RU2829811C1 - Interleukin 10 conjugates and applications thereof - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: present invention relates to biotechnology and can be used in medicine. Disclosed are novel interleukin 10 (IL-10) peptide conjugates, in which a PEG fragment is attached to IL-10 through a dibenzocyclooctine linker using click chemistry. Said conjugate is characterized by an increased half-life in blood plasma and, when dimerized, is able to enhance the effect of tumour-infiltrating immune cells on a set of tumour cells.

EFFECT: invention can be used to modulate T-cell responses in humans, particularly in treating patients suffering from cancer.

36 cl, 7 ex, 10 tbl, 14 dwg

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] This application claims priority to U.S. Provisional Patent Application No. 62/930,322, filed November 4, 2019, and U.S. Provisional Patent Application No. 62/953,095, filed December 23, 2019, the disclosures of each of which are hereby incorporated by reference in their entireties.

SEQUENCE LIST

[2] This application contains a sequence listing that was filed electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII-encoded copy of this file, created on November 2, 2020, is named 01183-0077-00PCT_sequence_listing.txt and is 135 KB in size.

LEVEL OF TECHNOLOGY

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

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

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

[6] In some cases, interleukin 10 (IL-10) signaling is used to modulate T cell responses. Accordingly, in one aspect, IL-10 conjugates and uses thereof are provided herein.

ESSENCE OF THE INVENTION

[7] In certain embodiments, interleukin 10 (IL-10) conjugates and uses thereof in the treatment of one or more indications are disclosed herein. In some embodiments, IL-10 conjugates for the treatment of cancer are disclosed herein. In additional instances, pharmaceutical compositions and kits that comprise an IL-10 conjugate described herein are disclosed herein.

[8] The following embodiments are covered.

[9] Embodiment A1. An IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced by a structure of formula (I),

Where

Z is CH ₂ and Y is ;

Y is CH ₂ , and Z is ;

Z is CH ₂ and Y is ; or

Y is CH ₂ , and Z is ;

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

q is equal to 1, 2, or 3;

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

[10] Embodiment A2. The IL-10 conjugate of Embodiment A1, wherein Z is CH ₂ and Y is .

[11] Embodiment A3. The IL-10 conjugate of Embodiment A1, wherein Y is CH ₂ , and Z is .

[12] Embodiment A4. The IL-10 conjugate of Embodiment A1, wherein Z is CH ₂ , and Y is .

[13] Embodiment A5. The IL-10 conjugate of Embodiment 1, wherein Y is CH ₂ , and Z is .

[14] Embodiment A6. The IL-10 conjugate of any one of Embodiments A1-5, wherein the PEG group has an average molecular weight selected from 5 kDa, 10 kDa, 20 kDa, and 30 kDa.

[15] Embodiment A7. The IL-10 conjugate of embodiment A6, wherein the PEG group has an average molecular weight selected from 10 kDa and 20 kDa.

[16] Embodiment A8. The IL-10 conjugate of any one of Embodiments A1-7, wherein the position of the structure of formula (I) is selected from N82, K88, A89, K99, K125, N126, N129, and K130.

[17] Embodiment A9. The IL-10 conjugate of embodiment A8, wherein the position of the structure of formula (I) is selected from N82 and N129.

[18] Embodiment A10. The IL-10 conjugate of Embodiment A1, wherein the structure of formula (I) has a structure of formula (X) or formula (XI) or is a mixture of formula (X) and formula (XI),

Where

q is equal to 1, 2, or 3;

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

Wavy lines indicate covalent bonds with amino acid residues within SEQ ID NO: 1 that are not replaced.

[19] Embodiment A11. The IL-10 conjugate of embodiment A10, wherein the position of the structure of formula (X) or formula (XI) in SEQ ID NO: 1 is selected from N82, K88, A89, K99, K125, N126, N129, and K130.

[20] Embodiment A12. The IL-10 conjugate of embodiment A11, wherein the position of the structure of formula (X) or formula (XI) in SEQ ID NO: 1 is selected from N82 and N129.

[21] Embodiment A13. The IL-10 conjugate of any one of Embodiments A10-12, wherein n is an integer such that -( _OCH2CH2 ) _n - _OCH3 has a _molecular weight of about 10 kDa or 20 kDa.

[22] Embodiment A14. An IL-10 conjugate according to embodiment A1, wherein the structure of formula (I) has a structure of formula (XII) or formula (XIII) or is a mixture of formula (XII) and formula (XIII),

Where

q is equal to 1, 2, or 3;

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

Wavy lines indicate covalent bonds with amino acid residues within SEQ ID NO: 1 that are not replaced.

[23] Embodiment A15. The IL-10 conjugate of embodiment A14, wherein the position of the structure of formula (XII) or formula (XIII) in SEQ ID NO: 1 is selected from N82, K88, A89, K99, K125, N126, N129, and K130.

[24] Embodiment A16. The IL-10 conjugate of embodiment A14, wherein the position of the structure of formula (XII) or formula (XIII) in SEQ ID NO: 1 is selected from N82 and N129.

[25] Embodiment A17. The IL-10 conjugate of any one of Embodiments A14-16, wherein n is an integer such that -( _OCH2CH2 ) _n - _OCH3 has a _molecular weight of about 10 kDa or 20 kDa.

[26] Embodiment A18. The IL-10 conjugate of any one of Embodiments A1-17, wherein q is 1.

[27] Embodiment A19. The IL-10 conjugate of any one of Embodiments A1-17, wherein q is 2.

[28] Embodiment A20. The IL-10 conjugate of any one of Embodiments A1-17, wherein q is 3.

[29] Embodiment A21. The IL-10 conjugate of any one of Embodiments A1-20, wherein the IL-10 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

[30] Embodiment A22: A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an IL-10 conjugate of any one of Embodiments A1-21.

[31] Embodiment A23. The method of embodiment A22, wherein the cancer is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin's lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability cancer, microsatellite stable cancer, microsatellite stable colorectal cancer, gastric cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), melanoma, small cell lung cancer (SCLC), esophageal cancer, glioblastoma, mesothelioma, breast cancer, triple negative breast cancer, prostate cancer, bladder cancer, ovarian cancer, tumors with moderate to low mutational burden, squamous cell carcinoma of the skin (CSCC), cutaneous squamous cell carcinoma (SCSC), tumors with low to absent PD-L1 expression, tumors systemically disseminating to the liver and CNS beyond their primary anatomical site of origin, and diffuse large B-cell lymphoma.

[32] Embodiment A24. The method of embodiment A22 or A23, wherein the IL-10 conjugate is administered to the subject once daily, twice daily, three times daily, once weekly, once every two weeks, once every three weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, or once every 8 weeks.

[33] Embodiment A25. The method of any one of embodiments A22-24, wherein the IL-10 conjugate is administered to the subject by intravenous administration.

[34] Embodiment A26. A method for producing an IL-10 conjugate comprising

carrying out a reaction of an IL-10 polypeptide containing an unnatural amino acid of the formula , wherein the IL-10 polypeptide comprises the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 polypeptide is replaced with a non-natural amino acid, position X-1 indicates the point of attachment to the previous amino acid residue; position X+1 indicates the point of attachment to the next amino acid residue, and position X indicates the position of the amino acid that is replaced by the non-natural amino acid, with mPEG-DBCO of the formula

or , where q is 1, 2, or 3, and n is such that mPEG-DBCO comprises PEG having a molecular weight of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, or 60 kDa,

thus obtaining an IL-10 conjugate.

[35] Embodiment A27. The method of embodiment A26, wherein q is 1.

[36] Embodiment A28. The method of embodiment A26, wherein q is 2.

[37] Embodiment A29. The method of embodiment A26, wherein q is 3.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[38] Various aspects of the present invention are particularly set forth in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description, which sets forth illustrative embodiments in which the principles of the present invention are utilized, and the accompanying drawings.

[39] Figure 1 illustrates an illustrative SDS-PAGE and Western blot analysis of Compound A under reducing conditions and illustrates homogeneous PEGylation of IL-10 monomers as described in Example 2.

[40] Fig. 2 illustrates an illustrative determination of the molecular weight of compound A, which is described in Example 2, using SEC-MALS.

[41] Figure 3 illustrates an illustrative stability analysis of the dimer of compound A, as described in Example 2, at low concentrations using size exclusion chromatography (SEC).

[42] Figure 4 illustrates the trace concentration of compound A (pg/mL) as a function of proliferation (OD ₄₅₀ ) in the proliferation assay using MC/9 from Example 3.

[43] Figure 5 illustrates the trace concentration of compound D (pg/mL) as a function of proliferation (OD ₄₅₀ ) in the proliferation assay using MC/9 from Example 3.

[44] Figure 6 illustrates the trace concentration of compound E (pg/mL) as a function of proliferation (OD ₄₅₀ ) in the proliferation assay using MC/9 from Example 3.

[45] Figure 7A illustrates the trace concentration of compound F (pg/mL) as a function of proliferation (OD ₄₅₀ ) in the proliferation assay using MC/9 from Example 3.

[46] Fig. 7B illustrates the concentration of compound G and compound H (pg/mL) as a function of proliferation (OD ₄₅₀ ) in the proliferation assay using MC/9 from Example 3.

[47] Figure 8 illustrates the measurement of the biological activity of wild-type IL-10 in the PathHunter assay from Example 3.

[48] Figure 9 illustrates the measurement of biological activity of compound A in the PathHunter assay of Example 3.

[49] Figures 10A-C illustrate pSTAT3 profiling in Balb/c mouse splenocytes against wild-type IL-10 (filled circles), Compound A (open triangles), and Compound D (open squares) from Example 4 in CD8+ T cells, NK cells, and B cells, respectively.

[50] Figures 11A-C illustrate the profiling of pSTAT3 in B57BL/6 mouse splenocytes against wild-type IL-10 (filled circles), Compound A (open triangles), and Compound D (open squares) from Example 4 in CD8+ T cells, NK cells, and B cells, respectively.

[51] Fig. 12A-C illustrate the concentration of wild-type His-IL-10, Compound A, and Compound D as a function of pSTAT3 MFI from Example 5 in CD8+ T cells, NK cells, and B cells, respectively.

[52] Figure 13A-B illustrates the release of IFNγ upon activation of antigen-specific TCR using wild-type His-IL-10 or compound A from Example 6. ND=not detected.

[53] Fig. 14A-B illustrates the upregulation of PD-1 following treatment with [His]-IL-10 or Compound A from Example 6 and demonstrates that this upregulation is independent of TCR activation.

DETAILED DESCRIPTION

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

[55] Interleukins are signaling proteins that modulate the development and differentiation of T and B lymphocytes, cells of the monocytic lineage, neutrophils, basophils, eosinophils, megakaryocytes, and hematopoietic cells. Interleukins are produced by CD4 helper T and B lymphocytes, monocytes, macrophages, endothelial cells, and other tissue-resident cells. In some cases, approximately 15 interleukins are present, interleukins 1–13, interleukin 15, and interleukin 17.

[56] IL-10 mediates an immune response against tumors by activating tumor-infiltrating CD8+ T cells, promoting CD8+ T cell proliferation, inducing IFN-γ, leading to increased MHC class I molecules on tumor cells and MHC class II on macrophages, and inducing cytotoxic proteins that mediate target cell lysis. Increased stimulation of the T cell receptor on CD8+ T cells provides anti-apoptotic and proliferative signals. An unexpected role of IL-10 in the tumor microenvironment (TME) is to downregulate proinflammatory Th17 cells and cytokines responsible for tumor-associated inflammation, resulting in suppression of antitumor effector cell responses. Preclinical studies have shown that IL-10 deficiency increases tumor incidence and impairs immune surveillance. Furthermore, treatment of Her2 transgenic mice with pegylated IL-10 resulted in tumor rejection, but this required the presence of IFN-γ expression and granzyme-expressing CD8+ T cells, which was accompanied by a significant increase in the number of CD8a/b+ T cells in the tumor.

[57] In vivo, IL-10 has a relatively short serum half-life. In fact, the half-life in mice, as measured by in vitro bioassays or by potency in a septic shock model system (see Smith et al., Cellular Immunology 173:207-214 (1996), the disclosure of which is incorporated herein by reference), is approximately 2 to 6 hours.

[58] In certain embodiments, disclosed herein is a modified IL-10 polypeptide that has an increased plasma half-life. In some embodiments, disclosed herein is also a modified IL-10 polypeptide that, when dimerized, enhances the effect of tumor-infiltrating immune cells on a population of tumor cells. In other embodiments, disclosed herein is further a modified IL-10 polypeptide that forms a biologically active IL-10 dimer. In some embodiments, disclosed herein is a modified IL-10 polypeptide that forms a biologically active modified IL-10 dimer.

[59] Additionally described herein are IL-10 conjugates, wherein the IL-10 conjugates are IL-10 or modified IL-10 polypeptides conjugated to at least one conjugation moiety. Also described herein are pharmaceutical compositions comprising one or more modified IL-10 polypeptides or IL-10 conjugates, and methods for treating a disease or indication.

Definitions

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

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

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

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

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

[65] As used herein, the term "subject(s)" or "patient(s)" means any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is non-human. In some embodiments, the subject does not have a disease. In some embodiments, the subject is not diagnosed with a disease. In some embodiments, the subject is diagnosed with a disease. In some embodiments, the subject is diagnosed with at least one disease. In some cases, the subject is a patient. Neither term requires or is limited to situations characterized by the supervision (e.g., ongoing or intermittent) of a healthcare professional (e.g., a physician, registered nurse, nurse practitioner, physician assistant, orderly, or hospice worker).

[66] As used herein, the terms "significant" and "substantially" when applied to receptor binding mean a change sufficient to affect the binding of the IL-10 polypeptide to the target receptor. In some cases, the term refers to a change of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. In some cases, the term refers to a change of at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or more.

[67] In some cases, the term "essentially" in relation to dimerization means a change sufficient to prevent IL-10 dimer formation.

