation of these selected 1H3-hIgG1-L6-hIL-2 fusion proteins in the TF1+IL-2Rβ cell line was also tested, demonstrating that some of these fusion proteins significantly attenuated IL-2 activity on intermediate-affinity receptors.

Example 6: Production of anti-hPD-1 antibodies

Several methods were used to generate a variety of different anti-hPD-1 antibodies with the desired properties.

In one approach, transgenic chickens (OmniChicken ^™ ) expressing human antibody genes (human light chain (VLCL or VKCK) and human VH) and the constant region of the chicken heavy chain were used to generate anti-hPD-1 human monoclonal antibodies (Ching et al., mAbs 2018). The transgenic chickens were immunized every 14 days with 100 μg of Fc-labeled human PD-1 protein (huPD-1-Fc) (SEQ ID NO:380) for 14 weeks. In another approach, transgenic chickens were genetically immunized six times with DNA encoding human PD-1 (SEQ ID NO:347), followed by a final boost with 100 μg of huPD-1-Fc (SEQ ID NO:380). Serum immune responses in each animal were monitored by an ELISA targeting biotinylated human PD-1 on streptavidin-coated plates.

Spleen cells were isolated from each immunized animal and tested for positive antibody clones using gel encapsulation microenvironment (GEM) assays (e.g., Mettler Izquierdo, S., Varela, S., Park, M., Collarini, E.J., Lu, D., Pramanick, S., Rucker, J., Lopalco, L., Etches, R., & Harriman, W. (2016. High-efficiency antibody discovery achieved with multiplexed microscopy. Microscopy (Oxford, UK), 65(4), 341–352)). Beads labeled with human PD-1 were screened. Positive clones were sequenced, and the variable regions of the heavy and light chains were cloned, assembled into single-chain variable fragments, and fused into the hinge and Fc regions of immunoglobulins (ScFv-Fc). These unique scFv-Fc fusion proteins were transiently expressed in Expi293 cells, and their binding activity was tested by ELISA on supernatants coated with huPD-1-Fc (SEQ ID NO:380) or cynomolgus monkey PD-1-Fc (SEQ ID NO:381). A total of 102 unique anti-human PD-1 variable weight and variable light pairings were identified using this method. 2H7-hIgG4 (SEQ ID NO:382-391, 424, and 425) and A2-hIgG4 (SEQ ID NO:402-411, 428, and 429) are antibodies identified by this method.

Other methods enabled the identification of an anti-hPD-1 antibody denoted as C51E6-hIgG4, which was germline optimized to become an antibody named C51E6-5-hIgG4 (SEQ ID NO: 392-401, 426, 427), and then humanized and further sequence optimized to become an antibody named Abz1mod-hIgG4 (SEQ ID NO: 449, 450).

As described in General Method A, anti-PD-1 variable region sequences were expressed using human IgG4κ antibodies, and their binding ability to PD-1-expressing cells was assessed using flow cytometry. First, test antibodies were screened for binding to human PD-1 using a Jurkat cell line expressing recombinant human PD-1 (Jurkat+hPD-1 cell line). Antibodies were serially diluted starting at a maximum concentration of 280 nM, and then allophycocyanin-conjugated anti-human IgG secondary antibody was added to the cells for detection. Of 92 hits, 79 tested anti-PD-1 antibodies showed _EC50 binding (by flow cytometry) <30 nM. 2H7-hIgG4 (SEQ ID NO:382-391, 424 and 425), C51E6-5-hIgG4 (SEQ ID NO:392-401, 426 and 427), A2-hIgG4 (SEQ ID NO:402-411, 428 and 429), OMC.1.B6-hIgG4 (SEQ ID NO:438 and 439), OMC.1.D6-hIgG4 (SEQ ID NO:442 and 443), OMC.2.C6-hIgG4 (SEQ ID NO:440 and 441), 1H9-hIgG4 (SEQ ID NO:576 and 525), 1D5-hIgG4 (SEQ ID NO:577 and 527), and 2A3.H7-hIgG4 (SEQ ID NO:424 and 523) are a group of antibodies used in Jurkat cell lines expressing human PD-1 (SEQ ID NO:382-391, 424 and 425), C51E6-5-hIgG4 (SEQ ID NO:392-401, 426 and 427), A2-hIgG4 (SEQ ID NO:402-411, 428 and 429), OMC.1.B6-hIgG4 (SEQ ID NO:438 and 439), OMC.1.D6-hIgG4 (SEQ ID NO:442 and 443), OMC.2.C6-hIgG4 (SEQ ID NO:440 and 441), 1H9-hIgG4 (SEQ ID NO:576 and 525), 1D5-hIgG4 (SEQ ID NO:577 and 527), and 2A3.H7-hIgG4 (SEQ ID NO:424 and 523). NO:346) was identified as an antibody with moderate to high affinity for binding to hPD-1. The EC50 for binding to recombinant hPD-1-expressing Jurkat cells, calculated by flow cytometry, was 0.1–0.3 nM for 2H7-hIgG4, 1H9-hIgG4, 1D5-hIgG4, and 2A3.H7-hIgG4 in multiple experiments. The _EC50 for binding to hPD-1-expressing Jurkat cells, calculated by flow cytometry, was 2–4 _nM for C51E6-5-hIgG4, and 3–16 nM for A2-hIgG4, OMC.1.B6-hIgG4, OMC.1.D6-hIgG4, and OMC.2.C6-hIgG4. Binding to hPD-1-specific antibodies was not observed because 2H7-hIgG4, C51E6-5-hIgG4, A2-hIgG4, 1H9-hIgG4, 1D5-hIgG4, 2A3.H7-hIgG4, OMC.1.B6-hIgG4, OMC.1.D6-hIgG4, and OMC.2.C6-hIgG4 antibodies did not bind to the parental Jurkat cell line (data not shown) that does not express hPD-1.

Example 7: Characterization of anti-hPD-1 antibody binding in the presence of anti-hPD-1#1-mIgG2b-N297A and anti-hPD-1#2-mIgG2b-N297A antibodies.

As described in General Method Protocol B, in the presence of anti-hPD-1#1-mIgG2b-N297A and anti-hPD-1#2-mIgG2b-N297A, the binding competition of 2H7-hIgG4, C51E6-5-hIgG4 and A2-hIgG4 with hPD-1 is evaluated.

As a control, titrations (nivolumab) were performed in the presence of a saturated concentration of 10 μM anti-hPD-1#1-mIgG2b-N297A (Figure 4A). Compared to the titration curves without an anti-hPD-1#1-mIgG2b-N297A competitor, the titration curves in the presence of the anti-hPD-1#1-mIgG2b-N297A competitor were significantly reduced (the titration curves shifted 100-fold to 1000-fold to the right of the graph). Adding saturated concentrations (10 μM) of anti-hPD-1#1-mIgG2b-N297A or anti-hPD-1#2-mIgG2b-N297A prior to exposure to 2H7-hIgG4, C51E6-5-hIgG4, or A2-hIgG4 did not eliminate the binding of 2H7-hIgG4, C51E6-5-hIgG4, or A2-hIgG4 to hPD-1, as shown by shifts of less than 10-fold in Figures 4B-4D. This indicates that 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 do not competitively bind to PD-1 in the presence of anti-hPD-1#1-mIgG2b-N297A or anti-hPD-1#2-mIgG2b-N297A.

Example 8: Characterization of non-antagonist hPD-1 antibody

As described in General Method Protocol C, the PD-1 antagonist activity of anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 was tested using an in vitro cell-based human PD-1/PD-L1 blocking bioassay. All antibodies except A2-hIgG4 were tested at a final concentration of 200 nM. A2-hIgG4 was tested at a final concentration of 500 nM.

None of the anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, A2-hIgG4, OMC.1.B6-hIgG4, OMC.1.D6-hIgG4, OMC.2.C6-hIgG4, 1H9-hIgG4, 1D5-hIgG4, and 2A3.H7-hIgG4 exhibited hPD-1 antagonist activity, as they all showed a mean luminescence level of 3000 relative luminescent units (RLU) and exhibited similar RLU levels to the negative control KLH-C3-hIgG4 (data not shown). In contrast, anti-hPD-1#1, a known hPD-1 antagonist that blocks the binding of hPD-L1 (SEQ ID NO:584) to hPD-1, exhibited a luminescence level exceeding 14,000 RLU (data not shown).

Example 9: hIL-2 fusion protein against hPD-1 attenuation binds to Jurkat cells expressing human PD-1

To construct expression vectors for various antibodies and antibody-attenuated hIL-2 fusion proteins, corresponding polynucleotides encoding antibody, cytokine, cytokine receptor, and adaptor sequences were generated and cloned into expression vectors. Antibodies or antibody fusion proteins were transiently expressed in human embryonic kidney (HEK) 293 cells and then purified by affinity chromatography using protein A or protein G agarose. The purified proteins were concentrated using ultracentrifugation filtration and buffer-exchanged to phosphate-buffered saline or phosphate-buffered saline containing 100 mM L-arginine and 10 mM L-histidine, after which protein concentrations were determined.

In some methods, 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 carrying stable S228P hinge mutations are directly fused to hIL-2 (df) or fused to hIL-2 at the C-terminus of the immunoglobulin heavy chain using an L6 linker. Figure 5 summarizes the illustration of these anti-PD-1-attenuation hIL-2 fusion proteins. As described in Example 2, various constructs were generated by substituting hIL-2 with hIL-2 that attenuates hIL-2 activity. As described in General Method Protocol A, the binding of the anti-hPD-1-attenuation hIL-2 fusion proteins listed in Table 20 to hPD-1 was tested using a Jurkat cell line expressing hPD-1. The variable region of 2H7-hIgG4 (SEQ ID NO: 384 and 385) was further optimized, and the isotype was converted to human IgG1 with zero-substituted L235A/G237A (LAGA, as described in WO1998/006248) to become H7-632-hIgG1-LAGA (SEQ ID NO: 414 and 415). The optimized H7-632-hIgG1-LAGA was also directly fused (df) with hIL-2 variants (hIL-2T3A/D20A/R38E/C125A; SEQ ID NO: 217) with attenuated hIL-2 activity to become H7-767 (SEQ ID NO: 412-413, 415-423, 532), and the binding of both H7-632-hIgG1-LAGA and H7-767 to hPD-1 was tested (Table 20). The _EC50 value was calculated using GraphPad Prism 7 software based on the geometric mean fluorescence intensity (gMFI) across titration concentrations.

The production of the anti-hPD-1-attenuated hIL-2 fusion protein did not reduce binding to hPD-1, and the anti-hPD-1-attenuated hIL-2 fusion protein was still able to bind to Jurkat cells expressing human PD-1. Table 20 summarizes the calculated _EC50 of the tested anti-hPD-1-attenuated hIL-2 fusion protein compared to the corresponding anti-hPD-1 antibody without the attenuated hIL-2 moiety.

Compared to anti-hPD-1 antibodies without the attenuated hIL-2 moiety, the binding of the anti-hPD-1-hIL-2 fusion protein to the _EC50 of Jurkat+hPD-1 cells increased by less than 2-fold, demonstrating that adding the attenuated hIL-2 moiety to the anti-hPD-1 antibody does not eliminate binding to human PD-1.

Example 10: Anti-hPD-1-attenuated hIL-2 fusion protein binds to hPD-1 in the presence of anti-hPD-1#1 and anti-hPD-1#2 antibodies.