[68] As used herein, the term "tumor-infiltrating immune cell(s)" refers to immune cells that have infiltrated an area containing tumor cells (e.g., the tumor microenvironment). In some cases, tumor-infiltrating immune cells are associated with tumor cell destruction, decreased tumor cell proliferation, decreased tumor burden, or combinations thereof. In some cases, tumor-infiltrating immune cells include tumor-infiltrating lymphocytes (TILs). In some cases, tumor-infiltrating immune cells include T cells, B cells, natural killer cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, or basophils. In some cases, tumor-infiltrating immune cells include CD4+ or CD8+ T cells.

[69] As used herein, the term "unnatural amino acid" refers to an amino acid other than one of the 20 naturally occurring amino acids. Illustrative unnatural amino acids are described in Young et al., "Beyond the canonical 20 amino acids: expanding the genetic lexicon," J. of Biological Chemistry 285 (15):11039-11044 (2010), the disclosure of which is incorporated herein by reference.

[70] As used herein, "nucleotide" refers to a compound containing a nucleoside moiety and a phosphate moiety. Illustrative naturally occurring nucleotides include, but are not limited to, adenosine triphosphate (ATP), uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), adenosine diphosphate (ADP), uridine diphosphate (UDP), cytidine diphosphate (CDP), guanosine diphosphate (GDP), adenosine monophosphate (AMP), uridine monophosphate (UMP), cytidine monophosphate (CMP) and guanosine monophosphate (GMP), deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyadenosine diphosphate (dADP), thymidine diphosphate (dTDP), deoxycytidine diphosphate (dCDP), deoxyguanosine diphosphate (dGDP), deoxyadenosine monophosphate (dAMP), deoxythymidine monophosphate (dTMP), deoxycytidine monophosphate (dCMP), and deoxyguanosine monophosphate (dGMP). Illustrative naturally occurring deoxyribonucleotides that contain deoxyribose as a sugar moiety include dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Illustrative naturally occurring ribonucleotides that contain ribose as a sugar moiety include ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, and GMP.

[71] As used herein, a "base" or "nucleobase" refers to at least a portion of a nucleoside or nucleotide (nucleoside and nucleotide include ribo- or deoxyribonucleotide variants) that is a nucleobase, which in some cases may contain additional modifications to the sugar portion of the nucleoside or nucleotide. In some cases, the term "base" is also used to refer to the entire nucleoside or nucleotide (e.g., a "base" may be incorporated by DNA polymerase into DNA or by RNA polymerase into RNA). However, the term "base" should not be interpreted as necessarily representing the entire nucleoside or nucleotide unless the context so requires. In the chemical structures of a base or nucleobase provided herein, only the base of the nucleoside or nucleotide is shown, and the sugar moiety and optionally any phosphate residues are omitted for clarity. As used in the chemical structures of a base or nucleobase provided herein, a wavy line represents a connection to a nucleoside or nucleotide, wherein the sugar portion of the nucleoside or nucleotide may be further modified. In some embodiments, a wavy line represents the attachment of a base or nucleobase to a sugar portion of a nucleoside or nucleotide, such as a pentose. In some embodiments, the pentose is ribose or deoxyribose.

[72] In some embodiments, the nucleobase is typically a heterocyclic base moiety of a nucleoside. Nucleobases may be naturally occurring, may be modified, may not resemble natural bases, and/or may be synthesized, such as by organic synthesis. In certain embodiments, a nucleobase comprises any atom or group of atoms in a nucleoside or nucleotide wherein the atom or group of atoms is capable of interacting with a base in another nucleic acid, with or without hydrogen bonding. In certain embodiments, a non-natural nucleobase is not a derivative of a natural nucleobase. It should be noted that non-natural nucleobases do not necessarily have basic properties, but for simplicity they are referred to as nucleobases. In some embodiments, when referring to a nucleobase, "(d)" indicates that the nucleobase may be attached to deoxyribose or ribose, whereas "d" without parentheses indicates that the nucleobase is attached to deoxyribose.

[73] As used herein, a "nucleoside" is a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (found in DNA and RNA), abasic nucleosides, modified nucleosides, and nucleosides having mimetic bases and/or sugar moieties. Nucleosides include nucleosides containing any variety of substituents. A nucleoside may be a glycosidic compound formed through a glycosidic linkage between a nucleic acid base and a reducing group on a sugar.

[74] As used herein, the term "analog" of a chemical structure refers to a chemical structure that retains substantial similarity to the parent structure, although it cannot be readily prepared synthetically from the parent structure. In some embodiments, a nucleotide analog is a non-natural nucleotide. In some embodiments, a nucleoside analog is a non-natural nucleoside. A related chemical structure that is readily prepared synthetically from the parent chemical structure is referred to as a "derivative."

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

Modified IL-10 polypeptides

[76] Disclosed herein, in some embodiments, are IL-10 polypeptides that are modified at an amino acid position. In some cases, the modification is to a naturally occurring amino acid. In some cases, the modification is to a non-naturally occurring amino acid. In some cases, described herein is an isolated and modified IL-10 polypeptide that comprises at least one non-naturally occurring amino acid. In some cases, the modified IL-10 polypeptide is an isolated and purified mammalian IL-10, such as a rodent IL-10 protein or a human IL-10 protein. In some cases, the modified IL-10 polypeptide is a human IL-10 protein. In some embodiments, the basis for the modification into a modified IL-10 polypeptide is a parental IL-10 sequence. In some cases, the parental IL-10 sequence is a wild-type IL-10 sequence. In some cases, the IL-10 parent sequence is SEQ ID NO: 1. In some embodiments, the modified IL-10 polypeptides described herein comprise an optional methionine at the N-terminus, shown as (M) in SEQ ID NOs: 1 and 3-73. In some embodiments, the modified IL-10 polypeptides comprise a methionine at the N-terminus of the wild-type or parent IL-10 sequence, followed by a serine. In some cases, the modified IL-10 polypeptides comprise a serine at the N-terminus of the wild-type or parent IL-10 sequence. In some embodiments, the modified IL-10 polypeptides comprise a methionine replacing and substituting for the serine at the N-terminus of the wild-type or parent IL-10 sequence. In some embodiments, the modified IL-10 polypeptides comprise a methionine at the N-terminus followed by a serine shown as (M) in SEQ ID NO: 1. In some cases, the modified IL-10 polypeptides comprise a serine at the N-terminus of SEQ ID NO: 1. In some embodiments, the modified IL-10 polypeptides comprise a methionine replacing and substituting for the serine at the N-terminus shown as (M) in SEQ ID NO: 1. In some cases, the parental IL-10 sequence is SEQ ID NO: 2.

[77] In some cases, the modified IL-10 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some cases, the modified IL-10 polypeptide has about 80% sequence identity to SEQ ID NO: 1. In some cases, the modified IL-10 polypeptide has about 85% sequence identity to SEQ ID NO: 1. In some cases, the modified IL-10 polypeptide has about 90% sequence identity to SEQ ID NO: 1. In some cases, the modified IL-10 polypeptide has about 95% sequence identity to SEQ ID NO: 1. In some cases, the modified IL-10 polypeptide has about 96% sequence identity to SEQ ID NO: 1. In some cases, the modified IL-10 polypeptide has about 97% sequence identity to SEQ ID NO: 1. In some cases, the modified IL-10 polypeptide has about 98% sequence identity to SEQ ID NO: 1. In some cases, the modified IL-10 polypeptide has about 99% sequence identity to SEQ ID NO: 1. In some cases, the modified IL-10 polypeptide comprises the sequence of SEQ ID NO: 1. In some cases, the modified IL-10 polypeptide consists of the sequence of SEQ ID NO: 1. In further cases, the modified IL-10 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 2. In further cases, the modified IL-10 polypeptide comprises the sequence of SEQ ID NO: 2. In further cases, the modified IL-10 polypeptide consists of the sequence of SEQ ID NO: 2.

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

[79] In some embodiments, the modified IL-10 polypeptide is a functionally active fragment. In some cases, the functionally active fragment comprises a region of IL-10 5-160, 10-160, 15-160, 20-160, 1-155, 5-155, 10-155, 15-155, 20-155, 1-150, 5-150, 10-150, 15-150, or 20-150, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some cases, the functionally active fragment provides a region of IL-10 5-160, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some cases, the functionally active fragment provides a region of IL-10 10-160, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some cases, the functionally active fragment provides a region of IL-10 15-160, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some cases, a functionally active fragment provides a region of IL-10 20-160, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some cases, a functionally active fragment provides a region of IL-10 1-155, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some cases, a functionally active fragment provides a region of IL-10 5-155, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some cases, a functionally active fragment provides a region of IL-10 10-155, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some cases, a functionally active fragment provides a region of IL-10 15-155, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some cases, a functionally active fragment provides a region of IL-10 20-155, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some cases, a functionally active fragment provides a region of IL-10 1-150, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some cases, the functionally active fragment provides a region of IL-10 5-150, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some cases, the functionally active fragment provides a region of IL-10 10-150, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some cases, the functionally active fragment provides a region of IL-10 15-150, wherein the residue positions are related to the positions of SEQ ID NO: 1. In some cases, the functionally active fragment provides a region of IL-10 20-150, wherein the residue positions are related to the positions of SEQ ID NO: 1.

[80] In some embodiments, described herein is an IL-10 polypeptide that comprises at least one unnatural amino acid. In some cases, the at least one unnatural amino acid is located in helix C, D, or E. In some cases, helix C comprises residues L60-N82, the positions of which are related to the positions of SEQ ID NO: 1. In some cases, helix D comprises residues I87-C108, the positions of which are related to the positions of SEQ ID NO: 1. In some cases, helix E comprises residues S118-L131, the positions of which are related to the positions of SEQ ID NO: 1. In some cases, the at least one unnatural amino acid is located at a surface exposed location in helix C, D, or E.

[81] In some embodiments, described herein is a modified IL-10 polypeptide that comprises at least one non-naturally occurring amino acid at a position selected from E67, Q70, E74, E75, Q79, N82, K88, A89, K99, K125, N126, N129, K130, or Q132, wherein the residue positions correspond to positions 67, 70, 74, 75, 79, 82, 88, 89, 99, 125, 126, 129, 130, and 132 set forth in SEQ ID NO: 1. In some cases, the position of the at least one non-naturally occurring amino acid is selected from E67, Q70, E74, E75, Q79, or N82, wherein the residue positions correspond to positions 67, 70, 74, 75, 79, and 82 as set forth in SEQ ID NO: 1. In some cases, the position of the at least one unnatural amino acid is selected from K88, A89, K99, K125, N126, N129, K130, or Q132, wherein the residue positions correspond to positions 88, 89, 99, 125, 126, 129, 130, and 132 as set forth in SEQ ID NO: 1. In some cases, the position of the at least one unnatural amino acid is selected from K125, N126, N129, K130, or Q132, wherein the residue positions correspond to positions 125, 126, 129, 130, and 132 as set forth in SEQ ID NO: 1. In some cases, the position of the at least one unnatural amino acid is selected from E67, Q70, E74, E75, Q79, N82, K88, A89, K99, K125, N126, N129, K130, or Q132, wherein the residue positions correspond to positions 67, 70, 74, 75, 79, 82, 88, 89, 99, 125, 126, 129, 130, and 132 as set forth in SEQ ID NO: 1. In some cases, the position of the at least one unnatural amino acid is E67. In some cases, the position of the at least one unnatural amino acid is Q70. In some cases, the position of the at least one unnatural amino acid is E74. In some cases, the position of the at least one unnatural amino acid is E75. In some cases, the position of the at least one unnatural amino acid is Q79. In some cases, the position of the at least one unnatural amino acid is N82. In some cases, the position of the at least one unnatural amino acid is K88. In some cases, the position of the at least one unnatural amino acid is A89. In some cases, the position of the at least one unnatural amino acid is K99. In some cases, the position of the at least one unnatural amino acid is K125. In some cases, the position of the at least one unnatural amino acid is N126. In some cases, the position of the at least one unnatural amino acid is N129. In some cases, the position of the at least one unnatural amino acid is K130. In some cases, the position of the at least one unnatural amino acid is Q132.

[82] In some embodiments, described herein are IL-10 polypeptides that are modified at an amino acid position. In some cases, the modification is to a naturally occurring amino acid. In some cases, the modification is to a non-naturally occurring amino acid. In some cases, described herein is an isolated and modified IL-10 polypeptide that comprises at least one non-naturally occurring amino acid. In some cases, the modified IL-10 polypeptide is an isolated and purified mammalian IL-10, such as a rodent IL-10 protein or a human IL-10 protein. In some cases, the modified IL-10 polypeptide is a human IL-10 protein.