As described in General Method Protocol B and Example 7, the binding of the anti-hPD-1-attenuated hIL-2 fusion protein to the hPD-1 receptor was tested in the presence of anti-hPD-1#1 and anti-hPD-1#2. The reverse experiment was also performed, in which the binding of anti-hPD-1#1-mIgG2b-N297A or anti-hPD-1#2-mIgG2b-N297A to hPD-1 was examined in the presence of saturated concentrations of the test antibody-attenuated hIL-2 fusion protein. In this manner, Jurkat cells expressing hPD-1 were seeded at 100,000 cells per well in FACS buffer, blocked with anti-human FcγR blocking agent (Mitenia Biosciences) at 4°C for 10 min, and washed. The tested antibody-attenuated hIL-2 fusion proteins 2H7-hIgG4-df-hIL-2(D20A/R38E), C51E6-5-hIgG4-L6-hIL-2(D20A/R38E), A2-hIgG4-df-hIL-2(D20A/R38E), H7-767, and the isotype control anti-DNase 1H3-hIgG4-df-hIL-2(D20A/R38E) were diluted to a final concentration of 280 nM in 100 μL FACS buffer and incubated on ice with Jurkat cells expressing hPD-1 for 1 hour. Cells were washed and resuspended on ice in FACS buffer containing six-fold sequential titrations of either anti-hPD-1#1-mIgG2b-N297A or anti-hPD-1#2-mIgG2b-N297A, starting at a maximum concentration of 50 nM. Cells were then washed and resuspended on ice in a 1:100 dilution of phycoerythrin-conjugated anti-mouse IgG light chain κ monoclonal antibody for 45 minutes. Cells were washed again and resuspended in FACS buffer containing a 1:1000 dilution of Sytox Green (Thermo Fisher Scientific). Flow cytometry analysis was performed using a BD FACS Canto II (BD Biosciences), and gMFI was calculated using FlowJo software version 10. _EC50 values were calculated using GraphPad Prism 7 software based on the gMFI of the phycoerythrin signal across titration concentrations.

In the presence of anti-hPD-1#1-mIgG2b-N297A or anti-hPD-1#2-mIgG2b-N297A, adding attenuated hIL-2 to anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, and A2-hIgG4 did not weaken the ability of anti-hPD-1 protein to bind to human PD-1, similar to the results described in Example 7 (Figures 16B-16D). H7-767 was also tested in this competitive assay, and Figure 13B shows that H7-767 continued to bind to the hPD-1 receptor in the presence of anti-hPD-1#1-mIgG2b-N297A or anti-hPD-1#2-mIgG2b-N297A. Conversely, in the presence of anti-hPD-1#1-mIgG2b-N297A or anti-hPD-1#2-mIgG2b-N297A, the binding of the positive control anti-hPD-1#1 was significantly reduced (Fig. 16A, 13A).

Figures 6A and 6B show that, for the reverse competition assay, in the presence of saturated (280 nM) anti-hPD-1-attenuated hIL-2 fusion proteins 2H7-hIgG4-df-hIL-2(D20A/R38E), C51E6-5-hIgG4-df-hIL-2(D20A/R38E), or A2-hIgG4-df-hIL-2(D20A/R38E), anti-hPD-1#1-mIgG2b-N297A (Figure 6A) and anti-hPD-1#2-mIgG2b-N297A (Figure 6B) can still bind to hPD-1 on Jurkat cells. Prior to exposure to the anti-hPD-1 fusion protein, the binding curves of these saturated anti-hPD-1-attenuated hIL-2 fusion proteins overlapped with the binding curves of anti-hPD-1#1-mIgG2b-N297A (non-competitive) or anti-hPD-1#2-mIgG2b-N297A (non-competitive). The binding curves also overlapped with the saturated negative control fusion protein 1H3-hIgG4-df-hIL-2 (D20A/R38E), which does not bind to hPD-1.

Example 11: Anti-hPD-1 attenuation hIL-2 fusion protein binds to recombinantly expressed cynomolgus monkey PD-1

The binding of anti-hPD-1-attenuated hIL-2 fusion protein to cynomolgus monkey PD-1 was tested by flow cytometry using a human embryonic kidney 293 cell line expressing SV40 large T cell antigen (HEK-293T), which was transiently transfected to recombinantly express cynomolgus monkey PD-1. For each transfection reaction, 2 million HEK-293T cells were transfected with 2 μg pCMV6-hygro-HA-cyno-PD-1(1-185) (SEQ ID NO:448) (which is a mammalian vector containing a cynomolgus monkey PD-1 extracellular domain labeled with human influenza hemagglutinin and a sequence encoding hygromycin resistance). Transfection was performed by electroporation. Transfected cells were blocked with human FcγR blocking agent and stained with a titration of anti-hPD-1-attenuated hIL-2 fusion protein. In addition, phycoerythrin-conjugated antihemagglutinin clone 15B12 was added to cells for staining transfected cells, and allophycocyanin-conjugated anti-human IgG Fc secondary clone HP6017 (BioLegend, catalog number 409306) was added to cells for staining with bound antibodies. Cells were analyzed on BD Canto II, and live, transfected (hemagglutinin-positive) cells were selected using FlowJo software version 10, and the gMFI of the allophycocyanin signal was calculated. _EC50 values were calculated based on the gMFI across titration concentrations using GraphPad Prism 7 software.

The hIL-2 fusion protein, which is anti-hPD-1 attenuation, binds to HEK-293T cells expressing cynomolgus monkey PD-1 in a similar manner to that observed on Jurkat T cells expressing human PD-1 (Fig. 17). The _EC50 values binding to HEK-293T cells expressing cynomolgus monkey PD-1 were 5 nM for 2H7-hIgG4-df-hIL-2 (D20A/R38E), 6 nM for C51E6-5-hIgG4-df-hIL-2 (D20A/R38E), and 11 nM for A2-hIgG4-df-hIL-2 (D20A/R38E). Anti-hPD-1#1 and anti-hPD-1#2 (formatted as comparative anti-hPD-1-attenuated hIL-2 fusion protein) also bound to cynomolgus PD-1 with _EC50 values of 9 nM and 2 nM, respectively, indicating that adding the attenuated hIL-2 moiety to the anti-hPD-1 antibody did not eliminate the binding to cynomolgus PD-1.

Example 12: Anti-hPD-1 attenuation hIL-2 fusion protein binds to activated primary human and cynomolgus monkey PD-1

The binding of anti-hPD-1 antibody and anti-hPD-1-attenuated hIL-2 fusion protein to activated primary T cells expressing hPD-1 was examined by flow cytometry. To test whether 2H7-hIgG4, C51E6-5-hIgG4, or A2-hIgG4 binds to native hPD-1, cryopreserved human peripheral blood mononuclear cells (PBMCs) were thawed and activated with 50 ng/mL phorbol 1,2-myristate 1,3-acetate (PMA) and 1 μg/mL iomycin to upregulate the hPD-1 receptor. Activated PBMCs were collected, blocked with a 1:50 dilution of human FcγR blocking agent (Mitenia Biotech) at 4°C for 10 min, and stained with titrated concentrations of anti-hPD-1 antibodies 2H7-hIgG4, C51E6-5-hIgG4, A2-hIgG4, anti-hPD-1#1, and isotype controls. Cells were then stained with 1:20 diluted allophycocyanin-conjugated anti-human IgG Fc to detect bound antibodies. To characterize immune subsets, a mixture of surface markers including anti-human CD3, anti-CD4, and anti-CD8 antibodies was used. Additionally, cellular expression of hPD-1, hCD25, hCD122, and hCD132 in sample fractions was examined. Cells were analyzed on a BD Fortessa (BD Biosciences), using FlowJo software version 10 to gating T cell subsets, and the gMFI of the allophycocyanin signal was calculated. _EC50 values were calculated based on the gMFI across titration concentrations using GraphPadPrism 7 software. To test binding to the anti-hPD-1-hIL-2 fusion protein, cryopreserved CD3+ T cells were activated with PMA/ionomycin and bound by flow cytometry in the same manner as described above.

The binding of human PD-1 antibody-attenuated hIL-2 fusion protein to activated cynomolgus T cells was also tested using flow cytometry. Cynomolgus PBMCs were activated with a mixture of 0.081 μM PMA and 1.34 μM iomycin. After 24 hours, cells were stained using the same procedure as for the human PD-1-bound primary cells described above, except for the use of cynomolgus cross-reactivity markers. FlowJo software version 10 was used to gate active CD3 ⁺ CD4 ⁺ or CD3 ⁺ CD8 ⁺ T cells, and the gMFI of the allophycocyanin signal was then calculated. _EC50 values were calculated using GraphPad Prism 7 software based on the gMFI across titrated concentrations of anti-hPD-1 antibody or hPD-1 antibody-attenuated hIL-2 fusion protein.

In some tested variants, the decayed hIL-2 also includes substitutions for T3A and C125A, which remove O-linked glycosylation sites and replace free cysteine residues, respectively.

Following activation with PMA and iomycin, 40–50% of CD4 ⁺ T cells were PD-1 ⁺ , while 30–40% of CD8 ⁺ T cells were PD-1 ⁺ (data not shown). The _EC50 for binding to activated human CD3 ⁺ CD4 ⁺ T cells, calculated by flow cytometry, was 0.1–0.7 nM for 2H7-hIgG4, 12 nM for C51E6-5-hIgG4, 30 nM for A2-hIgG4, and 0.04 nM for 2H7-hIgG4-df-hIL-2 (T3A/D20A/R38E/C125A). The _EC50 binding to activated human CD3 ⁺ CD8+ T cells was 0.1-0.8 nM for 2H7-hIgG4, 16 nM for C51E6-5-hIgG4, 22 nM for A2-hIgG4, and 0.03 nM for 2H7-hIgG4-df-hIL-2 (T3A/D20A/R38E/C125A). For H7-767, the _EC50 binding to activated human CD3 ⁺ CD4 ⁺ T cells was 0.19 nM, and for activated human CD3 ⁺ CD8 ⁺ T cells it was 0.12 nM. The _EC50 binding to activated cynomolgus monkey CD3 ⁺ CD4+ T cells was 0.09 nM for 2H7-hIgG4 and 0.04 nM for 2H7-hIgG4-df-hIL-2 (T3A/D20A/R38E/C125A). The _EC50 binding to activated cynomolgus monkey CD3 ⁺ CD4+ T cells was 0.08 nM for 2H7-hIgG4 and 0.03 nM for 2H7-hIgG4-df-hIL-2 (T3A/D20A/R38E/C125A). For H7-767, the _EC50 binding to activated cynomolgus monkey CD3 ⁺ CD4 ⁺ T cells was 0.26 nM, and for activated cynomolgus monkey CD3 ⁺ CD8 ⁺ T cells it was 0.24 nM. This data demonstrates that when the hPD-1 antibody is converted to an anti-hPD-1-attenuated hIL-2 fusion protein, the calculated _EC50 value for binding with activated hPD-1 remains similar to the calculated _EC50 value for binding with naked hPD-1 antibody.

The binding of H7-767 and H7-632-hIgG1-LAGA naked anti-PD-1 antibodies to primary, inactive human CD4 ⁺ and CD8 ⁺ T cells was tested by flow cytometry. Frozen human CD3 ⁺ T cells were thawed and flow cytometry was performed as described above. Neither H7-767 nor H7-632-hIgG1-LAGA naked anti-PD-1 antibodies bound to inactive human CD4 ⁺ and CD8 ⁺ T cells (data not shown).

Example 13: Quantification of the binding of anti-hPD-1 antibody and anti-hPD-1-attenuated hIL-2 fusion protein to recombinant human or cynomolgus monkey PD-1 by surface plasmon resonance (SPR).

Surface plasmon resonance binding assays were performed using a high-throughput SPR LSA ^™ to determine the binding affinity of the anti-hPD-1 antibody and the anti-hPD-1-attenuated hIL-2 fusion protein. The proteins were diluted to 2 or 10 μg/mL in 10 mM sodium acetate (pH 4.5) containing 0.01% Tween-20 and conjugated to an HC30M (Carterra Bio) chip using a sulfonyl-N-hydroxysuccinimide/1-ethyl-3-(3-dimethylamino)propylcarbodiimide (sulfonyl-NHS/EDC) conjugation chemistry, followed by blocking with ethanolamine. Non-regenerative kinetic conjugation processes were used to determine the binding kinetics with commercially available recombinant His-labeled human PD-1 and His-labeled cynomolgus monkey PD-1 (Acro Biosystems).

Anti-hPD-1 antibody and anti-hPD-1-attenuated hIL-2 fusion protein were expressed using modified human IgG1 or modified IgG4 isotypes with a κ light chain framework. Other alternatives, L235E or L235A/G237A (LAGA, as described in International Publication WO1998/006248) (numbered according to the EU numbering system), were introduced into the Fc region to eliminate the effector function of the immunoglobulin components.

The binding constants ( _ka ), dissociation constants (kd), and equilibrium constants ( _KD ) of various anti-hPD-1 antibodies and anti-hPD-1 antibody-attenuated hIL-2 fusion proteins binding to recombinant human or cynomolgus monkey PD- ₁ proteins were determined by titration curves and Carterra kinetic software. The maximum feasible SPR signal ( _Rmax ) and residual standard deviation (Res SD) were also calculated. Table 21 summarizes the results of the kinetic screening and demonstrates that adding the attenuated hIL-2 moiety to the anti-hPD-1 antibody does not modulate the binding of the PD-1 antibody to the human PD-1 or cynomolgus monkey PD-1 antigen. In individual experiments, H7-632-hIgG1-LAGA (SEQ ID NO: 414 and 415) were measured by SPR, and its steady-state equilibrium dissociation constant ( _KD ) was 1.23 × ^10⁻⁹ M, while _that of H7-767 was 1.93 × ^10⁻⁹ M.

Example 14: Determining whether anti-hPD-1 antibody and anti-hPD-1-attenuated hIL-2 fusion protein compete with anti-hPD-1#1 and anti-hPD-1#2 for binding to PD-1 by surface plasmon resonance (SPR).

The competition between anti-hPD-1 and anti-hPD-1-attenuated hIL-2 fusion proteins was determined using a sandwich assay. Antibodies and corresponding antibody-IL-2 cytokine fusion proteins were immobilized onto an HC30M chip using the amine coupling chemistry described in Example 13. Following the kinetic analysis described in Example 13, 80 nM human PD-1 (Bepsys, catalog PD-1-H5221-100 μg) was injected into the entire array. Competitive anti-hPD-1 and anti-hPD-1-attenuated hIL-2 fusion proteins (analytes) were diluted to 30 μg/mL and subsequently injected into the array, and binding parameters were assessed using SPR. Evaluation of all anti-hPD-1 and anti-hPD-1-hIL-2 fusion proteins was repeated. Some variants tested had modified human IgG1 or IgG4 light chain frameworks with additional L235E or L235A/G237A (LAGA) substitutions to eliminate the effector function of the immunoglobulins.

Screening for paired anti-hPD-1 or anti-hPD-1-attenuated hIL-2 fusion proteins allowed for the identification of two flasks, as shown in Table 22. In the presence of all antibodies and fusion proteins in group 2, antibodies and fusion proteins from group 1 were able to bind hPD-1, but competed with all members of the same group. In the presence of all antibodies and fusion proteins from group 1, antibodies and fusion proteins from group 2 were able to bind hPD-1, but competed with all members of the same group. None of the anti-hPD-1 antibodies listed in group 1 in Table 22 competed with hPD-1.

Table 22. Binning screen of anti-hPD-1 and anti-hPD-1-attenuation hIL-2 fusion proteins by SPR: Group 1 and Group 2

*Commercial source, no available sequences.

Example 15: Antagonistic effect of hPD-1-attenuating hIL-2 fusion protein against hPD-1 in the presence of anti-hPD-1#1 and anti-hPD-1#2

The antagonistic effect of the anti-hPD-1-attenuated hIL-2 fusion protein against hPD-1 was tested. The anti-hPD-1-attenuated hIL-2 fusion protein was characterized according to Universal Method Protocol C. Figure 7 illustrates these results. When compared with PD-1 antagonists, 2H7-hIgG4-df-hIL-2(D20A/R38E), C51E6-5-hIgG4-L6-hIL-2(D20A/R38E), and A2-hIgG4-df-hIL-2(D20A/R38E) were non-antagonistic to human PD-1, as evidenced by low levels of detectable luminescence. H7-632-hIgG1-LAGA and H7-767 were also tested for antagonistic activity as described in Universal Method Protocol C. Figure 15 illustrates that H7-632-hIgG1-LAGA and H7-767 do not block the interaction between hPD-L1 (SEQ ID NO:584) and the hPD-1 receptor.

For the competitive assay using the cell-based co-culture assay described in General Protocol C, some modifications were made. The anti-hPD-1-attenuated hIL-2 fusion protein sample was diluted to a fixed concentration of 400 nM, and 20 μL was added to 20 μL of titrated anti-hPD-1#1 or anti-hPD-1#2. 40 μL of the mixture was added to CHO cells. Forty (40) μL of Jurkat PD-1 effector cells were then placed over the CHO cells and the mixture of anti-hPD-1-attenuated hIL-2 fusion protein. In this competitive assay, the final concentration of saturated 100 nM anti-hPD-1-attenuated hIL-2 fusion protein was tested in combination with titrated anti-hPD-1#1 or anti-hPD-1#2. The remainder of the assay was performed as described in General Protocol C. Figures 18A and 18B demonstrate that the addition of 100 nM anti-hPD-1-attenuated hIL-2 fusion protein does not compete with the binding of anti-hPD-1#1 to hPD-L1 (SEQ ID NO: 584) during titration. The dose-titer curve of anti-hPD-1#1 remained unchanged compared to the curve without competing antibodies, indicating that the presence of anti-hPD-1-attenuated hIL-2 fusion protein does not compete with the function of anti-hPD-1#1 even at high concentrations. In the presence of 100 nM 2H7-hIgG4-df-hIL-2 (D20A/R38E) and 100 nM C51E6-5-hIgG4-L6-hIL-2 (D20A/R38E), anti-hPD-1#2 showed a 35% decrease in luminescence intensity (RLU) at higher concentrations (Figure 18B), but it is unclear whether this decrease is significant due to the extent of the standard deviation.

In the opposite experiment, anti-hPD-1#1 or anti-hPD-1#2 was diluted to a concentration of 400 nM, and 20 μL was combined with 20 μL of titrated anti-hPD-1-attenuated hIL-2 fusion protein. The anti-hPD-1-attenuated hIL-2 fusion protein was titrated sequentially, and 40 μL of the mixture was added to CHO cells, followed by coverage with 40 μL of JURKAT PD-1 effector cells. The remainder of the assay was performed as described in General Protocol C. Figures 18C and 18D demonstrate that the addition of 100 nM anti-hPD-1#1 (Figure 18C) or 100 nM anti-hPD-1#2 (Figure 18D) does not impair the ability of the anti-hPD-1-attenuated hIL-2 fusion protein as an antagonist. The observed flat curves with relative luminescent units (RLU) above 18,000 indicate no competition for antagonistic activity, and the tested anti-hPD-1-attenuation hIL-2 fusion protein still exhibits antagonistic function even in the presence of anti-hPD-1#1 or anti-hPD-1#2.

Example 16: Testing the attenuation of anti-hPD-1-attenuation hIL-2 fusion protein on high-affinity and intermediate-affinity hIL-2 receptors using cell-based proliferation assays

As described in General Protocol E, the attenuation level of hIL-2 activity of the anti-hPD-1-attenuated hIL-2 fusion protein was assessed using cell proliferation assays on NK-92 and TF1+IL-2Rβ cell lines. Control fusion proteins included those incorporating an anti-DNase I antibody (designated 1H3) having a human IgG4 or human IgG1 backbone directly fused to hIL-2 or having a linker (SEQ ID NO: 355) to demonstrate the effectiveness of the non-targeted attenuated hIL-2 fusion protein. As described in Example 2, the hIL-2 sequence of these constructs contained substitutions for attenuated hIL-2 activity. Similar to Example 3, complete, partial, or non-agonistic IL-2 activity (inactivity) was also evaluated. Some tested variants were expressed on human IgG1 or IgG4 isotypes modified with a κ light chain and had additional L235E or L235A/G237A (LAGA) substitutions in the Fc region to eliminate immunoglobulin effector function. In some antibody-cytokine fusion proteins, hIL-2 cytokine is fused to the C-terminus of the light chain (LC fusion).

The calculated _EC50 for each antibody-cytokine fusion protein was determined by relative luminescent units (RLU), and the fold change in _EC50 was calculated when compared to recombinant human IL-2 (rhIL-2). Table 23 summarizes the fold change and agonistic activity relative to rhIL-2. The agonistic activity was determined by the maximum luminescence of the antibody-attenuated hIL-2 fusion protein compared to the maximum luminescence of rhIL-2. Antibody-attenuated hIL-2 fusion proteins that reached maximum luminescence like rhIL-2 were considered variants with full activity based on their dose-titer profiles. The percentage of partial activity to full activity was calculated using the maximum luminescence of rhIL-2 as 100%. Antibody-attenuated hIL-2 fusion proteins with a maximum RLU of less than 10% of the maximum rhIL-2 RLU at the highest concentration of 1200 nM were considered to have no agonistic activity or were inactive. As shown in Table ^23a , _EC50 values were estimated only for some variants because maximum luminescence was not reached.

Example 17: Rescuing IL-2 activity of an anti-hPD-1-attenuated hIL-2 fusion protein in cell lines expressing intermediate-affinity hIL-2 receptor and hPD-1

The rescue of hIL-2 activity by the hPD-1-attenuated hIL-2 fusion protein was evaluated using a targeted cell line expressing hPD-1. Briefly, the TF1+IL-2Rβ cell line described in Universal Method Protocol D was modified to express the hPD-1 receptor (SEQ ID NO: 580) via lentiviral transduction. TF1+IL-2Rβ cells expressing hPD-1 were detected by flow cytometry using an hPD-1 antibody conjugated with Brilliant Blue 515 (BD Biosciences, catalog number 565936). Cells with low hPD-1 expression (intensity below 10³ on the Brilliant Blue 515 fluorophore) were sorted. The collections were then sorted twice more to collect cells with near-hPD-1 expression levels on activated primary cells. This cell line (TF1+IL-2Rβ+hPD-1) was aliquoted and frozen for cell-based proliferation assays. Proliferation assays were performed according to Universal Method Protocol E, with an incubation period of 3 days. Some of the tested variants have a modified human IgG1 or IgG4 light chain framework with additional L235E or L235A/G237A (LAGA) substitutions to eliminate the effector function of the immunoglobulin.

Table 24 summarizes the results of proliferation assays targeting TF1+IL-2Rβ+hPD-1 cell lines. The agonist activity was determined as complete, partial, or inactive by comparing the maximum luminescence of the antibody-attenuated hIL-2 fusion protein to the maximum luminescence of rhIL-2. A dose-titer curve of the antibody-attenuated hIL-2 fusion protein at maximum rhIL-2 luminescence was considered a variant with complete activity. The percentage of partial activity to complete activity was calculated using maximum rhIL-2 luminescence as 100%. Antibody-attenuated hIL-2 fusion proteins with less than 10% of the maximum rhIL-2 RLU at the highest concentration of 1200 nM were considered to have no agonist activity or be inactive. For some variants, the _EC50 values were estimates only because a complete curve was not reached. Many instances of anti-hPD-1-hIL-2 fusion proteins with attenuated hIL-2 showed rescued hIL-2 activity in the targeted cell lines, while the non-targeted antibody control (denoted as 1H3) did not demonstrate rescued hIL-2 activity. Complete recovery is demonstrated by reducing the factor of change relative to rhIL-2 to a value of 0 or 1.

Table 24. Folds of change relative to rhIL-2 and agonistic activity of the antibody-hIL-2 fusion protein on the TF1+IL-2Rβ+hPD-1 cell line (a human PD-1 expression cell line with intermediate affinity for IL-2R).

NC = Not computed using GraphPad Prism 7

^a = the change factor is only an estimate because a complete four-parameter logic curve has not been reached.