[83] In some cases, the modified IL-10 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1. In some cases, the modified IL-10 polypeptide comprises the sequence of SEQ ID NO: 1. In some cases, the modified IL-10 polypeptide consists of the sequence of SEQ ID NO: 1. In some cases, the modified IL-10 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3. In some cases, the modified IL-10 polypeptide comprises the sequence of SEQ ID NO: 3. In some cases, the modified IL-10 polypeptide consists of the sequence of SEQ ID NO: 3. In further cases, the modified polypeptide IL-10 has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 4. In further instances, the modified IL-10 polypeptide comprises the sequence of SEQ ID NO: 4. In further instances, the modified IL-10 polypeptide consists of the sequence of SEQ ID NO: 4. In some instances, the modified IL-10 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 5. In some instances, the modified IL-10 polypeptide comprises the sequence of SEQ ID NO: 5. In some instances, the modified IL-10 polypeptide consists of the sequence of SEQ ID NO: 5. In further instances, the modified IL-10 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 6. In further instances, the modified IL-10 polypeptide comprises the sequence of SEQ ID NO: 6. In further instances, the modified IL-10 polypeptide consists of the sequence of SEQ ID NO: 6. In some instances, the modified IL-10 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 7. In some instances, the modified IL-10 polypeptide comprises the sequence of SEQ ID NO: 7. In some instances, the modified IL-10 polypeptide consists of the sequence of SEQ ID NO: 7. In further instances, the modified IL-10 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 8. In further instances, the modified IL-10 polypeptide comprises the sequence of SEQ ID NO: 8. In further instances, the modified IL-10 polypeptide consists of the sequence of SEQ ID NO: 8. In some instances, the modified IL-10 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 9. In some instances, the modified IL-10 polypeptide comprises the sequence of SEQ ID NO: 9. In some instances, the modified IL-10 polypeptide consists of the sequence of SEQ ID NO: 9. In some instances, the modified IL-10 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 10. In some cases, the modified IL-10 polypeptide comprises the sequence of SEQ ID NO: 10. In some cases, the modified IL-10 polypeptide consists of the sequence of SEQ ID NO: 10. In some cases, the modified IL-10 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 11. In some cases, the modified IL-10 polypeptide comprises the sequence of SEQ ID NO: 11. In some cases, the modified IL-10 polypeptide consists of the sequence of SEQ ID NO: 11. In some cases, the modified IL-10 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12. In some cases, the modified IL-10 polypeptide comprises the sequence of SEQ ID NO: 12. In some cases, the modified IL-10 polypeptide consists of the sequence of SEQ ID NO: 12. In some cases, the modified IL-10 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13. In some cases, the modified IL-10 polypeptide comprises the sequence of SEQ ID NO: 13. In some cases, the modified IL-10 polypeptide consists of the sequence of SEQ ID NO: 13. In some cases, the modified IL-10 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 14. In some cases, the modified IL-10 polypeptide comprises the sequence of SEQ ID NO: 14. In some cases, the modified IL-10 polypeptide consists of the sequence of SEQ ID NO: 14. In some cases, the modified IL-10 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 15. In some cases, the modified IL-10 polypeptide comprises the sequence of SEQ ID NO: 15. In some cases, the modified IL-10 polypeptide consists of the sequence of SEQ ID NO: 15. In some cases, the modified IL-10 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to with SEQ ID NO: 16. In some cases, the modified IL-10 polypeptide comprises the sequence of SEQ ID NO: 16. In some cases, the modified IL-10 polypeptide consists of the sequence of SEQ ID NO: 16. In some cases, the modified IL-10 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 17. In some cases, the modified IL-10 polypeptide comprises the sequence of SEQ ID NO: 17. In some cases, the modified IL-10 polypeptide consists of the sequence of SEQ ID NO: 17. In some cases, the modified IL-10 polypeptide has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 18. In some cases, the modified IL-10 polypeptide comprises the sequence of SEQ ID NO: 18. In some cases, the modified IL-10 polypeptide consists of the sequence of SEQ ID NO: 18.

[84] In some cases, the at least one unnatural amino acid is located proximal to the N-terminus. As used herein, "proximal" refers to a residue located at a distance of from at least 1 residue from the N-terminal residue to about 50 residues from the N-terminal residue. In some cases, the at least one unnatural amino acid is located within the first 10, 20, 30, 40, or 50 residues from the N-terminal residue. In some cases, the at least one unnatural amino acid is located within the first 10 residues from the N-terminal residue. In some cases, the at least one unnatural amino acid is located within the first 20 residues from the N-terminal residue. In some cases, the at least one unnatural amino acid is located within the first 30 residues from the N-terminal residue. In some cases, at least one unnatural amino acid is located within the first 40 residues of the N-terminal residue. In some cases, at least one unnatural amino acid is located within the first 50 residues of the N-terminal residue.

[85] In some cases, at least one unnatural amino acid is present at the N-terminal residue.

[86] In some cases, the at least one unnatural amino acid is located proximal to the C-terminus. As used herein, "proximal" refers to a residue located at a distance of from at least 1 residue from the C-terminal residue to about 50 residues from the C-terminal residue. In some cases, the at least one unnatural amino acid is located within the first 10, 20, 30, 40, or 50 residues from the C-terminal residue. In some cases, the at least one unnatural amino acid is located within the first 10 residues from the C-terminal residue. In some cases, the at least one unnatural amino acid is located within the first 20 residues from the C-terminal residue. In some cases, the at least one unnatural amino acid is located within the first 30 residues from the C-terminal residue. In some cases, at least one unnatural amino acid is located within the first 40 residues from the C-terminal residue. In some cases, at least one unnatural amino acid is located within the first 50 residues from the C-terminal residue.

[87] In some cases, at least one unnatural amino acid is present at the C-terminal residue.

[88] In some embodiments, the modified IL-10 polypeptide is a functionally active monomer or a functionally active dimer that is capable of binding to an IL-10R and activating a signaling pathway. In some cases, the functionally active modified IL-10 monomer or dimer is characterized by an increased plasma half-life. In some cases, the increased plasma half-life is comparable to the plasma half-life of the wild-type IL-10 protein. In some cases, the increased plasma half-life of the modified IL-10 polypeptide is at least 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, 30 days, or longer than the plasma half-life of the wild-type IL-10 protein. In some cases, the increased plasma half-life of the modified IL-10 polypeptide is approximately 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, or 30 days compared to the plasma half-life of the wild-type IL-10 protein.

[89] In some embodiments, the modified IL-10 monomer or dimer has a plasma half-life that allows for proliferation and/or expansion of tumor infiltrating lymphocytes (TILs), T cells, B cells, natural killer cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, basophils, or CD4+ or CD8+ T cells.

[90] In some embodiments, a modified IL-10 monomer or dimer is administered to a subject. In some embodiments, the modified IL-10 monomer or dimer administered to the subject has reduced toxicity compared to the toxicity of wild-type IL-10 administered to the subject. In some embodiments, the modified IL-10 monomer or dimer has reduced toxicity that is reduced by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, or more compared to the wild-type IL-10 dimer. In some cases, the reduced toxicity is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more compared to wild-type IL-10 protein.

[91] In some embodiments, the modified IL-10 monomer or dimer is administered to the subject. In some embodiments, the modified IL-10 monomer or dimer administered to the subject does not cause grade 3 or 4 adverse events. In some embodiments, the modified IL-10 monomer or dimer administered to the subject has a reduced incidence or severity of grade 3 or 4 adverse events compared to the incidence or severity of grade 3 or 4 adverse events caused by administration of wild-type IL-10 protein to the subject. Illustrative grade 3 or 4 adverse events include anemia, leukopenia, thrombocytopenia, ALT increased, anorexia, arthralgia, back pain, chills, diarrhea, dyslipidemia, fatigue, fever, influenza-like symptoms, hypoalbuminemia, lipase increased, injection site reaction, myalgia, nausea, night sweats, pruritis, rash, rash erythematous, rash maculopapular, transaminitis, vomiting, and weakness.

[92] In some embodiments, the modified IL-10 monomer or dimer reduces the incidence of grade 3 or 4 adverse events in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% compared to administration of wild-type IL-10 protein to the subject. In some cases, the modified IL-10 monomer or dimer reduces the severity of grade 3 or 4 adverse events in the subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% compared to administration of wild-type IL-10 protein to the subject.

[93] In some embodiments, a modified IL-10 monomer or dimer as described herein has a reduced affinity for the IL-10R compared to the affinity of wild-type IL-10 protein for the IL-10R. In some embodiments, the affinity of the modified IL-10 monomer or dimer for the IL-10R compared to the affinity of wild-type IL-10 protein for the IL-10R is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or greater than 99%. In some cases, the reduction in affinity is about 10%. In some cases, the reduction in affinity is about 20%. In some cases, the reduction in affinity is about 30%. In some cases, the reduction in affinity is about 40%. In some cases, the decrease in affinity is approximately 50%. In some cases, the decrease in affinity is approximately 60%. In some cases, the decrease in affinity is approximately 70%. In some cases, the decrease in affinity is approximately 80%. In some cases, the decrease in affinity is approximately 90%. In some cases, the decrease in affinity is approximately 95%. In some cases, the decrease in affinity is approximately 99%. In some cases, the decrease in affinity is approximately 100%.

[94] In some embodiments, the decrease in affinity of the modified IL-10 monomer or dimer compared to wild-type IL-10 protein is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more. In some cases, the decrease in affinity is about 1-fold. In some cases, the decrease in affinity is about 2-fold. In some cases, the decrease in affinity is about 3-fold. In some cases, the decrease in affinity is about 4-fold. In some cases, the decrease in affinity is approximately 5-fold. In some cases, the decrease in affinity is approximately 6-fold. In some cases, the decrease in affinity is approximately 7-fold. In some cases, the decrease in affinity is approximately 8-fold. In some cases, the decrease in affinity is approximately 9-fold. In some cases, the decrease in affinity is approximately 10-fold. In some cases, the decrease in affinity is approximately 30-fold. In some cases, the decrease in affinity is approximately 50-fold. In some cases, the decrease in affinity is approximately 100-fold. In some cases, the decrease in affinity is approximately 200-fold. In some cases, the decrease in affinity is approximately 300-fold. In some cases, the decrease in affinity is approximately 400-fold. In some cases, the decrease in affinity is approximately 500-fold. In some cases, the decrease in affinity is approximately 1000-fold. In some cases, the reduction in affinity is more than 1000-fold.

[95] In some cases, the modified IL-10 monomer or dimer does not interact with IL-10R. In some cases, the modified IL-10 monomer or dimer has approximately the same affinity for IL-10R as the affinity of wild-type IL-10 for IL-10R.

[96] In some embodiments, a modified IL-10 monomer or dimer as described herein has an increased affinity for the IL-10R compared to the affinity of wild-type IL-10 protein for the IL-10R. In some embodiments, the affinity of the modified IL-10 monomer or dimer for the IL-10R compared to the affinity of wild-type IL-10 protein for the IL-10R is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or greater than 99%. In some cases, the increase in affinity is about 10%. In some cases, the increase in affinity is about 20%. In some cases, the increase in affinity is about 30%. In some cases, the increase in affinity is about 40%. In some cases, the increase in affinity is approximately 50%. In some cases, the increase in affinity is approximately 60%. In some cases, the increase in affinity is approximately 70%. In some cases, the increase in affinity is approximately 80%. In some cases, the increase in affinity is approximately 90%. In some cases, the increase in affinity is approximately 95%. In some cases, the increase in affinity is approximately 99%. In some cases, the increase in affinity is approximately 100%.

[97] In some embodiments, the increase in affinity of the modified IL-10 monomer or dimer compared to wild-type IL-10 protein is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more. In some cases, the increase in affinity is about 1-fold. In some cases, the increase in affinity is about 2-fold. In some cases, the increase in affinity is about 3-fold. In some cases, the increase in affinity is about 4-fold. In some cases, the increase in affinity is approximately 5-fold. In some cases, the increase in affinity is approximately 6-fold. In some cases, the increase in affinity is approximately 7-fold. In some cases, the increase in affinity is approximately 8-fold. In some cases, the increase in affinity is approximately 9-fold. In some cases, the increase in affinity is approximately 10-fold. In some cases, the increase in affinity is approximately 30-fold. In some cases, the increase in affinity is approximately 50-fold. In some cases, the increase in affinity is approximately 100-fold. In some cases, the increase in affinity is approximately 200-fold. In some cases, the increase in affinity is approximately 300-fold. In some cases, the increase in affinity is approximately 400-fold. In some cases, the increase in affinity is approximately 500-fold. In some cases, the increase in affinity is approximately 1000-fold. In some cases, the increase in affinity is more than 1000-fold.

[98] In some cases, the IL-10R signaling efficacy mediated by IL-10 is measured by a reduction in half the maximal effective concentration (EC50). In some embodiments, the EC50 of the modified IL-10 monomer or dimer is reduced compared to the EC50 of the wild-type IL-10 protein. In some embodiments, the reduction in the EC50 of the modified IL-10 monomer or dimer is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or greater than 99%. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 10%. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 20%. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 30%. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 40%. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 50%. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 60%. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 70%. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 80%. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 90%. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 95%. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 99%. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 100%.

[99] In some embodiments, the reduction in EC50 of the modified IL-10 monomer or dimer compared to the wild-type IL-10 protein is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 1-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 2-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 3-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 4-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 5-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 6-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 7-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 8-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 9-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 10-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 30-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 50-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 100-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 200-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 300-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 400-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 500-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by about 1000-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is reduced by more than 1000-fold.

[100] In some cases, the EC50 of the modified IL-10 monomer or dimer is approximately the same as the EC50 of the wild-type IL-10 protein.

[101] In some cases, a modified IL-10 monomer or dimer as described herein has an increased EC50 compared to the EC50 of wild-type IL-10 protein in activating signaling through IL-10R. In some embodiments, the increase in the EC50 of the modified IL-10 monomer or dimer is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or greater than 99%. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 10%. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 20%. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 30%. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 40%. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 50%. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 60%. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 70%. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 80%. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 90%. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 95%. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 99%. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 100%.

[102] In some embodiments, the increase in the EC50 of the modified IL-10 monomer or dimer compared to the EC50 of the wild-type IL-10 protein is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 1-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 2-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 3-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 4-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 5-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 6-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 7-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 8-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 9-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 10-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 30-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 50-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 100-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 200-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 300-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 400-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 500-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by about 1000-fold. In some cases, the EC50 of the modified IL-10 monomer or dimer is increased by more than 1000-fold.

[103] In some cases, the IL-10R signaling efficacy mediated by IL-10 is measured by the median effective dose (ED50). In some embodiments, a modified IL-10 monomer or dimer as described herein has a reduced ED50 compared to the ED50 of the wild-type IL-10 protein. In some embodiments, the reduction in the ED50 of the modified IL-10 monomer or dimer is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or greater than 99%. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 10%. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 20%. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 30%. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 40%. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 50%. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 60%. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 70%. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 80%. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 90%. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 95%. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 99%. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by approximately 100%.

[104] In some embodiments, the reduction in the ED50 of the modified IL-10 monomer or dimer compared to the ED50 of the wild-type IL-10 protein is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 1-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 2-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 3-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 4-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 5-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 6-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 7-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 8-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 9-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 10-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 30-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 50-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 100-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 200-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 300-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 400-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 500-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by about 1000-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is reduced by more than 1000-fold.

[105] In some cases, the ED50 of the modified IL-10 monomer or dimer is approximately the same as the ED50 of the wild-type IL-10 protein.