Example 18: Evaluation of alternative anti-hPD-1 attenuation hIL-2 fusion protein blocking or not blocking mouse PD-L1 in an in vivo mouse colon adenocarcinoma (MC38) model

Because there is no universally accepted model for exploring the in vivo efficacy of tumor therapy in primates, an alternative anti-mPD-1-attenuated hIL-2 fusion protein was developed and tested in a syngeneic mouse tumor model. This MC38 colon adenocarcinoma model is commonly used to test the efficacy of immuno-oncology therapies. To investigate the in vivo effects of the anti-PD-1-attenuated hIL-2 fusion protein, an alternative anti-mouse PD-1 antibody, RMP1-14 (known to block mouse PD-L1 binding) and RMP1-30 (described as a non-blocker of mouse PD-L1), were fused to the C-terminus of the mouse IgG2b-N297A heavy chain with attenuated hIL-2 and tested in the MC38 colon adenocarcinoma model. The hIL-2 moiety includes substitutions for F42K, Y45R, and V69R, which were tested in the IL-2-dependent mouse T-lymphoblastic cell line (CTLL-2) and demonstrated to attenuate mouse IL-2 activity. Human IL-2 can stimulate the proliferation of mouse T cells at similar concentrations; however, the same substitutions that weaken the activity of human IL-2-dependent cell lines do not weaken the activity of the CTLL-2 cell line (data not shown). Therefore, F42K/Y45R/V69R substitutions were used as alternatives to hIL-2 because they showed attenuated IL-2 activity in mouse cell lines. Sequences containing the heavy and light chain variable regions of anti-mouse PD-1 antibodies RMP1-14 and RMP1-30 (e.g., Matsumoto K et al., J Immunol. 2004 Feb 15; 172(4):2530-41) were also formed on a mouse IgG2b-N297A background to produce anti-mPD-1RMP1-14 mIgG2b-N297A (SEQ ID NO: 564 and 566) and anti-mPD-1RMP1-30 mIgG2b-N297A (SEQ ID NO: 567 and 568). The mouse IgG2b isotype with N297A substitution is the mouse equivalent of the Fc isotype with deactivated Fc immune effector function. Standard techniques were used to generate, express, and purify the alternative antibodies and antibody-attenuated hIL-2 fusion proteins.

In this mouse tumor model, 5 × ^10⁵ MC38 colorectal cancer cells were injected into the right ventral region of ten-week-old female C57BL/6NCrl (Charles River) mice. When the tumor reached 80-120 ^mm³ , the mice were sorted into groups (n=10 per group) and treatment began on day 1 of the study. Anti-mPD-1RMP1-14mIgG2b-N297A, anti-mPD-1RMP1-30mIgG2b-N297A, anti-mPD-1RMP1-14mIgG2b-N297A-L6-hIL-2(F42K/Y45R/V69R) (SEQ ID NO:565 and 566), and anti-mPD-1RMP1-30mIgG2b-N297A-L6-hIL-2(F42K/Y45R/V69R) (SEQ ID NO:568 and 569), along with a mediator control (phosphate-buffered saline), were administered intraperitoneally at a dose of 5 mg/kg twice weekly for 4 weeks. During the study period, tumor size was measured twice weekly using calipers using the formula ( ^w² × L)/2, where w = width and L = length. The study endpoint is a tumor volume of 1000 ^mm³ or survival on day 50, whichever comes first.

Figure 8 shows that although administration of anti-mPD-1RMP1-14-mIgG2b-N297A or anti-mPD-1RMP1-30-mIgG2b-N297A antibodies alone did not significantly improve efficacy compared to the mediator control, administration of the anti-mPD-1RMP1-14 mIgG2b-N297A-L6-hIL-2(F42K/Y45R/V69R) or anti-mPD-1RMP1-30 mIgG2b-N297A-L6-hIL-2(F42K/Y45R/V69R) anti-PD-1-attenuated hIL-2 fusion protein was associated with 90% and 100% complete tumor regression, respectively. These data demonstrate that the anti-tumor efficacy mediated by the anti-mPD-1-hIL-2(F42K/Y45R/V69R) fusion protein does not require PD-1 checkpoint blockade and that this efficacy depends on hIL-2 activity. The data further demonstrate that antibody-mediated T cells targeting PD-1 are sufficient to promote effective anti-tumor efficacy in the MC38 tumor model.

Example 19: Expansion of effector memory CD8+ T cells in an in vivo mouse colon adenocarcinoma model by replacing hIL-2 fusion protein with anti-hPD-1 attenuation alternative.

To understand the mechanism of action of the alternative anti-hPD-1 attenuation hIL-2 fusion protein in vivo, in vivo experiments similar to those in Example 18 were performed, followed by immunophenotypic analysis of T cell populations obtained from tumors, blood, spleen, and lymph nodes after three doses. Five × ^10⁵ mouse MC38 colon adenocarcinoma tumor cells were subcutaneously implanted into the right ventral region of ten-week-old female C57BL/6NCrl (Chasliva) mice, and tumor growth was monitored. Animals with tumors ranging from 150 to 260 ^mm³ were divided into four groups of 10 mice each for the study. Twenty-one days post-implantation, on days 1, 4, and 8, animals were intraperitoneally administered 0.2 mL/dose of phosphate-buffered saline (PBS) as a mediator control, 5 mg/kg of anti-KLH-C3-mIgG2b-N297A-L6-hIL-2 (F42K/Y45R/V69R), 5 mg/kg of anti-mPD-1RMP1-30mIgG2b-N297A, or 5 mg/kg of anti-mPD-1RMP1-30mIgG2b-N297A-L6-hIL-2 (F42K/Y45R/V69R). On day 9, tumors, spleens, and inguinal lymph nodes were collected from all mice and processed into single-cell suspensions for subsequent flow cytometry analysis.

Figure 9A plots tumor volume growth ( ^mm³ ) over 9 days from the first administration on day 1, where each point represents the mean of 10 mice. By day 8, tumor volume was reduced in the anti-mPD-1RMP1-30mIgG2b-N297A-L6-hIL-2 (F42K/Y45R/V69R) group compared to other treatment groups. Figure 9B summarizes the contributions of various CD8 ⁺ T cell subsets in tumors in each treatment group. The T central memory phenotype is CD45 ⁺ CD3 ⁺ CD4 ^- CD8 ⁺ CD44 ⁺ CD127 ⁺ CD69 ^- CD103, the T effector memory is CD45 ⁺ CD3 ⁺ CD4 ^- CD8 ⁺ CD44 ⁺ CD127 ⁺ CD69 ^- CD103 ^{- CD62L-} , the T resident memory is CD45 ⁺ CD3 ⁺ CD4 ^- CD8 ⁺ CD44 ⁺ CD127 ⁺ CD69 ⁺ CD103 ⁺ ,CD44 ^- CD62L- ^, the T cell type is CD45 ⁺ CD3 ⁺ CD4 ^- CD8 ⁺ CD44 ^{+ CD62L-} , and the primary T type is CD45 ⁺ CD3 ⁺ CD4 ^- CD8 ⁺ CD44 ^- CD62L ⁺ . Compared to other treatment groups, mice treated with anti-mPD-1RMP1-30 mIgG2b-N297A-L6-hIL-2 (F42K/Y45R/V69R) showed an increase in the CD8 ⁺ T effector memory subset, as indicated by the increase in the light gray slice in Figure 9B. This is also illustrated in Figure 9C by the absolute count (cells/μL) within the tumor cleaved by MC38. Furthermore, within the tumor, the absolute count (cells/μL) of regulatory T cells, defined as those expressing the CD45+CD3+CD4+CD8-CD25+FoxP3+ label, was reduced.

The expansion of CD8 ⁺ T effector cell memory and the reduction of regulatory T cells are associated with effective immunotherapy in mice and humans.

Example 20: In the NCG-PBMC model, the hIL-2 fusion protein resistant to hPD-1 attenuation is active in vivo.

Transplanting human immune cells into NOD-Prkdc ^em26Cd52 IL-2rg ^em26Cd22 /NjuCrl (NCG) mice lacking functional T, B, and NK cells has become a valuable tool for evaluating the efficacy of hypothetical therapies that stimulate human T cells. In this model, if the therapeutic agent activates human T cells, it leads to T cell proliferation and an acceleration of graft-versus-host disease (GvHD).

Over a 4-week period, the transplantation kinetics and expression of human PD-1 and human IL-2 receptors on T cells from three independent donors for human peripheral monocyte (hPBMC) transplantation were evaluated. Of the three donors tested, the one that induced the most T cells and had the GvHD intermediate window was selected. 1.5 x 10⁷ hPBMCs were intravenously injected into NCG mice, which were randomly divided into 8 groups of 8–16 mice each. Mice were intraperitoneally injected on days 7, 10, and 14 with three doses of 2H7-hIgG1-LAGA-df-hIL-2(T3A/D20A/R38E/C125A) (SEQ ID NO:471, 425) (2.5 mg/kg, 5 mg/kg, or 10 mg/kg), 1H3-hIgG1-LAGA-df-hIL-2(T3A/D20A/R38E/C125A) (SEQ ID NO:546, 374) (5 mg/kg or 10 mg/kg), 1H3-hIgG1-LAGA-df-hIL-2(T3A/C125A) (SEQ ID NO:563, 374) (10 mg/kg), or 2H7-hIgG1-LAGA-df-hIL-2.

(T3A/R38E/I92K/C125A)(SEQ ID NO:474,425)(5mg/kg). Anti-DNase fusion proteins of wild-type hIL-2 (1H3-hIgG1-LAGA-df-hIL-2(T3A/C125A)) and those with attenuated hIL-2 moieties (1H3-hIgG1-LAGA-df-hIL-2(T3A/D20A/R38E/C125A)) were used as non-targeted antibody controls. Although the 1H3-hIgG1-LAGA-df-hIL-2 (T3A/C125A) fusion protein does not differ from the hIL-2 moiety that reduces hIL-2 activity, it does contain T3A and C125A substitutions to remove the predicted O-linked glycosylation site on human IL-2 (see, for example, International Publication No. WO2012/107417) and unpaired cysteine residues (see, for example, International Publication No. WO2018/184964), respectively. These substitutions have not shown reduced hIL-2 potency clinically. On day 21, blood, spleen, and lungs were collected, with the blood and spleen processed for flow cytometry immunophenotypic analysis, while the lungs were weighed.

Twenty-one days later, flow cytometry immunophenotyping was performed on the animals' blood and spleen. Table 25 summarizes the markers used to characterize the human T cell population for subsequent analysis.

Table 25. Phenotypic markers defining human T cell subsets in NCG-PBMC mice

cell population Phenotypic markers pan-T cells CD3+ CD8+ Original CD3+CD4-CD8+CD45RO-CCR7+ CD8+ effector CD3+CD4-CD8+CD45RO-CCR7- CD8+ effector memory CD3+CD4-CD8+CD45RO+CCR7- CD8+ Central Memory CD3+CD4-CD8+CD45RO+CCR7+ CD4+primary CD3+CD4+CD8-CD45RO-CCR7+ CD4+ effector CD3+CD4+CD8-CD45RO-CCR7- CD4+ effector memory CD3+CD4+CD8-CD45RO+CCR7- CD4+ Central Memory CD3+CD4+CD8-CD45RO+CCR7+ Regulatory T cells CD3+CD4+CD8-CD25+Foxp3+ NK cells CD3-CD56+

As shown in Figure 10, the body weight of each individual animal was measured at 21 days and normalized to day 1 for assessment of graft-versus-host disease (GvHD). Accelerated GvHD was observed in mice treated with 10 mg/kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A/D20A/R38E/C125A). Slight weight loss was also observed in mice treated with 2.5 mg/kg, 5 mg/kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A/D20A/R38E/C125A), and 5 mg/kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A/R38E/I92K/C125A). Although weight loss was observed in mice treated with 1H3-hIgG1-LAGA-df-hIL-2 (T3A/C125A), this did not persist.

Flow cytometry analysis was associated with the observed accelerated graft-versus-host disease (GvHD). Using phenotypic markers depicting human T cell subsets provided in Table 25, flow cytometry analysis of peripheral blood showed only slight expansion of CD3+, CD4+, and CD8+ T cell subsets in mice treated with 2.5 mg/kg and 5 mg/kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A/D20A/R38E/C125A) and mice treated with 10 mg/kg of 1H3-hIgG1-LAGA-df-hIL-2 (T3A/C125A) (quantified by fold changes in CD3+ T cells relative to the mediator control between 10 and 50 folds). Furthermore, in mice treated with 10 mg/kg of 2H7-hIgG1-LAGA-df-hIL-2...

In the peripheral blood of mice treated with (T3A/D20A/R38E/C125A), CD3 ⁺ , CD4 ⁺ , and CD8 ⁺ T cell subsets were significantly expanded (for CD3 ⁺ T cells, the fold change relative to the drug control was greater than 50-fold). Table 26 summarizes the expanded human T cell subsets.

In addition to assessing CD3 ⁺ , CD4 ⁺ , and CD8 ⁺ T cells across treatment groups, memory and primary subsets of CD4 ⁺ and CD8 ⁺ T cells were also evaluated. Table 25 summarizes the phenotypic markers used to characterize primary, effector, effector memory, and central memory of CD4+ and CD8+ T cells. There were no changes in primary, effector, or central memory T cells across treatment groups (data not shown). However, mice treated with 10 mg/kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A/D20A/R38E/C125A) showed a significant expansion of CD4+ and CD8+ effector memory (EM) T cells in peripheral blood, with mean numbers of CD8+ T cells greater than 5 million per mL and CD4+ T cells greater than 50 million (Figures 11A and 11B). Box-and-whisker plots were drawn using boxes around the first and third quartiles, with horizontal lines representing median values and lines representing minimum and maximum points. In animals treated with 2.5 mg/kg and 5 mg/kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A/D20A/R38E/C125A) and 1H3-hIgG1-LAGA-df-hIL-2 (T3A/C125A), moderate expansion of CD8 ⁺ effector memory (EM)T cells was observed, defined as an average cell count of 1 million to 5 million per milliliter. In mice treated with 2.5 mg/kg and 5 mg/kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A/D20A/R38E/C125A), moderate expansion of CD4 ⁺ effector memory (EM)T cells was observed, with ^a count of 6 million to 13 million per milliliter.

In addition to stimulating effector T cells, IL-2 has also been described as stimulating NK cells and regulatory T cells (Tregs). These immune cell types were also evaluated because Tregs express high levels of CD25 and NK cells express CD122. Figure 12 illustrates that animals treated with the highest dose of 10 mg/kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A/D20A/R38E/C125A) did not expand human regulatory T cells, but instead had the lowest percentage of regulatory T cells in the animal's peripheral blood (as defined by phenotype in Table 25). A dose-dependent reduction in human regulatory T cells was observed, and compared to a mediator control with an average of 1.6% of human CD3+ T cells as tregs, 10 mg/kg of 2H7-hIgG1-LAGA-df-hIL-2 (T3A/D20A/R38E/C125A) had an average of 0.16% of human CD3 ⁺ T cells as tregs. Compared with the mediator control, the percentage of human NK cells in peripheral blood (phenotype as defined in Table 25) did not change in any of the treatment groups (data not shown).

Example 21: Non-clinical safety of hIL-2 fusion protein against hPD-1 attenuation

Cynomolgus monkeys have previously been used to evaluate the toxicity of unmodified IL-2. Lethality of exogenous recombinant IL-2 at doses as low as 50 μg/kg/day has been observed in cynomolgus monkeys. Since the binding of H7-767 to cynomolgus monkey hPD-1 on primary activated PBMCs was confirmed by flow cytometry (Example 12), single-dose studies were conducted to preliminarily assess the safety of variants of H7-767 (H7-02-hIgG1-LAGA-df-hIL-2)(T3A/D20A/R38E/C125A) (SEQ ID NO: 582 and 583) and H7-767. H7-02-hIgG1-LAGA-df-hIL-2 (T3A/D20A/R38E/C125A) was delivered to eight monkeys via intravenous infusion over 15 minutes at doses of 1 mg/kg (4 animals) or 10 mg/kg (4 animals). Sampling was performed 360 hours after infusion. No adverse reactions, total toxicity, weight loss, or lethality were observed (data not shown). Subsequent single-dose studies using H7-767 were conducted at higher doses of 5 mg/kg and 50 mg/kg, similar to the first study, with sampling at 360 hours after infusion. Again, no adverse reactions, total toxicity, weight loss, or lethality were observed (data not shown).

Example 22: Attenuation of IL-2 activity in modified hIL-2 protein

As described in Example 5 above, the attenuation of IL-2 activity of modified hIL-2 protein, comprising a substitution at amino acid position 20 (D20) and a substitution at amino acid position 38 (R38), was tested in proliferation assays of NK-92 and TF1+IL-2Rβ cell lines. Based on the maximum agonist activity of the modified hIL-2 protein and the level of potency attenuation against intermediate and high affinity receptors relative to unmodified recombinant hIL-2 (Table 27), the modified hIL-2 proteins were divided into 7 groups (1 to 7). The criteria used for grouping the modified hIL-2 proteins were:

●Group 1: Variants with the highest attenuation (i.e., >10,000-fold) and at least about 80% activity to intermediate affinity receptors, but also with high attenuation and at least about 70% activity to high affinity receptors.

●Group 2: Variants with at least about 70% activity and >1,000-fold attenuation to intermediate affinity receptors and about 20% to about 30% activity to high affinity receptors.

●Group 3: Variants with approximately 50% to 70% activity and >1,000-fold attenuation to intermediate affinity receptors and approximately 20% activity to high affinity receptors.

●Group 4: Variants with at least about 70% activity but only >500-fold attenuation to intermediate affinity receptors and about 50% activity to high affinity receptors.

●Group 5: Variants that have at least about 70% activity against both receptors but exhibit a decrease in activity of >10 to >300 times against intermediate affinity receptors and a decrease in activity of 70 to 1500 times against high affinity receptors.

●Group 6: Variants with only about 30% activity and >2,500-fold attenuation to intermediate affinity receptors and no activity to high affinity receptors.

●Group 7: Variants that are inactive against both intermediate-affinity and high-affinity receptors.

Table 27. Fold change relative to rhIL-2 and agonistic activity of modified hIL-2 protein in cell-based proliferation assays, including substitutions at amino acid position 20 (D20) and amino acid position 38 (R38).

Example 23: Activity of Alternative Fusion Proteins in a Mouse MC38 Colorectal Tumor Model

Ten-week-old female C57BL/6NCrl mice were injected with 5 × ^10⁵ syngeneic MC38 colorectal cancer cells into the right ventral region. When tumors reached 80–120 ^mm³ , mice were sorted into groups (n=10/group) and treatment began on day 1 of the study. From day 1, all drugs except hIL-2 were administered intraperitoneally at a dose of 5 mg/kg twice weekly for 4 weeks. From days 1–5, hIL-2 was administered intraperitoneally at 36,000 IU once daily. Tumor size was measured twice weekly using calipers during the study. The study endpoint was a tumor volume of 1000 ^mm³ , survival at day 50, or progression-free survival at day 70, whichever came first.

All tested agents, including antibody molecules and antibody-hIL-2 fusion proteins, were produced using the mouse IgG2b Fc region with a single N297A amino acid substitution at position 297. This prevents Fc region glycosylation and significantly reduces the function of any Fc region-mediated immune effectors, thereby preventing cellular exhaustion in vivo. Anti-mPD-1RMP1-14 is a monoclonal antibody antagonist of the mouse PD-1 receptor (Matsumoto, *Journal of Immunology* 172:2530-2541, 2004). Anti-mPD-1RMP1-14-hIL-2F42K/Y45R/V69R is a bifunctional fusion protein composed of a monoclonal RMP1-14 antibody antagonist of the mouse PD-1 receptor, fused at its C-terminus to hIL-2F42K/Y45R/V69R (SEQ ID NO: 621), a reduced-potency IL-2 variant, via a flexible six-amino acid glycine/serine linker. This molecule is engineered to directly target the reduced-potency hIL-2 variant to PD-1-expressing T cells in mice. Anti-KLH-hIL-2F42K,Y45R,V69R is a control fusion protein composed of an isotype control monoclonal antibody recognizing a non-mammalian antigen (keyhole cyanin, KLH), fused at its C-terminus to the reduced-potency IL-2 variant hIL-2F42K,Y45R,V69R via a flexible six-amino acid glycine/serine linker.

The results are presented in Figure 19. The MC38 colorectal tumor model was particularly sensitive to antibody-mediated PD-1 receptor inhibition. Although tumors rapidly reached the study endpoint in mice treated with the drug, 50% of mice treated with anti-mPD-1RMP1-14 experienced complete tumor regression. In contrast, 100% of mice treated with the anti-mPD-1RMP1-14-hIL-2F42K,Y45R,V69R fusion protein experienced durable long-term tumor regression. Mice treated with various combinations of the components of the anti-mPD-1RMP1-14-hIL-2F42K, Y45R, V69R fusion protein, including combinations of anti-mPD-1RMP1-14 with free hIL-2 cytokines (administered at doses and regimens comparable to human treatment) or anti-mPD-1RMP1-14 with the non-targeted anti-KLH-hIL-2F42K, Y45R, V69R fusion protein, did not summarize the efficacy observed with anti-mPD-1RMP1-14-hIL-2F42K, Y45R, V69R. These data suggest that targeting PD-1-expressing cells with reduced hIL-2 potency significantly improves antitumor efficacy compared to anti-PD-1 receptor antagonists, and that the activity of the fusion protein is not due to the additive effect of the individual components of the molecule.

Example 24: Evaluation of protective antitumor immunity induced by alternative anti-mPD-1RMP1-14-hIL-2F42K, Y45R, and V69R in an MC38 colorectal tumor model

Mice that experienced complete tumor regression and survived to day 50 in the primary tumor study described in Example 23 were subjected to secondary tumor challenge without any additional drug treatment. For tumor re-challenge, the mice were transplanted into the left ventral side of the primary tumor site, contralateral to the site of the primary tumor, which contained 5 × ^10⁵ MC38 tumor cells. As a control group, 10 age-matched tumor-naïve mice were also implanted with MC38 tumor cells.

Figure 20 shows that all mice that had previously experienced complete tumor regression in the primary tumor study and survived to day 50 after treatment with anti-mPD-1RMP1-14-hIL-2F42K, Y45R, and V69R were completely protected from secondary tumor development. In contrast, all tumor-free mice transplanted with MC38 tumor cells continued to develop tumors, rapidly reaching the study endpoint of a tumor volume of 100 ^mm³ . The development of protective anti-tumor immunity in the absence of continuous drug treatment indicates that anti-mPD-1RMP1-14-hIL-2F42K, Y45R, and V69R induced an anti-tumor memory T cell response.

Those skilled in the art will understand that many changes and modifications can be made to the preferred embodiments disclosed herein, and that these changes and modifications can be made without departing from the spirit of the invention. Therefore, the appended claims are intended to cover all such equivalent changes that fall within the true spirit and scope of the invention.

The disclosure of each patent, patent application, and publication cited or described in this document is hereby incorporated in its entirety by reference.

Example

The following list of examples is intended to supplement, rather than replace or supersede, the previous descriptions.

Example 1. A modified human interleukin-2 (hIL-2) protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, comprising a substitution at amino acid position 20 and a substitution at amino acid position 38, wherein, relative to the unmodified hIL-2, the modified hIL-2 protein exhibits reduced potency against both high-affinity and intermediate-affinity hIL-2 receptors.

Example 2. The modified hIL-2 protein according to Example 1, wherein the substitution at amino acid position 20 is selected from D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution.

Example 3. The modified hIL-2 protein according to Example 1 or 2, wherein the substitution at amino acid position 38 is selected from R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D and R38K substitutions.

Example 4. The modified hIL-2 protein according to any one of the preceding examples, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, further comprises a deletion or substitution at amino acid position 3.

Example 5. The modified hIL-2 protein according to Example 4, wherein the substitution at position 3 of amino acid is T3A.

Example 6. The modified hIL-2 protein according to any one of the preceding examples, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, further comprises a deletion or substitution at amino acid position 125.

Example 7. The modified hIL-2 protein according to Example 6, wherein the substitution at amino acid position 125 is C125A.