[106] In some cases, a modified IL-10 monomer or dimer described herein has an increased ED50 compared to the ED50 of a wild-type IL-10 protein. In some embodiments, the increase in the ED50 of the modified IL-10 monomer or dimer is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or greater than 99%. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 10%. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 20%. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 30%. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 40%. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 50%. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 60%. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 70%. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 80%. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 90%. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 95%. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 99%. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 100%.

[107] In some embodiments, the increase in the ED50 of the modified IL-10 monomer or dimer compared to the ED50 of the wild-type IL-10 protein is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 1-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 2-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 3-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 4-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 5-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 6-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 7-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 8-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 9-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 10-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 30-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 50-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 100-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 200-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 300-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 400-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 500-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by about 1000-fold. In some cases, the ED50 of the modified IL-10 monomer or dimer is increased by more than 1000-fold.

IL-10 conjugates

[108] Described herein in some embodiments are IL-10 conjugates. In some embodiments, the modified IL-10 polypeptides described herein are IL-10 conjugates. In some embodiments, an IL-10 conjugate comprises an IL-10 polypeptide comprising at least one unnatural amino acid and at least one conjugable moiety linked to the at least one unnatural amino acid. In some cases, the at least one conjugable moiety is directly linked to the at least one unnatural amino acid. In other cases, the at least one conjugable moiety is indirectly linked to the at least one unnatural amino acid via a linker described herein.

[109] In some embodiments, the IL-10 conjugate comprises at least one mutation comprising at least one unnatural amino acid and at least one conjugated moiety linked to at least one unnatural amino acid at at least one of any one of the positions in SEQ ID NOs: 1-66 ( Table 1 ). In some embodiments, the IL-10 conjugates described herein comprise an optional methionine at the N-terminus, shown as (M) in SEQ ID NOs: 1 and 3-73. In some embodiments, the IL-10 conjugates comprise a methionine at the N-terminus of a wild-type or parental IL-10 sequence, followed by a serine. In some cases, the IL-10 conjugates described herein comprise a serine at the N-terminus of the wild-type IL-10 sequence or the parental IL-10 sequence. In some embodiments, the modified IL-10 conjugates comprise a methionine replacing and substituting for the serine at the N-terminus of the wild-type IL-10 sequence or the parental IL-10 sequence. In some embodiments, the IL-10 conjugates comprise a methionine at the N-terminus followed by the serine shown as (M) in SEQ ID NO: 1. In some cases, the IL-10 conjugates comprise a serine at the N-terminus of SEQ ID NO: 1. In some embodiments, the IL-10 conjugates comprise a methionine replacing and substituting for the serine at the N-terminus shown as (M) in SEQ ID NO: 1.

(M) indicates that a methionine residue may optionally be added to the N-terminus of the modified IL-10 polypeptides and IL-10 conjugates shown under SEQ ID NOs: 1 and 3-73. Alternatively, a methionine residue may replace and substitute for serine at the N-terminus.

X denotes a site containing an unnatural amino acid.

[AzK] stands for N6-((2-azidoethoxy)-carbonyl)-L-lysine. The compound has a Chemical Abstracts accession number of 1167421-25-1.

[AzK_PEG] denotes N6-((2-azidoethoxy)-carbonyl)-L-lysine stably conjugated to PEG via DBCO-mediated click chemistry to form a compound having a structure of formula (II) or formula (III) or formula (X) or formula (XI). In some examples, the compound has a structure of formula (II), formula (III), formula (X) or formula (XI), wherein the q substituent is present and q is 1. In some examples, the compound has a structure of formula (II), formula (III), formula (X) or formula (XI), wherein the q substituent is present and q is 2. In some examples, the compound has a structure of formula (II), formula (III), formula (X) or formula (XI), wherein the q substituent is present and q is 3. For example, when specified, PEG20kDa means, in the case of a compound having a structure of formula (II) or formula (III), a linear polyethylene glycol chain with an average molecular weight of 20 kilodaltons, capped with a methoxy group. In another example, if indicated, PEG20kDa denotes, in the case of a compound providing a structure of formula (X) or formula (XI), a compound wherein n is a value providing a PEG group with a mass of 20 kilodaltons. The ratio of regioisomers formed as a result of the click reaction is about 1:1 or greater than 1:1. The term "DBCO" denotes a chemical moiety containing a dibenzocyclooctyne group, such as, for example, containing the compound mPEG-DBCO.

[AzK_L1_PEG] denotes N6-((2-azidoethoxy)-carbonyl)-L-lysine stably conjugated to PEG via DBCO-mediated click chemistry to form a compound having a structure of formula (IV) or formula (V) or formula (XII) or formula (XIII). In some examples, the compound has a structure of formula (IV), formula (V), formula (XII) or formula (XIII), wherein the q substituent is present and q is 1. In some examples, the compound has a structure of formula (IV), formula (V), formula (XII) or formula (XIII), wherein the q substituent is present and q is 2. In some examples, the compound has a structure of formula (IV), formula (V), formula (XII) or formula (XIII), wherein the q substituent is present and q is 3. For example, when specified, PEG20kDa means, in the case of a compound having a structure of formula (IV) or formula (V), a linear polyethylene glycol chain with an average molecular weight of 20 kilodaltons, capped with a methoxy group. In another example, if indicated, PEG20kDa denotes, in the case of a compound providing a structure of formula (XII) or formula (XIII), a compound wherein n is a value providing a PEG group with a mass of 20 kilodaltons. The ratio of regioisomers formed as a result of the click reaction is about 1:1 or greater than 1:1. The term "DBCO" denotes a chemical moiety containing a dibenzocyclooctyne group, such as containing the compound mPEG-DBCO.

[His] is an amino acid sequence containing a histidine tag and a TEV recognition site having the sequence HHHHHHGSSENLYFQ (residues 1-15 of SEQ ID NO: 67-73). This sequence can be cleaved from the expressed IL-10 conjugate by methods described herein and known to those skilled in the art to produce an IL-10 conjugate lacking the amino acid sequence HHHHHHGSSENLYFQ (residues 1-15 of SEQ ID NO: 67-73). For example, the histidine tag and TEV recognition site contained in the IL-10 conjugate of SEQ ID NO: 68 can be cleaved to yield the IL-10 conjugate of SEQ ID NO: 27. More generally, "[His]-SEQ ID NO: X" indicates that the sequence containing the histidine tag and TEV recognition site shown above is present at the N-terminus of said sequence, immediately following the original methionine, if present.

[110] As described herein, at least one unnatural amino acid is optionally located in helix C, D, or E, such as in an exposed residue position on the surface. In some cases, the residues include E67, Q70, E74, E75, Q79, N82, K88, A89, K99, K125, N126, N129, K130, or Q132, wherein the residue positions correspond to positions 67, 70, 74, 75, 79, 82, 88, 89, 99, 125, 126, 129, 130, and 132 as set forth in SEQ ID NO: 1. In some cases, the residues include E67, Q70, E74, E75, Q79, or N82, wherein the residue positions correspond to positions 67, 70, 74, 75, 79, and 82 as set forth in SEQ ID NO: 1. In some cases, the residue includes K88, K125, N126, N129, K130 or Q132, wherein the residue positions correspond to positions 88, 125, 126, 129, 130 and 132 as set forth in SEQ ID NO: 1. In some cases, the residue provides K125, N126, N129, K130 or Q132, wherein the residue positions correspond to positions 125, 126, 129, 130 and 132 as set forth in SEQ ID NO: 1. In some cases, the residue provides Q70, E74, N82, K88, N126, K130 or Q132, wherein the residue positions correspond to positions 70, 74, 82, 88, 126, 130 and 132 as set forth in SEQ ID NO: 1. In some cases, the residue provides A89 and K99, wherein the residue positions correspond to positions 89 and 99 as set forth in SEQ ID NO: 1.

[111] In some cases, the position of the at least one unnatural amino acid is E67 of SEQ ID NO: 1. In some cases, the position of the at least one unnatural amino acid is Q70 of SEQ ID NO: 1. In some cases, the position of the at least one unnatural amino acid is E74 of SEQ ID NO: 1. In some cases, the position of the at least one unnatural amino acid is E75 of SEQ ID NO: 1. In some cases, the position of the at least one unnatural amino acid is Q79 of SEQ ID NO: 1. In some cases, the position of the at least one unnatural amino acid is N82 of SEQ ID NO: 1. In some cases, the position of the at least one unnatural amino acid is K88 of SEQ ID NO: 1. In some cases, the position of the at least one unnatural amino acid is A89 of SEQ ID NO: 1. In some cases, the position of the at least one unnatural amino acid is K99 of SEQ ID NO: 1. In some cases, the position of the at least one unnatural amino acid is K125 of SEQ ID NO: 1. In some cases, the position of the at least one unnatural amino acid is N126 of SEQ ID NO: 1. In some cases, the position of the at least one unnatural amino acid is N129 of SEQ ID NO: 1. In some cases, the position of the at least one unnatural amino acid is K130 of SEQ ID NO: 1. In some cases, the position of the at least one unnatural amino acid is Q132 of SEQ ID NO: 1.

[112] In some cases, the at least one unnatural amino acid residue is selected from E85, Q88, E92, E93, Q97, N100, K106, A107, K117, K143, N144, N147, K148, or Q150, wherein the residue positions correspond to positions 85, 88, 92, 93, 97, 100, 106, 107, 117, 143, 144, 147, 148, and 150 as set forth in the IL-10 precursor of SEQ ID NO: 2. In some cases, the position of the at least one unnatural amino acid is E85 of SEQ ID NO: 2. In some cases, the position of the at least one unnatural amino acid is Q88 of SEQ ID NO: 2. In some cases, the position of the at least one unnatural amino acid is is E92 of SEQ ID NO: 2. In some cases, the position of the at least one unnatural amino acid is E93 of SEQ ID NO: 2. In some cases, the position of the at least one unnatural amino acid is Q97 of SEQ ID NO: 2. In some cases, the position of the at least one unnatural amino acid is N100 of SEQ ID NO: 2. In some cases, the position of the at least one unnatural amino acid is K106 of SEQ ID NO: 2. In some cases, the position of the at least one unnatural amino acid is A107 of SEQ ID NO: 2. In some cases, the position of the at least one unnatural amino acid is K117 of SEQ ID NO: 2. In some cases, the position of the at least one unnatural amino acid is K143 of SEQ ID NO: 2. In some cases, the position of the at least one unnatural amino acid is N144 of SEQ ID NO: 2. In some cases, the position of the at least one unnatural amino acid is N147 of SEQ ID NO: 2. In some cases, the position of the at least one unnatural amino acid is K148 of SEQ ID NO: 2. In some cases, the position of the at least one unnatural amino acid is Q150 of SEQ ID NO: 2.

[113] In some embodiments, this document describes an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (I),

Where

Z is CH ₂ and Y is ;

Y is CH ₂ , and Z is ;

Z is CH ₂ and Y is ; or

Y is CH ₂ , and Z is ;

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

X has the structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

[114] In other embodiments, described herein is an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (I),

Where

Z is CH ₂ and Y is ;

Y is CH ₂ , and Z is ;

Z is CH ₂ and Y is ; or

Y is CH ₂ , and Z is ;

q is equal to 1, 2, or 3;

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

X has the structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

[115] Here and throughout this document, the term "IL-10 conjugate" includes pharmaceutically acceptable salts, solvates and hydrates of the structure indicated.

[116] Here and throughout this document, the structure of formula (I) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. In some embodiments, the structure of formula (I) or any embodiment or variation thereof is provided as a pharmaceutically acceptable salt thereof. In some embodiments, the structure of formula (I) or any embodiment or variation thereof is provided as a solvate thereof. In some embodiments, the structure of formula (I) or any embodiment or variation thereof is provided as a hydrate thereof. In some embodiments, the structure of formula (I) or any embodiment or variation thereof is provided as a free base.

[117] In some embodiments of an IL-10 conjugate comprising formula (I), Z is CH ₂ and Y is

[118] . In some embodiments of the IL-10 conjugate comprising formula (I), Y is CH ₂ and Z is . In some embodiments of an IL-10 conjugate comprising formula (I), Z is CH ₂ and Y is . In some embodiments of the IL-10 conjugate comprising formula (I), Y is CH ₂ and Z is . In some embodiments of an IL-10 conjugate comprising formula (I), Z is CH ₂ and Y is . In some embodiments of the IL-10 conjugate comprising formula (I), Y is CH ₂ and Z is . In some embodiments of the IL-10 conjugate comprising formula (I), Z is CH ₂ and Y is . In some embodiments of the IL-10 conjugate comprising formula (I), Y is CH ₂ and Z is Here and throughout this document, embodiments of Z and Y also encompass a pharmaceutically acceptable salt, solvate or hydrate thereof.

[119] In some embodiments of an IL-10 conjugate comprising formula (I), q is 1. In some embodiments of an IL-10 conjugate comprising formula (I), q is 2. In some embodiments of an IL-10 conjugate comprising formula (I), q is 3.

[120] In some embodiments of the IL-10 conjugate of formula (I), the PEG moiety has an average molecular weight selected from 500 daltons, 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 100 kDa. In some embodiments, the PEG moiety has an average molecular weight selected from 5 kDa, 10 kDa, 20 kDa, and 30 kDa. In some embodiments of the IL-10 conjugate of formula (I), the PEG moiety has an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate of formula (I), the PEG group has an average molecular weight of 30 kDa.