Example 8. The modified hIL-2 protein according to any one of the preceding examples, wherein the modified hIL-2 protein exhibits approximately 1,000-fold reduced potency against the high-affinity IL-2 receptor (hIL-2Rαβγ).

Example 9. A modified hIL-2 protein according to any one of the preceding examples, wherein the modified hIL-2 protein exhibits approximately 10,000-fold reduced potency against the intermediate-affinity IL-2 receptor (hIL-2Rβγ).

Example 10. The modified hIL-2 protein according to any one of Examples 1 to 9, wherein the modified hIL-2 protein is fused with an anti-PD-1 antibody or its antigen-binding fragment.

Example 11. The modified hIL-2 protein according to Example 10, wherein the modified hIL-2 protein is fused to the antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment.

Example 12. The modified hIL-2 protein according to Example 10 or 11, wherein the modified hIL-2 protein is fused directly to the antibody or its antigen-binding fragment via a peptide bond.

Example 13. The modified hIL-2 protein according to Example 12, wherein the modified hIL-2 is fused directly to the C-terminal amino acid residue of the antibody heavy chain via a peptide bond.

Example 14. The modified hIL-2 protein according to Example 10 or 11, wherein the modified hIL-2 protein is fused to the antibody or its antigen-binding fragment via a linker.

Example 15. A modified human interleukin-2 (hIL-2) protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, comprising a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution at amino acid position 20 and an R38E substitution at amino acid position 38.

Example 16. The modified hIL-2 protein according to Example 15, comprising the amino acid sequence of any one of SEQ ID NO: 307, 607-611, 614, 617 or 620.

Example 17. The modified hIL-2 protein according to Example 15 or 16, comprising D20A substitution and R38E substitution.

Example 18. The modified hIL-2 protein according to Example 17, comprising the amino acid sequence of SEQ ID NO:149.

Example 19. The modified hIL-2 protein according to any one of Examples 15 to 18, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, further comprises a deletion or substitution at amino acid position 3.

Example 20. The modified hIL-2 protein according to Example 19, wherein the substitution at position 3 of amino acid is T3A.

Example 21. The modified hIL-2 protein according to Example 20, comprising the amino acid sequence of SEQ ID NO:216.

Example 22. The modified hIL-2 protein according to Example 19, comprising the amino acid sequence of SEQ ID NO:218.

Example 23. The modified hIL-2 protein according to any one of Examples 15 to 22, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, further comprises a deletion or substitution at amino acid position 125.

Example 24. The modified hIL-2 protein according to Example 23, wherein the substitution at amino acid position 125 is C125A.

Example 25. The modified hIL-2 protein according to Example 24, comprising the amino acid sequence of SEQ ID NO: 215, 217 or 219.

Example 26. The modified hIL-2 protein according to Example 25, comprising the amino acid sequence of SEQ ID NO:217.

Example 27. The modified hIL-2 protein according to any one of Examples 15 to 26, wherein the modified hIL-2 protein is fused with an anti-PD-1 antibody or its antigen-binding fragment.

Example 28. The modified hIL-2 protein according to Example 27, wherein the modified hIL-2 protein is fused to the antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment.

Example 29. The modified hIL-2 protein according to Example 27 or 28, wherein the modified hIL-2 protein is fused directly to the antibody or its antigen-binding fragment via a peptide bond.

Example 30. The modified hIL-2 protein according to Example 29, wherein the modified hIL-2 is fused directly to the C-terminal amino acid residue of the antibody heavy chain via a peptide bond.

Example 31. The modified hIL-2 protein according to Example 27 or 28, wherein the modified hIL-2 protein is fused to the antibody or its antigen-binding fragment via a linker.

Example 32. A human antibody molecule or its antigen-binding fragment thereof, wherein the human antibody molecule or its antigen-binding fragment binds specifically to human programmed cell death protein-1 (hPD-1), wherein the human antibody molecule or its antigen-binding fragment comprises:

a) Heavy chain complementarity-determining region 1 (CDR1) containing the amino acid sequence of SEQ ID NO:418, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:419, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:420, light chain CDR1 containing the amino acid sequence of SEQ ID NO:421, light chain CDR2 containing the amino acid sequence of SEQ ID NO:422, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:423;

b) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:388, light chain CDR1 containing the amino acid sequence of SEQ ID NO:389, light chain CDR2 containing the amino acid sequence of SEQ ID NO:390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:391;

c) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:398, light chain CDR1 containing the amino acid sequence of SEQ ID NO:399, light chain CDR2 containing the amino acid sequence of SEQ ID NO:400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:401; or

d) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:406, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:407, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:408, light chain CDR1 containing the amino acid sequence of SEQ ID NO:409, light chain CDR2 containing the amino acid sequence of SEQ ID NO:410, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:411.

Example 33. The human antibody molecule or its antigen-binding fragment according to Example 32 comprises:

a) The heavy chain variable region containing the amino acid sequence of SEQ ID NO:416 and the light chain variable region containing the amino acid sequence of SEQ ID NO:417;

b) The heavy chain variable region containing the amino acid sequence of SEQ ID NO:384 and the light chain variable region containing the amino acid sequence of SEQ ID NO:385;

c) The heavy chain variable region containing the amino acid sequence of SEQ ID NO:394 and the light chain variable region containing the amino acid sequence of SEQ ID NO:395; or

d) The heavy chain variable region containing the amino acid sequence of SEQ ID NO:404 and the light chain variable region containing the amino acid sequence of SEQ ID NO:405.

Example 34. The human antibody molecule or its antigen-binding fragment according to Example 32 or 33 contains the constant region of the human IgG1 heavy chain.

Example 35. The human antibody molecule or its antigen-binding fragment according to Example 34, according to the EU designation, contains L235A substitution and G237A substitution.

Example 36. A human antibody molecule or antigen-binding fragment thereof according to any one of Examples 32 to 35, comprising:

a) The heavy chain containing the amino acid sequence of SEQ ID NO:414 and the light chain containing the amino acid sequence of SEQ ID NO:415;

b) The heavy chain containing the amino acid sequence of SEQ ID NO:424 and the light chain containing the amino acid sequence of SEQ ID NO:425;

c) The heavy chain containing the amino acid sequence of SEQ ID NO:426 and the light chain containing the amino acid sequence of SEQ ID NO:427; or

d) The heavy chain containing the amino acid sequence of SEQ ID NO:428 and the light chain containing the amino acid sequence of SEQ ID NO:429.

Example 37. The human antibody molecule or its antigen-binding fragment according to Example 36, comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO:414 and a light chain comprising the amino acid sequence of SEQ ID NO:415.

Example 38. A human antibody molecule or antigen-binding fragment thereof according to any one of Examples 32 to 37, fused with a modified human interleukin-2 (hIL-2) protein, wherein the modified hIL-2 protein comprises a substitution at amino acid position 20 and a substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345.

Example 39. The human antibody molecule or its antigen-binding fragment according to Example 38, wherein the modified hIL-2 protein comprises the amino acid sequence of any one of SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617 or 620.

Example 40. The human antibody molecule or its antigen-binding fragment according to Example 39, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:149.

Example 41. A human antibody molecule or antigen-binding fragment thereof according to any one of Examples 38 to 40, wherein the modified hIL-2 protein further comprises a deletion or substitution at amino acid position 3 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345.

Example 42. The human antibody molecule or its antigen-binding fragment according to Example 41, wherein the substitution at position 3 of the amino acid is T3A.

Example 43. The human antibody molecule or its antigen-binding fragment according to Example 42, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:216.

Example 44. The human antibody molecule or its antigen-binding fragment according to Example 41, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:218.

Example 45. A human antibody molecule or antigen-binding fragment thereof according to any one of Examples 38 to 44, wherein the modified hIL-2 protein further comprises a deletion or substitution at amino acid position 125 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345.

Example 46. The human antibody molecule or its antigen-binding fragment according to Example 45, wherein the substitution at position 125 of the amino acid is C125A.

Example 47. The human antibody molecule or its antigen-binding fragment according to Example 46, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 215, 217 or 219.

Example 48. The human antibody molecule or its antigen-binding fragment according to Example 47, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:217.

Example 49. A human antibody molecule or antigen-binding fragment thereof according to any one of Examples 38 to 48, wherein the modified hIL-2 protein is fused to the antibody or antigen-binding fragment thereof at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment.

Example 50. A human antibody molecule or antigen-binding fragment thereof according to any one of Examples 38 to 49, wherein the modified hIL-2 protein is directly fused to the antibody or antigen-binding fragment thereof via a peptide bond.

Example 51. The human antibody molecule or its antigen-binding fragment according to Example 50, wherein the modified hIL-2 protein is directly fused to the C-terminal amino acid residue of the antibody heavy chain via a peptide bond.

Example 52. A human antibody molecule or antigen-binding fragment thereof according to any one of Examples 38 to 49, wherein the modified hIL-2 protein is fused to the antibody or antigen-binding fragment via a linker.

Example 53. An immunoconjugate comprising:

(a) A modified human interleukin-2 (hIL-2) protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, comprising a substitution at amino acid position 20 and a substitution at amino acid position 38; and

(b) A human antibody molecule or its antigen-binding fragment thereof, said human antibody molecule or its antigen-binding fragment binding specifically to human programmed cell death protein-1 (hPD-1), said human antibody molecule or its antigen-binding fragment comprising:

(i) the heavy chain complementarity determination region 1 (CDR1) containing the amino acid sequence of SEQ ID NO:418, the heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:419, the heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:420, the light chain CDR1 containing the amino acid sequence of SEQ ID NO:421, the light chain CDR2 containing the amino acid sequence of SEQ ID NO:422, and the light chain CDR3 containing the amino acid sequence of SEQ ID NO:423;

(ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:388, light chain CDR1 containing the amino acid sequence of SEQ ID NO:389, light chain CDR2 containing the amino acid sequence of SEQ ID NO:390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:391;

(iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:398, light chain CDR1 containing the amino acid sequence of SEQ ID NO:399, light chain CDR2 containing the amino acid sequence of SEQ ID NO:400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:401; or

(iv) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:406, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:407, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:408, light chain CDR1 containing the amino acid sequence of SEQ ID NO:409, light chain CDR2 containing the amino acid sequence of SEQ ID NO:410, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:411.

Example 54. The immunoconjugate according to Example 53, wherein the substitution at amino acid position 20 of the modified hIL-2 protein is selected from D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution.

Example 55. The immunoconjugate according to Example 53 or 54, wherein the substitution at amino acid position 38 of the modified hIL-2 protein is selected from R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D, and R38K substitutions.

Example 56. The immunoconjugate according to any one of Examples 53 to 55, wherein the substitution at amino acid position 20 of the modified hIL-2 protein is selected from D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution, and the amino acid substitution at amino acid position 38 of the modified hIL-2 protein is R38E.

Example 57. An immunoconjugate according to any one of Examples 53 to 56, wherein the modified hIL-2 protein comprises the amino acid sequence of any one of SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617 or 620.

Example 58. An immunoconjugate according to any one of Examples 53 to 56, wherein the modified hIL-2 protein comprises D20A and R38E substitutions.

Example 59. The immunoconjugate according to Example 58, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:149.

Example 60. An immunoconjugate according to any one of Examples 53 to 57, comprising the amino acid sequence of any one of SEQ ID NO: 608, 614, 611, 620, 607, 610, 617, 609 or 307.

Example 61. An immunoconjugate according to any one of Examples 53 to 60, wherein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345, the modified hIL-2 protein further comprises a deletion or substitution at amino acid position 3.

Example 62. The immunoconjugate according to Example 61, wherein the amino acid substitution at position 3 of the modified hIL-2 protein is T3A.

Example 63. The immunoconjugate according to Example 62, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:216.

Example 64. The immunoconjugate according to Example 61, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:218.

Example 65. An immunoconjugate according to any one of Examples 53 to 64, wherein the modified hIL-2 protein further comprises a deletion or substitution at amino acid position 125 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345.

Example 66. The immunoconjugate according to Example 65, wherein the substitution at position 125 of the amino acid is C125A.

Example 67. The immunoconjugate according to Example 66, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 215, 217 or 219.