[121] In some embodiments of an IL-10 conjugate comprising formula (I), the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is selected from E67, Q70, E74, E75, Q79, N82, K88, A89, K99, K125, N126, N129, K130, and Q132, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is among the positions in SEQ ID NO: 1. In some embodiments of an IL-10 conjugate comprising formula (I), the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is selected from N82, K88, A89, K99, K125, N126, N129, and K130, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate refers to the positions in SEQ ID NO: 1. In some embodiments of the IL-10 conjugate providing formula (I), the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is E67, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate refers to the positions in SEQ ID NO: 1. In some embodiments of the IL-10 conjugate providing formula (I), the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is Q70, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate refers to the positions in SEQ ID NO: 1. In some embodiments of the IL-10 conjugate providing formula (I), the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is E74, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate refers to the positions in SEQ ID NO: 1. In some embodiments of the IL-10 conjugate comprising formula (I), the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is E75, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate refers to the positions in SEQ ID NO: 1. In some embodiments of the IL-10 conjugate comprising formula (I), the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is Q79, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate refers to the positions in SEQ ID NO: 1. In some embodiments of the IL-10 conjugate comprising formula (I), the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is N82, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is between the positions in SEQ ID NO: 1 and in SEQ ID NO: 3. In some embodiments of the IL-10 conjugate comprising formula (I), the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is K88, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is between the positions in SEQ ID NO: 1 and in SEQ ID NO: 4. In some embodiments of the IL-10 conjugate comprising formula (I), the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is A89, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is between the positions in SEQ ID NO: 1 and in SEQ ID NO: 5. In some embodiments of the conjugate IL-10 comprising formula (I), the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is K99, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is related to the positions in SEQ ID NO: 1 and in SEQ ID NO: 6. In some embodiments of the IL-10 conjugate comprising formula (I), the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is K125, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is related to the positions in SEQ ID NO: 1 and in SEQ ID NO: 7. In some embodiments of the IL-10 conjugate comprising formula (I), the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is N126, wherein the position of the structure of formula (I) in the amino acid sequence of the conjugate IL-10 refers to the positions of SEQ ID NO: 1 and SEQ ID NO: 8. In some embodiments of an IL-10 conjugate comprising formula (I), the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is N129, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate refers to the positions of SEQ ID NO: 1 and SEQ ID NO: 9. In some embodiments of an IL-10 conjugate comprising formula (I), the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is K130, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate refers to the positions of SEQ ID NO: 1 and SEQ ID NO: 10. In some embodiments of an IL-10 conjugate comprising formula (I), the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is Q132, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate refers to the positions in SEQ ID NO: 1.

[122] In some embodiments, this document describes an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 19-26, wherein [AzK_PEG] has a structure of formula (II), formula (III), or is a mixture of formula (II) and formula (III),

Where

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

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

[123] In some embodiments, this document describes an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 19-26, wherein [AzK_PEG] has a structure of formula (II), formula (III), or is a mixture of formula (II) and formula (III),

Where

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

q is equal to 1, 2, or 3; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

[124] Here and throughout this document, the structure of formula (II) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. Here and throughout this document, the structure of formula (III) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof.

[125] In some embodiments, [AzK_PEG] has a structure of formula (II). In some embodiments, [AzK_PEG] has a structure of formula (III). In some embodiments, [AzK_PEG] is a mixture of formula (II) and formula (III).

[126] In some embodiments of an IL-10 conjugate having formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 19. In some embodiments of an IL-10 conjugate having formula (II) and having the amino acid sequence of SEQ ID NO: 19, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate having formula (II) and having the amino acid sequence of SEQ ID NO: 19, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate comprising formula (II) and having the amino acid sequence of SEQ ID NO: 19, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate comprising formula (II) and having the amino acid sequence of SEQ ID NO: 19, W is a PEG moiety having an average molecular weight of 30 kDa.

[127] In some embodiments of an IL-10 conjugate having formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 20. In some embodiments of an IL-10 conjugate having formula (II) and having the amino acid sequence of SEQ ID NO: 20, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate having formula (II) and having the amino acid sequence of SEQ ID NO: 20, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate comprising formula (II) and having the amino acid sequence of SEQ ID NO: 20, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate comprising formula (II) and having the amino acid sequence of SEQ ID NO: 20, W is a PEG moiety having an average molecular weight of 30 kDa.

[128] In some embodiments of an IL-10 conjugate having formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 21. In some embodiments of an IL-10 conjugate having formula (II) and having the amino acid sequence of SEQ ID NO: 21, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate having formula (II) and having the amino acid sequence of SEQ ID NO: 10, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate having formula (II) and the amino acid sequence of SEQ ID NO: 21, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate having formula (II) and the amino acid sequence of SEQ ID NO: 21, W is a PEG moiety having an average molecular weight of 30 kDa.

[129] In some embodiments of an IL-10 conjugate having formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 22. In some embodiments of an IL-10 conjugate having formula (II) and having the amino acid sequence of SEQ ID NO: 22, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate having formula (II) and having the amino acid sequence of SEQ ID NO: 22, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate having formula (II) and the amino acid sequence of SEQ ID NO: 22, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate having formula (II) and the amino acid sequence of SEQ ID NO: 22, W is a PEG moiety having an average molecular weight of 30 kDa.

[130] In some embodiments of an IL-10 conjugate having formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 23. In some embodiments of an IL-10 conjugate having formula (II) and having the amino acid sequence of SEQ ID NO: 23, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate having formula (II) and having the amino acid sequence of SEQ ID NO: 23, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate having formula (II) and the amino acid sequence of SEQ ID NO: 23, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate having formula (II) and the amino acid sequence of SEQ ID NO: 23, W is a PEG moiety having an average molecular weight of 30 kDa.

[131] In some embodiments of an IL-10 conjugate having formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 24. In some embodiments of an IL-10 conjugate having formula (II) and having the amino acid sequence of SEQ ID NO: 24, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate having formula (II) and having the amino acid sequence of SEQ ID NO: 24, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate of formula (II) and having the amino acid sequence of SEQ ID NO: 24, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate of formula (II) and having the amino acid sequence of SEQ ID NO: 24, W is a PEG moiety having an average molecular weight of 30 kDa.

[132] In some embodiments of an IL-10 conjugate having formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 25. In some embodiments of an IL-10 conjugate having formula (II) and having the amino acid sequence of SEQ ID NO: 25, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate having formula (II) and having the amino acid sequence of SEQ ID NO: 25, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate having formula (II) and the amino acid sequence of SEQ ID NO: 25, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate having formula (II) and the amino acid sequence of SEQ ID NO: 25, W is a PEG moiety having an average molecular weight of 30 kDa.

[133] In some embodiments of an IL-10 conjugate having formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 26. In some embodiments of an IL-10 conjugate having formula (II) and having the amino acid sequence of SEQ ID NO: 26, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate having formula (II) and having the amino acid sequence of SEQ ID NO: 26, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate having formula (II) and the amino acid sequence of SEQ ID NO: 26, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate having formula (II) and the amino acid sequence of SEQ ID NO: 26, W is a PEG moiety having an average molecular weight of 30 kDa.

[134] In some embodiments of an IL-10 conjugate having formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 19. In some embodiments of an IL-10 conjugate having formula (III) and having the amino acid sequence of SEQ ID NO: 19, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate having formula (III) and having the amino acid sequence of SEQ ID NO: 19, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate comprising formula (III) and having the amino acid sequence of SEQ ID NO: 19, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate comprising formula (III) and having the amino acid sequence of SEQ ID NO: 19, W is a PEG moiety having an average molecular weight of 30 kDa.

[135] In some embodiments of an IL-10 conjugate having formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 20. In some embodiments of an IL-10 conjugate having formula (III) and having the amino acid sequence of SEQ ID NO: 20, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate having formula (III) and having the amino acid sequence of SEQ ID NO: 20, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate comprising formula (III) and having the amino acid sequence of SEQ ID NO: 20, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate comprising formula (III) and having the amino acid sequence of SEQ ID NO: 20, W is a PEG moiety having an average molecular weight of 30 kDa.

[136] In some embodiments of an IL-10 conjugate having formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 21. In some embodiments of an IL-10 conjugate having formula (III) and having the amino acid sequence of SEQ ID NO: 21, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate having formula (III) and having the amino acid sequence of SEQ ID NO: 10, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate comprising formula (III) and having the amino acid sequence of SEQ ID NO: 21, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate comprising formula (III) and having the amino acid sequence of SEQ ID NO: 21, W is a PEG moiety having an average molecular weight of 30 kDa.

[137] In some embodiments of an IL-10 conjugate having formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 22. In some embodiments of an IL-10 conjugate having formula (III) and having the amino acid sequence of SEQ ID NO: 22, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate having formula (III) and having the amino acid sequence of SEQ ID NO: 22, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate comprising formula (III) and having the amino acid sequence of SEQ ID NO: 22, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate comprising formula (III) and having the amino acid sequence of SEQ ID NO: 22, W is a PEG moiety having an average molecular weight of 30 kDa.

[138] In some embodiments of an IL-10 conjugate having formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 23. In some embodiments of an IL-10 conjugate having formula (III) and having the amino acid sequence of SEQ ID NO: 23, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate having formula (III) and having the amino acid sequence of SEQ ID NO: 23, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate comprising formula (III) and having the amino acid sequence of SEQ ID NO: 23, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate comprising formula (III) and having the amino acid sequence of SEQ ID NO: 23, W is a PEG moiety having an average molecular weight of 30 kDa.

[139] In some embodiments of an IL-10 conjugate having formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 24. In some embodiments of an IL-10 conjugate having formula (III) and having the amino acid sequence of SEQ ID NO: 24, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate having formula (III) and having the amino acid sequence of SEQ ID NO: 24, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate comprising formula (III) and having the amino acid sequence of SEQ ID NO: 24, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate comprising formula (III) and having the amino acid sequence of SEQ ID NO: 24, W is a PEG moiety having an average molecular weight of 30 kDa.

[140] In some embodiments of an IL-10 conjugate having formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 25. In some embodiments of an IL-10 conjugate having formula (III) and having the amino acid sequence of SEQ ID NO: 25, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate having formula (III) and having the amino acid sequence of SEQ ID NO: 25, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate comprising formula (III) and having the amino acid sequence of SEQ ID NO: 25, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate comprising formula (III) and having the amino acid sequence of SEQ ID NO: 25, W is a PEG moiety having an average molecular weight of 30 kDa.

[141] In some embodiments of an IL-10 conjugate having formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 26. In some embodiments of an IL-10 conjugate having formula (III) and having the amino acid sequence of SEQ ID NO: 26, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate having formula (III) and having the amino acid sequence of SEQ ID NO: 26, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate comprising formula (III) and having the amino acid sequence of SEQ ID NO: 26, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate comprising formula (III) and having the amino acid sequence of SEQ ID NO: 26, W is a PEG moiety having an average molecular weight of 30 kDa.

[142] In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 19. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 19, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 19, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 19, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 19, W is a PEG moiety having an average molecular weight of 30 kDa.

[143] In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has an amino acid sequence of SEQ ID NO: 20. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having an amino acid sequence of SEQ ID NO: 20, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 20, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 20, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 20, W is a PEG moiety having an average molecular weight of 30 kDa.

[144] In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 21. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 21, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 10, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 21, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 21, W is a PEG moiety having an average molecular weight of 30 kDa.

[145] In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 22. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 22, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 22, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 22, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 22, W is a PEG moiety having an average molecular weight of 30 kDa.

[146] In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has an amino acid sequence of SEQ ID NO: 23. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having an amino acid sequence of SEQ ID NO: 23, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 23, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 23, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 23, W is a PEG moiety having an average molecular weight of 30 kDa.

[147] In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 24. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 24, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 24, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 24, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 24, W is a PEG moiety having an average molecular weight of 30 kDa.

[148] In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 25. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 25, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 25, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 25, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 25, W is a PEG moiety having an average molecular weight of 30 kDa.

[149] In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 26. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 26, W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 26, W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 26, W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having the amino acid sequence of SEQ ID NO: 26, W is a PEG moiety having an average molecular weight of 30 kDa.

[150] In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having one or more amino acid sequences of SEQ ID NOs: 19-26, W is a linear or branched PEG moiety. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having one or more amino acid sequences of SEQ ID NOs: 19-26, W is a linear PEG moiety. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having one or more amino acid sequences of SEQ ID NOs: 19-26, W is a branched PEG moiety. In some embodiments of the IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having one or more amino acid sequences of SEQ ID NOs: 19-26, W is a methoxy-PEG group. In some embodiments of the IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having one or more amino acid sequences of SEQ ID NOs: 19-26, the methoxy-PEG group is linear or branched. In some embodiments of the IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having one or more amino acid sequences of SEQ ID NOs: 19-26, the methoxy-PEG group is linear. In some embodiments of an IL-10 conjugate comprising formula (II), formula (III), or a mixture of formula (II) and formula (III) and having one or more amino acid sequences of SEQ ID NO: 19-26, the methoxy-PEG group is branched.

[151] In some embodiments, this document describes an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 27-34, wherein [AzK_PEG20kDa] has a structure of formula (II), formula (III), or is a mixture of formula (II) and formula (III),

Where

W is a PEG group having an average molecular weight of 20 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

[152] In some embodiments, this document describes an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 27-34, wherein [AzK_PEG20kDa] has a structure of formula (II), formula (III), or is a mixture of formula (II) and formula (III),

Where

W is a PEG group with an average molecular weight of 20 kDa;

q is equal to 1, 2, or 3; and

X has the structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

[153] In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 27. In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 28. In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 29. In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 30. In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 31. In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 32. In some embodiments of the conjugate IL-10 comprising [AzK_PEG20kDa] and having a structure of formula (II), formula (III), or being a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 33. In some embodiments of the IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (II), formula (III), or being a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 34.

[154] In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 27. In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 28. In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 29. In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 30. In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 31. In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 32. In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 33. In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 34.

[155] In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 27. In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 28. In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 29. In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 30. In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 31. In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 32. In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 33. In some embodiments of an IL-10 conjugate comprising [AzK_PEG20kDa] and having a structure of formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 34.

[156] In some embodiments, this document describes an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 35-42, wherein [AzK_PEG30kDa] has a structure of formula (II), formula (III), or is a mixture of formula (II) and formula (III),

Where

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

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

[157] In some embodiments, this document describes an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 35-42, wherein [AzK_PEG30kDa] has a structure of formula (II), formula (III), or is a mixture of formula (II) and formula (III),

Where

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

q is equal to 1, 2, or 3; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

[158] In some embodiments of an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 35. In some embodiments of an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 36. In some embodiments of an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 37. In some embodiments, an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 38. In some embodiments, an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 39. In some embodiments, an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (II), formula (III), or a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 40. In some embodiments, the conjugate IL-10 comprising [AzK_PEG30kDa] and having a structure of formula (II), formula (III), or being a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 41. In some embodiments of the IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (II), formula (III), or being a mixture of formula (II) and formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 42.

[159] In some embodiments of an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 35. In some embodiments of an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 36. In some embodiments of an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 37. In some embodiments of an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 38. In some embodiments of an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 39. In some embodiments of an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 40. In some embodiments of an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 41. In some embodiments of an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (II), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 42.

[160] In some embodiments of an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 35. In some embodiments of an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 36. In some embodiments of an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 37. In some embodiments of an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 38. In some embodiments of an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 39. In some embodiments of an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 40. In some embodiments of an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 41. In some embodiments of an IL-10 conjugate comprising [AzK_PEG30kDa] and having a structure of formula (III), the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 42.

[161] In some embodiments, this document describes an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 19-26, wherein [AzK_PEG] is a mixture of the structures of formula (II) and formula (III),

Where

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

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

[162] In some embodiments, this document describes an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 19-26, wherein [AzK_PEG] is a mixture of the structures of formula (II) and formula (III),

Where

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

q is equal to 1, 2, or 3; and

X has the structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

[163] In some embodiments, the IL-10 conjugate comprises one or more amino acid sequences of SEQ ID NO: 19-26, wherein [AzK_PEG] is a mixture of structures of formula (II) and formula (III). In some embodiments of the IL-10 conjugate comprising one or more amino acid sequences of SEQ ID NO: 19-26 and having [AzK_PEG] as a mixture of structures of formula (II) and formula (III), the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) making up the total amount of [AzK_PEG] in the IL-10 conjugate is about 1:1. In some embodiments of the IL-10 conjugate comprising one or more amino acid sequences of SEQ ID NO: 19-26 and having [AzK_PEG] as a mixture of structures of formula (II) and formula (III), the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) constituting the total amount of [AzK_PEG] in the IL-10 conjugate is greater than 1:1. In some embodiments of the IL-10 conjugate comprising one or more amino acid sequences of SEQ ID NO: 19-26 and having [AzK_PEG] as a mixture of structures of formula (II) and formula (III), the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) constituting the total amount of [AzK_PEG] in the IL-10 conjugate is less than 1:1.

[164] In some embodiments of an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 19-26 and having [AzK_PEG] as a mixture of structures of formula (II) and formula (III), W is a linear or branched PEG moiety. In some embodiments of an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 19-26 and having [AzK_PEG] as a mixture of structures of formula (II) and formula (III), W is a linear PEG moiety. In some embodiments of an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 19-26 and having [AzK_PEG] as a mixture of structures of formula (II) and formula (III), W is a branched PEG moiety. In some embodiments of the IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 19-26 and having [AzK_PEG] as a mixture of structures of formula (II) and formula (III), W is a methoxy-PEG group. In some embodiments of the IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 19-26 and having [AzK_PEG] as a mixture of structures of formula (II) and formula (III), the methoxy-PEG group is linear or branched. In some embodiments of the IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 19-26 and having [AzK_PEG] as a mixture of structures of formula (II) and formula (III), the methoxy-PEG group is linear. In some embodiments of an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 19-26 and having [AzK_PEG] as a mixture of structures of formula (II) and formula (III), the methoxy-PEG group is branched.

[165] In some embodiments, this document describes an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 27-34, wherein [AzK_PEG20kDa] is a mixture of the structures of formula (II) and formula (III),

Where

W is a PEG group having an average molecular weight of 20 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

[166] In some embodiments, this document describes an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 27-34, wherein [AzK_PEG20kDa] is a mixture of the structures of formula (II) and formula (III),

Where

W is a PEG group with an average molecular weight of 20 kDa;

q is equal to 1, 2, or 3; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

[167] In some embodiments, the IL-10 conjugate comprises one or more amino acid sequences of SEQ ID NO: 27-34, wherein [AzK_PEG20kDa] is a mixture of structures of formula (II) and formula (III). In some embodiments of the IL-10 conjugate comprising one or more amino acid sequences of SEQ ID NO: 27-34 and having [AzK_PEG20kDa] as a mixture of structures of formula (II) and formula (III), the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) making up the total amount of [AzK_PEG20kDa] in the IL-10 conjugate is about 1:1. In some embodiments of the IL-10 conjugate comprising one or more amino acid sequences of SEQ ID NOs: 27-34 and having [AzK_PEG20kDa] as a mixture of structures of formula (II) and formula (III), the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) that make up the total amount of [AzK_PEG20kDa] in the IL-10 conjugate is greater than 1:1. In some embodiments of the IL-10 conjugate comprising one or more amino acid sequences of SEQ ID NOs: 27-34 and having [AzK_PEG20kDa] as a mixture of structures of formula (II) and formula (III), the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) that make up the total amount of [AzK_PEG20kDa] in the IL-10 conjugate is less than about 1:1.

[168] In some embodiments, this document describes an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 35-42, wherein [AzK_PEG30kDa] is a mixture of the structures of formula (II) and formula (III),

Where

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

X has the structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

[169] In some embodiments, this document describes an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 35-42, wherein [AzK_PEG30kDa] is a mixture of the structures of formula (II) and formula (III),

Where

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

q is equal to 1, 2, or 3; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

[170] In some embodiments, the IL-10 conjugate comprises one or more amino acid sequences of SEQ ID NO: 35-42, wherein [AzK_PEG30kDa] is a mixture of structures of formula (II) and formula (III). In some embodiments of the IL-10 conjugate comprising one or more amino acid sequences of SEQ ID NO: 35-42 and having [AzK_PEG30kDa] as a mixture of structures of formula (II) and formula (III), the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) making up the total amount of [AzK_PEG30kDa] in the IL-10 conjugate is about 1:1. In some embodiments of the IL-10 conjugate comprising one or more amino acid sequences of SEQ ID NO: 35-42 and having [AzK_PEG30kDa] as a mixture of structures of formula (II) and formula (III), the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) that make up the total amount of [AzK_PEG30kDa] in the IL-10 conjugate is greater than 1:1. In some embodiments of the IL-10 conjugate comprising one or more amino acid sequences of SEQ ID NO: 35-42 and having [AzK_PEG30kDa] as a mixture of structures of formula (II) and formula (III), the ratio of the amount of structure of formula (II) to the amount of structure of formula (III) that make up the total amount of [AzK_PEG30kDa] in the IL-10 conjugate is less than about 1:1.

[171] In some embodiments of formula (II), formula (III), or a mixture of formula (II) and formula (III) described herein, q is 1. In some embodiments of formula (II), formula (III), or a mixture of formula (II) and formula (III) described herein, q is 2. In some embodiments of formula (II), formula (III), or a mixture of formula (II) and formula (III) described herein, q is 3. In some embodiments, the IL-10 conjugate is of formula (II), and q is 1. In some embodiments, the IL-10 conjugate is of formula (II), and q is 2. In some embodiments, the IL-10 conjugate is of formula (II), and q is 3. In some embodiments, the IL-10 conjugate is of formula (III), and q is 1. In some embodiments, the IL-10 conjugate is of formula (III), and q is 2. In some embodiments, the IL-10 conjugate provides formula (III), and q is 3. In some embodiments, the IL-10 conjugate provides a mixture of formula (II) and formula (III), and q is 1. In some embodiments, the IL-10 conjugate provides a mixture of formula (II) and formula (III), and q is 2. In some embodiments, the IL-10 conjugate provides a mixture of formula (II) and formula (III), and q is 3.

[172] In some embodiments, this document describes an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 59-66, wherein [AzK_L1_PEG] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V),

Where

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

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

[173] In some embodiments, this document describes an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 59-66, wherein [AzK_L1_PEG] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V),

Where

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

q is equal to 1, 2, or 3; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

[174] Here and throughout this document, the structure of formula (IV) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. Here and throughout this document, the structure of formula (V) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof.

[175] In some embodiments, the methods use an IL-10 conjugate in which [AzK_L1_PEG] is represented by formula (IV). In some embodiments, the methods use an IL-10 conjugate in which [AzK_L1_PEG] is represented by formula (V). In some embodiments, the methods use an IL-10 conjugate in which [AzK_L1_PEG] is a mixture of formula (IV) and formula (V).

[176] In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 59 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 500 Daltons, 1 kDa, 2 kDa, 3 Da, 4 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 100 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 59 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 59 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 59 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 59 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight of 30 kDa. In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 60 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG group having an average molecular weight selected from 500 daltons, 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 100 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 60 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 60 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 60 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 60 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight of 30 kDa.

[177] In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 61 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 500 Daltons, 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 100 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 61 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 61 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 61 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 61 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight of 30 kDa.

[178] In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 62 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 500 Daltons, 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 100 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 62 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 62 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 62 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 62 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight of 30 kDa.

[179] In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 63 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 500 Daltons, 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 100 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 63 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 63 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 63 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 63 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight of 30 kDa.

[180] In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 64 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 500 Daltons, 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 100 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 64 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 64 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 64 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 64 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight of 30 kDa.

[181] In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 65 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 500 Daltons, 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 100 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 65 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 65 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 65 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 65 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight of 30 kDa.

[182] In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 66 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 500 Daltons, 1 kDa, 2 kDa, 3 kDa, 4 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, and 100 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 66 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 66 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight selected from 20 kDa and 30 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 66 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight of 20 kDa. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 66 and [AzK_L1_PEG] having a structure of formula (IV), formula (V), or a mixture of formula (IV) and formula (V), W is a PEG moiety having an average molecular weight of 30 kDa.

[183] In some embodiments, this document describes an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 43-50, wherein [AzK_L1_PEG20kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V),

Where

W is a PEG group having an average molecular weight of 20 kDa; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

[184] In some embodiments, this document describes an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 43-50, wherein [AzK_L1_PEG20kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V),

Where

W is a PEG group with an average molecular weight of 20 kDa;

q is equal to 1, 2, or 3; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

[185] In some embodiments, the IL-10 conjugate comprises the amino acid sequence of SEQ ID NO: 43, wherein [AzK_L1_PEG20kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V).

[186] In some embodiments, the IL-10 conjugate comprises the amino acid sequence of SEQ ID NO: 44, wherein [AzK_L1_PEG20kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V). In some embodiments, the IL-10 conjugate comprises the amino acid sequence of SEQ ID NO: 45, wherein [AzK_L1_PEG20kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V). In some embodiments, the IL-10 conjugate comprises the amino acid sequence of SEQ ID NO: 46, wherein [AzK_L1_PEG20kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V). In some embodiments, the IL-10 conjugate comprises the amino acid sequence of SEQ ID NO: 47, wherein [AzK_L1_PEG20kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V). In some embodiments, the IL-10 conjugate comprises the amino acid sequence of SEQ ID NO: 48, wherein [AzK_L1_PEG20kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V). In some embodiments, the IL-10 conjugate comprises the amino acid sequence of SEQ ID NO: 49, wherein [AzK_L1_PEG20kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V). In some embodiments, the IL-10 conjugate comprises the amino acid sequence of SEQ ID NO: 50, wherein [AzK_L1_PEG20kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V).

[187] In some embodiments, the IL-10 conjugate comprises one or more amino acid sequences of SEQ ID NO: 43-50, wherein [AzK_L1_PEG20kDa] has a structure that is a mixture of formula (IV) and formula (V). In some embodiments of the IL-10 conjugate comprising one or more amino acid sequences of SEQ ID NO: 43-50 and [AzK_L1_PEG20kDa] having a structure that is a mixture of formula (IV) and formula (V), the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) that make up the total amount of [AzK_L1_PEG20kDa] in the IL-10 conjugate is about 1:1. In some embodiments of the IL-10 conjugate comprising one or more amino acid sequences of SEQ ID NOs: 43-50 and [AzK_L1_PEG20kDa] having a structure that is a mixture of formula (IV) and formula (V), the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) that make up the total amount of [AzK_L1_PEG20kDa] in the IL-10 conjugate is greater than 1:1. In some embodiments of the IL-10 conjugate comprising one or more amino acid sequences of SEQ ID NOs: 43-50 and [AzK_L1_PEG20kDa] having a structure that is a mixture of formula (IV) and formula (V), the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) that make up the total amount of [AzK_L1_PEG20kDa] in the IL-10 conjugate is less than 1:1.

[188] In some embodiments, this document describes an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 51-58, wherein [AzK_L1_PEG30kDa] has a structure of formula (IV), formula (V), or is a mixture of structures of formula (IV) and formula (V),

Where

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

X has the structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

[189] In some embodiments, this document describes an IL-10 conjugate comprising an amino acid sequence of any one of SEQ ID NOs: 51-58, wherein [AzK_L1_PEG30kDa] has a structure of formula (IV), formula (V), or is a mixture of structures of formula (IV) and formula (V),

Where

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

q is equal to 1, 2, or 3; and

X has a structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

[190] In some embodiments, the IL-10 conjugate comprises the amino acid sequence of SEQ ID NO: 51, wherein [AzK_L1_PEG30kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V). In some embodiments, the IL-10 conjugate comprises the amino acid sequence of SEQ ID NO: 52, wherein [AzK_L1_PEG30kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V). In some embodiments, the IL-10 conjugate comprises the amino acid sequence of SEQ ID NO: 53, wherein [AzK_L1_PEG30kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V). In some embodiments, the IL-10 conjugate comprises the amino acid sequence of SEQ ID NO: 54, wherein [AzK_L1_PEG30kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V). In some embodiments, the IL-10 conjugate comprises the amino acid sequence of SEQ ID NO: 55, wherein [AzK_L1_PEG30kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V). In some embodiments, the IL-10 conjugate comprises the amino acid sequence of SEQ ID NO: 56, wherein [AzK_L1_PEG30kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V). In some embodiments, the IL-10 conjugate comprises the amino acid sequence of SEQ ID NO: 57, wherein [AzK_L1_PEG30kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V). In some embodiments, the IL-10 conjugate comprises the amino acid sequence of SEQ ID NO: 58, wherein [AzK_L1_PEG30kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V).