Example 68. The immunoconjugate according to Example 67, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:217.

Example 69. An immunoconjugate according to any one of Examples 53 to 68, wherein the modified hIL-2 protein is fused to the antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment.

Example 70. An immunoconjugate according to any one of Examples 53 to 69, wherein the modified hIL-2 protein is fused directly to the antibody or its antigen-binding fragment via a peptide bond.

Example 71. The immunoconjugate according to Example 70, wherein the modified hIL-2 protein is fused directly to the C-terminal amino acid residue of the antibody heavy chain via a peptide bond.

Example 72. An immunoconjugate according to any one of Examples 53 to 69, wherein the modified hIL-2 protein is fused to the antibody or its antigen-binding fragment via a linker.

Example 73. An immunoconjugate according to any one of Examples 53 to 72, wherein the human antibody molecule or its antigen-binding fragment comprises:

a) The heavy chain variable region containing the amino acid sequence of SEQ ID NO:416 and the light chain variable region containing the amino acid sequence of SEQ ID NO:417;

b) The heavy chain variable region containing the amino acid sequence of SEQ ID NO:384 and the light chain variable region containing the amino acid sequence of SEQ ID NO:385;

c) The heavy chain variable region containing the amino acid sequence of SEQ ID NO:394 and the light chain variable region containing the amino acid sequence of SEQ ID NO:395; or

d) The heavy chain variable region containing the amino acid sequence of SEQ ID NO:404 and the light chain variable region containing the amino acid sequence of SEQ ID NO:405.

Example 74. An immunoconjugate according to any one of Examples 53 to 73, wherein the human antibody molecule or its antigen-binding fragment comprises the IgG1 heavy chain constant region.

Example 75. The immunoconjugate according to Example 74, wherein, according to EU designation, the human antibody molecule or its antigen-binding fragment comprises L235A substitution and G237A substitution.

Example 76. An immunoconjugate according to any one of Examples 53 to 75, wherein the human antibody molecule or its antigen-binding fragment comprises:

a) The heavy chain containing the amino acid sequence of SEQ ID NO:414 and the light chain containing the amino acid sequence of SEQ ID NO:415;

b) The heavy chain containing the amino acid sequence of SEQ ID NO:424 and the light chain containing the amino acid sequence of SEQ ID NO:425;

c) The heavy chain containing the amino acid sequence of SEQ ID NO:426 and the light chain containing the amino acid sequence of SEQ ID NO:427; or

d) The heavy chain containing the amino acid sequence of SEQ ID NO:428 and the light chain containing the amino acid sequence of SEQ ID NO:429.

Example 77. The immunoconjugate according to Example 76, wherein the human antibody molecule or its antigen-binding fragment comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO:414 and a light chain comprising the amino acid sequence of SEQ ID NO:415.

Example 78. An immunoconjugate according to any one of Examples 53 to 77, comprising...

A light chain comprising the amino acid sequence of SEQ ID NO:415; and

A heavy-chain modified hIL-2 protein fusion containing the amino acid sequence SEQ ID NO:532.

Example 79. A pharmaceutical composition comprising the modified hIL-2 protein according to any one of Examples 1 to 31, a human antibody molecule or its antigen-binding fragment according to any one of Examples 32 to 52, or an immunoconjugate according to any one of Examples 53 to 78.

Example 80. A polynucleotide comprising a nucleic acid sequence encoding a modified hIL-2 protein according to any one of Examples 1 to 31, a human antibody molecule or its antigen-binding fragment according to any one of Examples 32 to 52, or an immunoconjugate according to any one of Examples 53 to 78.

Example 81. A vector comprising a polynucleotide, said polynucleotide comprising a nucleic acid sequence encoding a modified hIL-2 protein according to any one of Examples 1 to 31, a human antibody molecule or its antigen-binding fragment according to any one of Examples 32 to 52, or an immunoconjugate according to any one of Examples 53 to 78.

Example 82. A transformed cell comprising the vector according to Example 81.

Example 83. A method for treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of the modified hIL-2 protein according to any one of Examples 10 to 14 and 27 to 31, the immunoconjugate according to any one of Examples 53 to 78, or the pharmaceutical composition according to Example 79, thereby treating the disease or condition.

Example 84. The method according to Example 83, wherein the disease or symptom is cancer.

Example 85. The method according to Example 84, wherein the cancer is melanoma.

Example 86. The method according to Example 84, wherein the cancer is non-small cell lung cancer.

Example 87. Use of the modified hIL-2 protein according to any one of Examples 10 to 14 and 27 to 31, the immunoconjugate according to any one of Examples 53 to 78, or the pharmaceutical composition according to Example 79, for the preparation of a medicament for treating a disease or condition.

Example 88. The use according to Example 87, wherein the disease or symptom is cancer.

Example 89. The use according to Example 88, wherein the cancer is melanoma.

Example 90. The use according to Example 88, wherein the cancer is non-small cell lung cancer.

Example 91. Use of a modified hIL-2 protein according to any one of Examples 10 to 14 and 27 to 31, an immunoconjugate according to any one of Examples 53 to 78, or a pharmaceutical composition according to Example 79, for the treatment of a disease or condition.

Example 92. The use according to Example 91, wherein the disease or symptom is cancer.

Example 93. The use according to Example 92, wherein the cancer is melanoma.

Example 94. The use according to Example 92, wherein the cancer is non-small cell lung cancer.

Claims (94)