[191] In some embodiments, the IL-10 conjugate comprises one or more amino acid sequences of SEQ ID NO: 51-58, wherein [AzK_L1_PEG30kDa] has a structure that is a mixture of formula (IV) and formula (V). In some embodiments of the IL-10 conjugate comprising one or more amino acid sequences of SEQ ID NO: 51-58 and [AzK_L1_PEG30kDa] having a structure that is a mixture of formula (IV) and formula (V), the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) that make up the total amount of [AzK_L1_PEG30kDa] in the IL-10 conjugate is about 1:1. In some embodiments of the IL-10 conjugate comprising one or more amino acid sequences of SEQ ID NOs: 51-58 and [AzK_L1_PEG30kDa] having a structure that is a mixture of formula (IV) and formula (V), the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) that make up the total amount of [AzK_L1_PEG30kDa] in the IL-10 conjugate is greater than 1:1. In some embodiments of the IL-10 conjugate comprising one or more amino acid sequences of SEQ ID NOs: 51-58 and [AzK_L1_PEG30kDa] having a structure that is a mixture of formula (IV) and formula (V), the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) that make up the total amount of [AzK_L1_PEG30kDa] in the IL-10 conjugate is less than 1:1.

[192] In some embodiments of formula (IV), formula (V), or a mixture of formula (IV) and formula (V) described herein, q is 1. In some embodiments of formula (IV), formula (V), or a mixture of formula (IV) and formula (V) described herein, q is 2. In some embodiments of formula (IV), formula (V), or a mixture of formula (IV) and formula (V) described herein, q is 3. In some embodiments, the IL-10 conjugate is of formula (IV), and q is 1. In some embodiments, the IL-10 conjugate is of formula (IV), and q is 2. In some embodiments, the IL-10 conjugate is of formula (IV), and q is 3. In some embodiments, the IL-10 conjugate is of formula (V), and q is 1. In some embodiments, the IL-10 conjugate is of formula (V), and q is 2. In some embodiments, the IL-10 conjugate provides formula (V), and q is 3. In some embodiments, the IL-10 conjugate provides a mixture of formula (IV) and formula (V), and q is 1. In some embodiments, the IL-10 conjugate provides a mixture of formula (IV) and formula (V), and q is 2. In some embodiments, the IL-10 conjugate provides a mixture of formula (IV) and formula (V), and q is 3.

[193] In some embodiments, this document describes an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII),

Where

n is an integer such that the molecular weight of the PEG group is from about 5,000 daltons to about 60,000 daltons; and

X has the structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

[194] In some embodiments, this document describes an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII),

Where

n is an integer such that the molecular weight of the PEG group is from about 5,000 Daltons to about 60,000 Daltons;

q is equal to 1, 2, or 3; and

X has the structure

;

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

[195] Here and throughout this document, the structure of formula (VI) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. Here and throughout this document, the structure of formula (VII) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof.

[196] In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII), the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is selected from E67, Q70, E74, E75, Q79, N82, K88, A89, K99, K125, N126, N129, K130, and Q132.

[197] In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII), the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is selected from N82, K88, A89, K99, K125, N126, N129, and K130. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII), the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is E67. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII), the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is Q70. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII), the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is E74. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII), the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is E75. In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII), the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is Q79. In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII), the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is N82. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII), the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is K88. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII), the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is A89. In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII), the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is K99. In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII), the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is K125. In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII), the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is N126. In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII), the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is N129.

[198] In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII), the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is K130. In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII), the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is Q132.

[199] In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one of E67, Q70, E74, E75, Q79, N82, K88, A89, K99, K125, N126, N129, K130, and Q132 in the IL-10 conjugate is replaced with a structure that is a mixture of formula (VI) and formula (VII), the ratio of the amount of the structure of formula (VI) to the amount of the structure of formula (VII) contained in the total amount of the IL-10 conjugate is about 1:1. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one of E67, Q70, E74, E75, Q79, N82, K88, A89, K99, K125, N126, N129, K130 and Q132 in the IL-10 conjugate is replaced by a structure that is a mixture of formula (VI) and formula (VII), the ratio of the amount of the structure of formula (VI) to the amount of the structure of formula (VII) contained in the total amount of the IL-10 conjugate is greater than 1:1. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one of E67, Q70, E74, E75, Q79, N82, K88, A89, K99, K125, N126, N129, K130 and Q132 in the IL-10 conjugate is replaced by a structure that is a mixture of formula (VI) and formula (VII), the ratio of the amount of the structure of formula (VI) to the amount of the structure of formula (VII) contained in the total amount of the IL-10 conjugate is less than 1:1.

[200] In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII), the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is selected from E67, Q70, E74, E75, Q79, N82, K88, A89, K99, K125, N126, N129, K130, and Q132, n is an integer such that the molecular weight of the PEG group is from about 1,000 Daltons to about 100,000 Daltons, from about 5,000 Daltons to about 50,000 daltons, from about 5,000 daltons to about 40,000 daltons, from about 5,000 daltons to about 30,000 daltons, from about 5,000 daltons to about 25,000 daltons, from about 5,000 daltons to about 20,000 daltons, from about 5,000 daltons to about 15,000 daltons, or from about 5,000 daltons to about 10,000 daltons.

[201] In some embodiments, n is an integer such that the molecular weight of the PEG group is about 1,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 5,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 10,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 15,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 20,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 25,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 30,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 40,000 daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 50,000 daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 100,000 daltons.

[202] In some embodiments of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) described herein, q is 1. In some embodiments of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) described herein, q is 2. In some embodiments of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) described herein, q is 3. In some embodiments, the IL-10 conjugate is of formula (VI), and q is 1. In some embodiments, the IL-10 conjugate is of formula (VI), and q is 2. In some embodiments, the IL-10 conjugate is of formula (VI), and q is 3. In some embodiments, the IL-10 conjugate is of formula (VII), and q is 1. In some embodiments, the conjugate IL-10 provides formula (VII), and q is 2. In some embodiments, the IL-10 conjugate provides formula (VII), and q is 3. In some embodiments, the IL-10 conjugate provides a mixture of formula (VI) and formula (VII), and q is 1. In some embodiments, the IL-10 conjugate provides a mixture of formula (VI) and formula (VII), and q is 2. In some embodiments, the IL-10 conjugate provides a mixture of formula (VI) and formula (VII), and q is 3.

[203] In some embodiments, this document describes an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII) or formula (IX) or a mixture of formula (VIII) and formula (IX),

Where

n is an integer such that the molecular weight of the PEG group is from about 5,000 daltons to about 60,000 daltons; and

X has a structure

,

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue.

[204] In some embodiments, this document describes an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII) or formula (IX) or a mixture of formula (VIII) and formula (IX),

Where

n is an integer such that the molecular weight of the PEG group is from about 5,000 Daltons to about 60,000 Daltons;

q is equal to 1, 2, or 3; and

X has a structure

,

X-1 indicates the point of attachment to the preceding amino acid residue; and

X+1 indicates the point of attachment to the next amino acid residue. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

[205] Here and throughout this document, the structure of formula (VIII) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. Here and throughout this document, the structure of formula (IX) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof.

[206] In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX), the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is selected from E67, Q70, E74, E75, Q79, N82, K88, A89, K99, K125, N126, N129, K130, and Q132. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced by a structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX), the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is selected from N82, K88, A89, K99, K125, N126, N129, and K130.

[207] In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX), the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is E67. In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX), the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is Q70. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX), the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is E74. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX), the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is E75. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX), the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is Q79. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX), the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is N82. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX), the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is K88. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX), the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is A89. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX), the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is K99. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX), the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is K125. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX), the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is N126. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX), the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is N129. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX), the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is K130. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX), the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is Q132.

[208] In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one of E67, Q70, E74, E75, Q79, N82, K88, A89, K99, K125, N126, N129, K130, and Q132 in the IL-10 conjugate is replaced with a structure that is a mixture of formula (VIII) and formula (IX), the ratio of the amount of the structure of formula (VIII) to the amount of the structure of formula (IX) contained in the total amount of the IL-10 conjugate is about 1:1. In some embodiments of the IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one of E67, Q70, E74, E75, Q79, N82, K88, A89, K99, K125, N126, N129, K130 and Q132 in the IL-10 conjugate is replaced by a structure that is a mixture of formula (VIII) and formula (IX), the ratio of the amount of the structure of formula (VIII) to the amount of the structure of formula (IX) contained in the total amount of the IL-10 conjugate is greater than 1:1. In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one of E67, Q70, E74, E75, Q79, N82, K88, A89, K99, K125, N126, N129, K130 and Q132 in the IL-10 conjugate is replaced by a structure that is a mixture of formula (VIII) and formula (IX), the ratio of the amount of the structure of formula (VIII) to the amount of the structure of formula (IX) contained in the total amount of the IL-10 conjugate is less than 1:1.

[209] In some embodiments of an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX), the position of the structure of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is selected from E67, Q70, E74, E75, Q79, N82, K88, A89, K99, K125, N126, N129, K130, and Q132, n is an integer such that the molecular weight of the PEG group is from about 1,000 Daltons to about 100,000 Daltons, from about 5,000 Daltons to about 50,000 daltons, from about 5,000 daltons to about 40,000 daltons, from about 5,000 daltons to about 30,000 daltons, from about 5,000 daltons to about 25,000 daltons, from about 5,000 daltons to about 20,000 daltons, from about 5,000 daltons to about 15,000 daltons, or from about 5,000 daltons to about 10,000 daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 1,000 daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 5,000 daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 10,000 daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 15,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 20,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 25,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 30,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 40,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 50,000 Daltons. In some embodiments, n is an integer such that the molecular weight of the PEG group is about 100,000 Daltons.

[210] In some embodiments of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) described herein, q is 1. In some embodiments of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) described herein, q is 2. In some embodiments of formula (VIII), formula (IX), or a mixture of formula (VIII) and formula (IX) described herein, q is 3. In some embodiments, the IL-10 conjugate is of formula (VIII), and q is 1. In some embodiments, the IL-10 conjugate is of formula (VIII), and q is 2. In some embodiments, the IL-10 conjugate is of formula (VIII), and q is 3. In some embodiments, the IL-10 conjugate is of formula (IX), and q is 1. In some embodiments The IL-10 conjugate has formula (IX), and q is 2. In some embodiments, the IL-10 conjugate has formula (IX), and q is 3. In some embodiments, the IL-10 conjugate has a mixture of formula (VIII) and formula (IX), and q is 1. In some embodiments, the IL-10 conjugate has a mixture of formula (VIII) and formula (IX), and q is 2. In some embodiments, the IL-10 conjugate has a mixture of formula (VIII) and formula (IX), and q is 3.

[211] In some embodiments, this document describes an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (X) or formula (XI) or a mixture of formula (X) and formula (XI),

Where

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

Wavy lines indicate covalent bonds with amino acid residues within SEQ ID NO: 1 that are not replaced.

[212] In some embodiments, this document describes an IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (X) or formula (XI) or a mixture of formula (X) and formula (XI),

Where

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

q is equal to 1, 2, or 3; and

wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 1 that are not substituted. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

[213] Herein and throughout this document, the structure of formula (X) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. Herein and throughout this document, the structure of formula (XI) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. In some embodiments, the IL-10 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

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

[215] In some embodiments of the IL-10 conjugate described herein, n in the compounds of Formula (X) or Formula (XI) or a mixture of Formula (X) and Formula (XI) is in the range of from about 5 to about 4600, or from about 10 to about 4000, or from about 20 to about 3000, or from about 100 to about 3000, or from about 100 to about 2900, or from about 150 to about 2900, or from about 125 to about 2900, or from about 100 to about 2500, or from about 100 to about 2000, or from about 100 to about 1900, or from about 100 to about 1850, or from about 100 to about 1750, or from about 100 to about 1650, or from about 100 to about 1500, or from about 100 to about 1400, or from about 100 to about 1300, or from about 100 to about 1250, or from about 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or from about 341 to about 1136, or from about 341 to about 1023, or from about 341 to about 910, or from about 341 to about 796, or from about 341 to about 682, or from about 341 to about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575. In some embodiments of the IL-10 conjugate described herein, n in the compounds of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), is an integer selected from of 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545 and 4546. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (X) or formula (XI) or a mixture of formula (X) and formula (XI) in the amino acid sequence of the IL-10 conjugate is selected from N82, K88, A89, K99, K125, N126, N129 and K130, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate refers to the positions in SEQ ID NO: 1. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (X) or formula (XI) or a mixture of formula (X) and formula (XI) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is selected from N82, K88, A89, K99, K125, N126, N129 and K130. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (X) or formula (XI) or a mixture of formula (X) and formula (XI) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position N82. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (X) or formula (XI) or a mixture of formula (X) and formula (XI) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position K88. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (X) or formula (XI) or a mixture of formula (X) and formula (XI) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position A89. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (X) or formula (XI) or a mixture of formula (X) and formula (XI) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position K99. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (X) or formula (XI) or a mixture of formula (X) and formula (XI) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position K125. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (X) or formula (XI) or a mixture of formula (X) and formula (XI) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position N126. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (X) or formula (XI) or a mixture of formula (X) and formula (XI) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position N129. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (X) or formula (XI) or a mixture of formula (X) and formula (XI) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position K130. In some embodiments of the IL-10 conjugate described herein, the ratio of the amount of the structure of formula (X) to the amount of the structure of formula (XI) contained in the total amount of the IL-10 conjugate is about 1:1. In some embodiments of the IL-10 conjugate described herein, the ratio of the amount of the structure of formula (X) to the amount of the structure of formula (XI) contained in the total amount of the IL-10 conjugate is greater than 1:1. In some embodiments of the IL-10 conjugate described herein, the ratio of the amount of the structure of formula (X) to the amount of the structure of formula (XI) contained in the total amount of the IL-10 conjugate is less than 1:1.