1. A modified human interleukin-2 (hIL-2) protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, comprising a substitution at amino acid position 20 and a substitution at amino acid position 38, wherein, relative to the unmodified hIL-2, the modified hIL-2 protein exhibits reduced potency against both high-affinity and intermediate-affinity hIL-2 receptors. 2. The modified hIL-2 protein according to claim 1, wherein the substitution at amino acid position 20 is selected from D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution. 3. The modified hIL-2 protein according to claim 1 or 2, wherein the substitution at amino acid position 38 is selected from R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D, and R38K substitutions. 4. The modified hIL-2 protein according to any one of the preceding claims, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, further comprises a deletion or substitution at amino acid position 3. 5. The modified hIL-2 protein according to claim 4, wherein the substitution at position 3 of amino acid is T3A. 6. The modified hIL-2 protein according to any one of the preceding claims, further comprising a deletion or substitution at amino acid position 125 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345. 7. The modified hIL-2 protein according to claim 6, wherein the substitution at amino acid position 125 is C125A. 8. The modified hIL-2 protein according to any one of the preceding claims, wherein the modified hIL-2 protein exhibits approximately 1,000-fold reduced potency against the high-affinity IL-2 receptor (hIL-2Rαβγ). 9. The modified hIL-2 protein according to any one of the preceding claims, wherein the modified hIL-2 protein exhibits approximately 10,000-fold reduced potency against the intermediate-affinity IL-2 receptor (hIL-2Rβγ). 10. The modified hIL-2 protein according to any one of claims 1 to 9, wherein the modified hIL-2 protein is fused with an anti-PD-1 antibody or an antigen-binding fragment thereof. 11. The modified hIL-2 protein according to claim 10, wherein the modified hIL-2 protein is fused to the antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment. 12. The modified hIL-2 protein according to claim 10 or 11, wherein the modified hIL-2 protein is fused directly to the antibody or its antigen-binding fragment via a peptide bond. 13. The modified hIL-2 protein according to claim 12, wherein the modified hIL-2 is fused directly to the C-terminal amino acid residue of the antibody heavy chain via a peptide bond. 14. The modified hIL-2 protein according to claim 10 or 11, wherein the modified hIL-2 protein is fused to the antibody or its antigen-binding fragment via a linker. 15. A modified human interleukin-2 (hIL-2) protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, comprising a D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution at amino acid position 20 and an R38E substitution at amino acid position 38. 16. The modified hIL-2 protein according to claim 15, comprising the amino acid sequence of any one of SEQ ID NO: 307, 607-611, 614, 617 or 620. 17. The modified hIL-2 protein according to claim 15 or 16, comprising D20A substitution and R38E substitution. 18. The modified hIL-2 protein according to claim 17, comprising the amino acid sequence of SEQ ID NO:149. 19. The modified hIL-2 protein according to any one of claims 15 to 18, further comprising a deletion or substitution at amino acid position 3 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345. 20. The modified hIL-2 protein according to claim 19, wherein the substitution at position 3 of amino acid is T3A. 21. The modified hIL-2 protein according to claim 20, comprising the amino acid sequence of SEQ ID NO:216. 22. The modified hIL-2 protein according to claim 19, comprising the amino acid sequence of SEQ ID NO:218. 23. The modified hIL-2 protein according to any one of claims 15 to 22, further comprising a deletion or substitution at amino acid position 125 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345. 24. The modified hIL-2 protein according to claim 23, wherein the substitution at amino acid position 125 is C125A. 25. The modified hIL-2 protein according to claim 24, comprising the amino acid sequence of SEQ ID NO: 215, 217 or 219. 26. The modified hIL-2 protein according to claim 25, comprising the amino acid sequence of SEQ ID NO:217. 27. The modified hIL-2 protein according to any one of claims 15 to 26, wherein the modified hIL-2 protein is fused with an anti-PD-1 antibody or an antigen-binding fragment thereof. 28. The modified hIL-2 protein of claim 27, wherein the modified hIL-2 protein is fused to the antibody or its antigen-binding fragment at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment. 29. The modified hIL-2 protein according to claim 27 or 28, wherein the modified hIL-2 protein is fused directly to the antibody or its antigen-binding fragment via a peptide bond. 30. The modified hIL-2 protein according to claim 29, wherein the modified hIL-2 is fused directly to the C-terminal amino acid residue of the antibody heavy chain via a peptide bond. 31. The modified hIL-2 protein according to claim 27 or 28, wherein the modified hIL-2 protein is fused to the antibody or its antigen-binding fragment via a linker. 32. A human antibody molecule or an antigen-binding fragment thereof, said human antibody molecule or antigen-binding fragment thereof binding specifically to human programmed cell death protein-1 (hPD-1), wherein said human antibody molecule or antigen-binding fragment thereof comprises: a) Heavy chain complementarity-determining region 1 (CDR1) containing the amino acid sequence of SEQ ID NO:418, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:419, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:420, light chain CDR1 containing the amino acid sequence of SEQ ID NO:421, light chain CDR2 containing the amino acid sequence of SEQ ID NO:422, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:423; b) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:388, light chain CDR1 containing the amino acid sequence of SEQ ID NO:389, light chain CDR2 containing the amino acid sequence of SEQ ID NO:390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:391; c) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:398, light chain CDR1 containing the amino acid sequence of SEQ ID NO:399, light chain CDR2 containing the amino acid sequence of SEQ ID NO:400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:401; or d) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:406, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:407, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:408, light chain CDR1 containing the amino acid sequence of SEQ ID NO:409, light chain CDR2 containing the amino acid sequence of SEQ ID NO:410, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:411. 33. The human antibody molecule or its antigen-binding fragment according to claim 32, comprising: a) The heavy chain variable region containing the amino acid sequence of SEQ ID NO:416 and the light chain variable region containing the amino acid sequence of SEQ ID NO:417; b) The heavy chain variable region containing the amino acid sequence of SEQ ID NO:384 and the light chain variable region containing the amino acid sequence of SEQ ID NO:385; c) The heavy chain variable region containing the amino acid sequence of SEQ ID NO:394 and the light chain variable region containing the amino acid sequence of SEQ ID NO:395; or d) The heavy chain variable region containing the amino acid sequence of SEQ ID NO:404 and the light chain variable region containing the amino acid sequence of SEQ ID NO:405. 34. The human antibody molecule or antigen-binding fragment thereof according to claim 32 or 33, comprising a constant region of human IgG1 heavy chain. 35. The human antibody molecule or its antigen-binding fragment according to claim 34, wherein, according to EU designation, it comprises L235A substitution and G237A substitution. 36. The human antibody molecule or its antigen-binding fragment according to any one of claims 32 to 35, comprising: a) The heavy chain containing the amino acid sequence of SEQ ID NO:414 and the light chain containing the amino acid sequence of SEQ ID NO:415; b) The heavy chain containing the amino acid sequence of SEQ ID NO:424 and the light chain containing the amino acid sequence of SEQ ID NO:425; c) The heavy chain containing the amino acid sequence of SEQ ID NO:426 and the light chain containing the amino acid sequence of SEQ ID NO:427; or d) The heavy chain containing the amino acid sequence of SEQ ID NO:428 and the light chain containing the amino acid sequence of SEQ ID NO:429. 37. The human antibody molecule or antigen-binding fragment thereof according to claim 36, comprising: a heavy chain comprising the amino acid sequence of SEQ ID NO:414 and a light chain comprising the amino acid sequence of SEQ ID NO:415. 38. The human antibody molecule or antigen-binding fragment thereof according to any one of claims 32 to 37, fused to a modified human interleukin-2 (hIL-2) protein, wherein the modified hIL-2 protein comprises a substitution at amino acid position 20 and a substitution at amino acid position 38 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345. 39. The human antibody molecule or its antigen-binding fragment according to claim 38, wherein the modified hIL-2 protein comprises the amino acid sequence of any one of SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617 or 620. 40. The human antibody molecule or its antigen-binding fragment according to claim 39, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:149. 41. The human antibody molecule or antigen-binding fragment thereof according to any one of claims 38 to 40, wherein the modified hIL-2 protein further comprises a deletion or substitution at amino acid position 3 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345. 42. The human antibody molecule or its antigen-binding fragment according to claim 41, wherein the substitution at position 3 of the amino acid is T3A. 43. The human antibody molecule or its antigen-binding fragment according to claim 42, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:216. 44. The human antibody molecule or its antigen-binding fragment according to claim 41, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:218. 45. The human antibody molecule or antigen-binding fragment thereof according to any one of claims 38 to 44, wherein the modified hIL-2 protein further comprises a deletion or substitution at amino acid position 125 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO: 345. 46. The human antibody molecule or its antigen-binding fragment according to claim 45, wherein the substitution at position 125 of the amino acid is C125A. 47. The human antibody molecule or its antigen-binding fragment according to claim 46, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 215, 217 or 219. 48. The human antibody molecule or its antigen-binding fragment according to claim 47, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:217. 49. The human antibody molecule or antigen-binding fragment thereof according to any one of claims 38 to 48, wherein the modified hIL-2 protein is fused to the antibody or antigen-binding fragment thereof at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment. 50. The human antibody molecule or antigen-binding fragment thereof according to any one of claims 38 to 49, wherein the modified hIL-2 protein is directly fused to the antibody or antigen-binding fragment thereof via a peptide bond. 51. The human antibody molecule or its antigen-binding fragment according to claim 50, wherein the modified hIL-2 protein is directly fused to the C-terminal amino acid residue of the antibody heavy chain via a peptide bond. 52. The human antibody molecule or antigen-binding fragment thereof according to any one of claims 38 to 49, wherein the modified hIL-2 protein is fused to the antibody or antigen-binding fragment via a linker. 53. An immunoconjugate comprising: (a) A modified human interleukin-2 (hIL-2) protein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, comprising a substitution at amino acid position 20 and a substitution at amino acid position 38; and (b) A human antibody molecule or its antigen-binding fragment thereof, said human antibody molecule or its antigen-binding fragment binding specifically to human programmed cell death protein-1 (hPD-1), said human antibody molecule or its antigen-binding fragment comprising: (i) the heavy chain complementarity determination region 1 (CDR1) containing the amino acid sequence of SEQ ID NO:418, the heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:419, the heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:420, the light chain CDR1 containing the amino acid sequence of SEQ ID NO:421, the light chain CDR2 containing the amino acid sequence of SEQ ID NO:422, and the light chain CDR3 containing the amino acid sequence of SEQ ID NO:423; (ii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:386, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:387, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:388, light chain CDR1 containing the amino acid sequence of SEQ ID NO:389, light chain CDR2 containing the amino acid sequence of SEQ ID NO:390, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:391; (iii) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:396, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:397, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:398, light chain CDR1 containing the amino acid sequence of SEQ ID NO:399, light chain CDR2 containing the amino acid sequence of SEQ ID NO:400, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:401; or (iv) Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO:406, heavy chain CDR2 containing the amino acid sequence of SEQ ID NO:407, heavy chain CDR3 containing the amino acid sequence of SEQ ID NO:408, light chain CDR1 containing the amino acid sequence of SEQ ID NO:409, light chain CDR2 containing the amino acid sequence of SEQ ID NO:410, and light chain CDR3 containing the amino acid sequence of SEQ ID NO:411. 54. The immunoconjugate according to claim 53, wherein the substitution at amino acid position 20 of the modified hIL-2 protein is selected from D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution. 55. The immunoconjugate according to claim 53 or 54, wherein the substitution at amino acid position 38 of the modified hIL-2 protein is selected from R38E, R38N, R38G, R38H, R38I, R38L, R38M, R38F, R38P, R38S, R38T, R38W, R38Y, R38V, R38A, R38Q, R38D, and R38K substitutions. 56. The immunoconjugate according to any one of claims 53 to 55, wherein the substitution at amino acid position 20 of the modified hIL-2 protein is selected from D20A, D20S, D20Q, D20M, D20I, D20V, D20N, D20G, D20T or D20E substitution, and the amino acid substitution at amino acid position 38 of the modified hIL-2 protein is R38E. 57. The immunoconjugate according to any one of claims 53 to 56, wherein the modified hIL-2 protein comprises the amino acid sequence of any one of SEQ ID NO: 134-150, 307, 344, 607-611, 614, 617 or 620. 58. The immunoconjugate according to any one of claims 53 to 56, wherein the modified hIL-2 protein comprises D20A and R38E substitutions. 59. The immunoconjugate of claim 58, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:149. 60. The immunoconjugate according to any one of claims 53 to 57, comprising the amino acid sequence of any one of SEQ ID NO: 608, 614, 611, 620, 607, 610, 617, 609 or 307. 61. The immunoconjugate according to any one of claims 53 to 60, wherein, relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345, the modified hIL-2 protein further comprises a deletion or substitution at amino acid position 3. 62. The immunoconjugate according to claim 61, wherein the substitution at amino acid position 3 of the modified hIL-2 protein is T3A. 63. The immunoconjugate according to claim 62, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:216. 64. The immunoconjugate of claim 61, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:218. 65. The immunoconjugate according to any one of claims 53 to 64, wherein the modified hIL-2 protein further comprises a deletion or substitution at amino acid position 125 relative to the unmodified hIL-2 amino acid sequence of SEQ ID NO:345. 66. The immunoconjugate according to claim 65, wherein the substitution at position 125 of the amino acid is C125A. 67. The immunoconjugate of claim 66, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO: 215, 217 or 219. 68. The immunoconjugate of claim 67, wherein the modified hIL-2 protein comprises the amino acid sequence of SEQ ID NO:217. 69. The immunoconjugate according to any one of claims 53 to 68, wherein the modified hIL-2 protein is fused to the antibody or an antigen-binding fragment thereof at the N-terminus of the antibody light chain, the C-terminus of the antibody light chain, the N-terminus of the antibody heavy chain, the C-terminus of the antibody heavy chain, the N-terminus of the antigen-binding fragment, or the C-terminus of the antigen-binding fragment. 70. The immunoconjugate according to any one of claims 53 to 69, wherein the modified hIL-2 protein is fused directly to the antibody or its antigen-binding fragment via a peptide bond. 71. The immunoconjugate according to claim 70, wherein the modified hIL-2 protein is fused directly to the C-terminal amino acid residue of the antibody heavy chain via a peptide bond. 72. The immunoconjugate according to any one of claims 53 to 69, wherein the modified hIL-2 protein is fused to the antibody or its antigen-binding fragment via a linker. 73. The immunoconjugate according to any one of claims 53 to 72, wherein the human antibody molecule or its antigen-binding fragment comprises: a) The heavy chain variable region containing the amino acid sequence of SEQ ID NO:416 and the light chain variable region containing the amino acid sequence of SEQ ID NO:417; b) The heavy chain variable region containing the amino acid sequence of SEQ ID NO:384 and the light chain variable region containing the amino acid sequence of SEQ ID NO:385; c) The heavy chain variable region containing the amino acid sequence of SEQ ID NO:394 and the light chain variable region containing the amino acid sequence of SEQ ID NO:395; or d) The heavy chain variable region containing the amino acid sequence of SEQ ID NO:404 and the light chain variable region containing the amino acid sequence of SEQ ID NO:405. 74. The immunoconjugate according to any one of claims 53 to 73, wherein the human antibody molecule or its antigen-binding fragment comprises the IgG1 heavy chain constant region. 75. The immunoconjugate of claim 74, wherein, according to EU designation, the human antibody molecule or its antigen-binding fragment comprises L235A substitution and G237A substitution. 76. The immunoconjugate according to any one of claims 53 to 75, wherein the human antibody molecule or its antigen-binding fragment comprises: a) The heavy chain containing the amino acid sequence of SEQ ID NO:414 and the light chain containing the amino acid sequence of SEQ ID NO:415; b) The heavy chain containing the amino acid sequence of SEQ ID NO:424 and the light chain containing the amino acid sequence of SEQ ID NO:425; c) The heavy chain containing the amino acid sequence of SEQ ID NO:426 and the light chain containing the amino acid sequence of SEQ ID NO:427; or d) The heavy chain containing the amino acid sequence of SEQ ID NO:428 and the light chain containing the amino acid sequence of SEQ ID NO:429. 77. The immunoconjugate of claim 76, wherein the human antibody molecule or its antigen-binding fragment comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO:414 and a light chain comprising the amino acid sequence of SEQ ID NO:415. 78. The immunoconjugate according to any one of claims 53 to 77, comprising: A light chain comprising the amino acid sequence of SEQ ID NO:415; and A heavy-chain modified hIL-2 protein fusion containing the amino acid sequence SEQ ID NO:532. 79. A pharmaceutical composition comprising the modified hIL-2 protein according to any one of claims 1 to 31, a human antibody molecule or its antigen-binding fragment according to any one of claims 32 to 52, or an immunoconjugate according to any one of claims 53 to 78. 80. A polynucleotide comprising a nucleic acid sequence encoding a modified hIL-2 protein according to any one of claims 1 to 31, a human antibody molecule or its antigen-binding fragment according to any one of claims 32 to 52, or an immunoconjugate according to any one of claims 53 to 78. 81. A vector comprising a polynucleotide, said polynucleotide comprising a nucleic acid sequence encoding a modified hIL-2 protein according to any one of claims 1 to 31, a human antibody molecule or its antigen-binding fragment according to any one of claims 32 to 52, or an immunoconjugate according to any one of claims 53 to 78. 82. A transformed cell comprising the vector according to claim 81. 83. A method of treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of the modified hIL-2 protein according to any one of claims 10 to 14 and 27 to 31, the immunoconjugate according to any one of claims 53 to 78, or the pharmaceutical composition according to claim 79, thereby treating the disease or condition. 84. The method of claim 83, wherein the disease or symptom is cancer. 85. The method of claim 84, wherein the cancer is melanoma. 86. The method of claim 84, wherein the cancer is non-small cell lung cancer. 87. Use of a modified hIL-2 protein according to any one of claims 10 to 14 and 27 to 31, an immunoconjugate according to any one of claims 53 to 78, or a pharmaceutical composition according to claim 79, for the preparation of a medicament for treating a disease or condition. 88. The use according to claim 87, wherein the disease or condition is cancer. 89. The use according to claim 88, wherein the cancer is melanoma. 90. The use according to claim 88, wherein the cancer is non-small cell lung cancer. 91. Use of a modified hIL-2 protein according to any one of claims 10 to 14 and 27 to 31, an immunoconjugate according to any one of claims 53 to 78, or a pharmaceutical composition according to claim 79, for the treatment of a disease or condition. 92. The use according to claim 91, wherein the disease or condition is cancer. 93. The use according to claim 92, wherein the cancer is melanoma. 94. The use according to claim 92, wherein the cancer is non-small cell lung cancer.