[216] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is selected from N82, K88, A89, K99, K125, N126, N129, and K130, and wherein n is an integer from 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or from about 341 to about 1136, or from about 341 to about 1023, or from about 341 to about 910, or from about 341 to about 796, or from about 341 to about 682, or from about 341 to about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575. In some embodiments of the IL-10 conjugate described herein, n in the compounds of Formula (X) or Formula (XI) or a mixture of Formula (X) and Formula (XI) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 32, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3978, 4544, 4545 and 4546.

[217] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (X) or formula (XI), or a mixture of formula (X) and formula (XI), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is selected from N82, K88, A89, K99, K125, N126, N129, and K130, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described herein, n in the compounds of formula (X) or formula (XI) or a mixture of formula (X) and formula (XI) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137 and 1249.

[218] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (X) or formula (XI), or a mixture of formula (X) and formula (XI), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is selected from N82, K88, A89, K99, K125, N126, N129, and K130, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described herein, n in the compounds of formula (X) or formula (XI) or a mixture of formula (X) and formula (XI) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910.

[219] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is N82, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described herein, n in the compounds of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 113, 227, 340, 454, 568, or 681.

[220] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is K88, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described herein, n in the compounds of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 113, 227, 340, 454, 568, or 681.

[221] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is A89, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described herein, n in the compounds of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 113, 227, 340, 454, 568, or 681.

[222] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is K99, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described herein, n in the compounds of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 113, 227, 340, 454, 568, or 681.

[223] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is K125, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described herein, n in the compounds of Formula (X) and Formula (XI) or a mixture of Formula (X) and Formula (XI) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 113, 227, 340, 454, 568, or 681.

[224] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is N126, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described herein, n in the compounds of Formula (X) and Formula (XI) or a mixture of Formula (X) and Formula (XI) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 113, 227, 340, 454, 568, or 681.

[225] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is N129, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described herein, n in the compounds of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 113, 227, 340, 454, 568, or 681.

[226] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is K130, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described herein, n in the compounds of Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI), is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 113, 227, 340, 454, 568, or 681.

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

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

[229] In some embodiments of formula (X), formula (XI), or a mixture of formula (X) and formula (XI) described herein, q is 1. In some embodiments of formula (X), formula (XI), or a mixture of formula (X) and formula (XI) described herein, q is 2. In some embodiments of formula (X), formula (XI), or a mixture of formula (X) and formula (XI) described herein, q is 3. In some embodiments, the IL-10 conjugate provides formula (X), and q is 1. In some embodiments, the IL-10 conjugate provides formula (X), and q is 2. In some embodiments, the IL-10 conjugate provides formula (X), and q is 3. In some embodiments, the IL-10 conjugate provides formula (XI), and q is 1. In some embodiments, the conjugate IL-10 provides formula (XI), and q is 2. In some embodiments, the IL-10 conjugate provides formula (XI), and q is 3. In some embodiments, the IL-10 conjugate provides a mixture of formula (X) and formula (XI), and q is 1. In some embodiments, the IL-10 conjugate provides a mixture of formula (X) and formula (XI), and q is 2. In some embodiments, the IL-10 conjugate provides a mixture of formula (X) and formula (XI), and q is 3.

[230] This document describes IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced by a structure of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII),

Where

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

Wavy lines indicate covalent bonds with amino acid residues within SEQ ID NO: 1 that are not replaced.

[231] This document describes IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced by a structure of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII),

Where

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

q is equal to 1, 2, or 3; and

wavy lines indicate covalent bonds to amino acid residues within SEQ ID NO: 1 that are not substituted. In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3.

[232] Herein and throughout this document, the structure of formula (XII) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. Herein and throughout this document, the structure of formula (XIII) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. In some embodiments, the IL-10 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

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

[234] In some embodiments of the IL-10 conjugate described herein, n in the compounds of Formula (XII) or Formula (XIII) or a mixture of Formula (XII) and Formula (XIII) is in the range of from about 5 to about 4600, or from about 10 to about 4000, or from about 20 to about 3000, or from about 100 to about 3000, or from about 100 to about 2900, or from about 150 to about 2900, or from about 125 to about 2900, or from about 100 to about 2500, or from about 100 to about 2000, or from about 100 to about 1900, or from about 100 to about 1850, or from about 100 to about 1750, or from about 100 to about 1650, or from about 100 to about 1500, or from about 100 to about 1400, or from about 100 to about 1300, or from about 100 to about 1250, or from about 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or from about 341 to about 1136, or from about 341 to about 1023, or from about 341 to about 910, or from about 341 to about 796, or from about 341 to about 682, or from about 341 to about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575. In some embodiments of the IL-10 conjugate described herein, n in the compounds of Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula (XIII), is an integer, selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545 and 4546. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII) in the amino acid sequence of the IL-10 conjugate is selected from N82, K88, A89, K99, K125, N126, N129 and K130, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate refers to the positions in SEQ ID NO: 1. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is selected from N82, K88, A89, K99, K125, N126, N129 and K130. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position N82. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position K88. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position A89. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position K99. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position K125. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position N126. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position N129. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XII) or formula (XIII) or a mixture of formula (XII) and formula (XIII) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position K130.

[235] In some embodiments of the IL-10 conjugate described herein, the ratio of the amount of the structure of formula (XII) to the amount of the structure of formula (XIII) contained in the total amount of the IL-10 conjugate is about 1:1. In some embodiments of the IL-10 conjugate described herein, the ratio of the amount of the structure of formula (XII) to the amount of the structure of formula (XIII) contained in the total amount of the IL-10 conjugate is greater than 1:1. In some embodiments of the IL-10 conjugate described herein, the ratio of the amount of the structure of formula (XII) to the amount of the structure of formula (XIII) contained in the total amount of the IL-10 conjugate is less than 1:1.

[236] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (XII) or formula (XIII), or a mixture of formula (XII) and formula (XIII), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is selected from N82, K88, A89, K99, K125, N126, N129, and K130, and wherein n is an integer from 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or from about 341 to about 1136, or from about 341 to about 1023, or from about 341 to about 910, or from about 341 to about 796, or from about 341 to about 682, or from about 341 to about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575. In some embodiments of the IL-10 conjugate described herein, n in the compounds of Formula (XII) or Formula (XIII) or a mixture of Formula (XII) and Formula (XIII) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545 and 4546.

[237] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (XII) or formula (XIII), or a mixture of formula (XII) and formula (XIII), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is selected from N82, K88, A89, K99, K125, N126, N129, and K130, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described herein, n in the compounds of formula (XII) or formula (XIII), or a mixture of formula (XII) and formula (XIII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137 and 1249.

[238] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (XII) or formula (XIII), or a mixture of formula (XII) and formula (XIII), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is selected from N82, K88, A89, K99, K125, N126, N129, and K130, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described herein, n in the compounds of formula (XII) or formula (XIII), or a mixture of formula (XII) and formula (XIII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 113, 227, 340, 454, 568, or 681.

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

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

[241] In some embodiments of formula (XII), formula (XIII), or a mixture of formula (XII) and formula (XIII) described herein, q is 1. In some embodiments of formula (XII), formula (XIII), or a mixture of formula (XII) and formula (XIII) described herein, q is 2. In some embodiments of formula (XII), formula (XIII), or a mixture of formula (XII) and formula (XIII) described herein, q is 3. In some embodiments, the IL-10 conjugate is of formula (XII), and q is 1. In some embodiments, the IL-10 conjugate is of formula (XII), and q is 2. In some embodiments, the IL-10 conjugate is of formula (XII), and q is 3. In some embodiments, the IL-10 conjugate is of formula (XIII), and q is 1. In some embodiments, the IL-10 conjugate is of formula (XIII), and q is 2. In some embodiments, the IL-10 conjugate is of formula (XIII), and q is 3. In some embodiments, the IL-10 conjugate is a mixture of formula (XII) and formula (XIII), and q is 1. In some embodiments, the IL-10 conjugate is a mixture of formula (XII) and formula (XIII), and q is 2. In some embodiments, the IL-10 conjugate is a mixture of formula (XII) and formula (XIII), and q is 3.

[242] This document describes IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced by a structure of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV),

Where

m is an integer between 0 and 20;

p is an integer between 0 and 20;

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

Wavy lines indicate covalent bonds with amino acid residues within SEQ ID NO: 1 that are not replaced.

[243] Herein and throughout this document, the structure of formula (XIV) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. Herein and throughout this document, the structure of formula (XV) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. In some embodiments, the IL-10 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

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

[245] In some embodiments of the IL-10 conjugate. described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is from 0 to 20, or from 1 to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 1. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 2. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 3. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 4. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 5. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 6. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 7. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 8. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 9. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 10. In some embodiments of the IL-10 conjugate described in herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 11. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 12. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 13. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 14. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 15. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 16. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 17. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 18. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 19. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 20.

[246] In some embodiments of the IL-10 conjugate. described herein, p in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is from 1 to 20, or from 1 to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2. In some embodiments of the IL-10 conjugate described herein, p in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 1. In some embodiments of the IL-10 conjugate described herein, p in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 2. In some embodiments of the IL-10 conjugate described herein, p in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 3. In some embodiments of the IL-10 conjugate described herein, p in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 4. In some embodiments of the IL-10 conjugate described herein, p in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 5. In some embodiments of the IL-10 conjugate described herein, p in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 6. In some embodiments of the IL-10 conjugate described herein, p in the compounds of Formula (XIV) or Formula (XV) or a mixture of Formula (XIV) and Formula (XV) is 7. In some embodiments of the IL-10 conjugate described herein, p in the compounds of Formula (XIV) or Formula (XV) or a mixture of Formula (XIV) and Formula (XV) is 8. In some embodiments of the IL-10 conjugate described herein, p in the compounds of Formula (XIV) or Formula (XV) or a mixture of Formula (XIV) and Formula (XV) is 9. In some embodiments of the IL-10 conjugate described herein, p in the compounds of Formula (XIV) or Formula (XV) or a mixture of Formula (XIV) and Formula (XV) is 10. In some embodiments of the IL-10 conjugate described in herein, p in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 11. In some embodiments of the IL-10 conjugate described herein, p in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 12. In some embodiments of the IL-10 conjugate described herein, p in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 13. In some embodiments of the IL-10 conjugate described herein, p in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 14. In some embodiments of the IL-10 conjugate described herein, p in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 15. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 16. In some embodiments of the IL-10 conjugate described herein, p in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 17. In some embodiments of the IL-10 conjugate described herein, p in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 18. In some embodiments of the IL-10 conjugate described herein, p in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 19. In some embodiments of the IL-10 conjugate described herein, p in the compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) is 20.

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

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

[249] In some embodiments of the IL-10 conjugate described herein, n in the compounds of Formula (XIV) or Formula (XV) or a mixture of Formula (XIV) and Formula (XV) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545 and 4546. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) in the amino acid sequence of the IL-10 conjugate is selected from N82, K88, A89, K99, K125, N126, N129 and K130. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position N82. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position K88. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position A89. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position K99. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position K125. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position N126. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position N129. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position K130.

[250] In some embodiments of the IL-10 conjugate described herein, the ratio of the amount of the structure of formula (XIV) to the amount of the structure of formula (XV) contained in the total amount of the IL-10 conjugate is about 1:1. In some embodiments of the IL-10 conjugate described herein, the ratio of the amount of the structure of formula (XIV) to the amount of the structure of formula (XV) contained in the total amount of the IL-10 conjugate is greater than 1:1. In some embodiments of the IL-10 conjugate described herein, the ratio of the amount of the structure of formula (XIV) to the amount of the structure of formula (XV) contained in the total amount of the IL-10 conjugate is less than 1:1.

[251] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (XIV) or formula (XV), or a mixture of formula (XIV) and formula (XV), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is selected from N82, K88, A89, K99, K125, N126, N129, and K130, and wherein n is an integer from 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or from about 341 to about 1136, or from about 341 to about 1023, or from about 341 to about 910, or from about 341 to about 796, or from about 341 to about 682, or from about 341 to about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575. In some embodiments of the IL-10 conjugate described herein, n in the compounds of Formula (XIV) or Formula (XV) or a mixture of Formula (XIV) and Formula (XV) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545 and 4546.

[252] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (XIV) or formula (XV), or a mixture of formula (XIV) and formula (XV), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is selected from N82, K88, A89, K99, K125, N126, N129, and K130, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described herein, n in the compounds of formula (XIV) or formula (XV), or a mixture of formula (XIV) and formula (XV) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137 and 1249.

[253] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (XIV) or formula (XV), or a mixture of formula (XIV) and formula (XV), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is selected from N82, K88, A89, K99, K125, N126, N129, and K130, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described herein, n in the compounds of formula (XIV) or formula (XV), or a mixture of formula (XIV) and formula (XV) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910.

[254] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (XIV) or formula (XV), or a mixture of formula (XIV) and formula (XV), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is selected from N82, K88, A89, K99, K125, N126, N129, and K130, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described herein, n in the compounds of formula (XIV) or formula (XV), or a mixture of formula (XIV) and formula (XV) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

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

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

[257] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (XIV) or formula (XV), or a mixture of formula (XIV) and formula (XV), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is selected from N82, K88, A89, K99, K125, N126, N129, and K130, m is an integer from 1 to 6, p is an integer from 1 to 6, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described In this document, in compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV), m is 2, p is 2, and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137 and 1249.

[258] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (XIV) or formula (XV), or a mixture of formula (XIV) and formula (XV), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is selected from N82, K88, A89, K99, K125, N126, N129, and K130, and wherein m is an integer from 1 to 6, p is an integer from 1 to 6, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate, described herein, in compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV), m is 2, p is 2, and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910.

[259] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (XIV) or formula (XV), or a mixture of formula (XIV) and formula (XV), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is selected from N82, K88, A89, K99, K125, N126, N129, and K130, and wherein m is an integer from 1 to 6, p is an integer from 1 to 6, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate, described herein, in compounds of formula (XIV) or formula (XV) or a mixture of formula (XIV) and formula (XV), m is 2, p is 2, and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

[260] This document describes IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced by a structure of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII),

Where

m is an integer between 0 and 20;

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

Wavy lines indicate covalent bonds with amino acid residues within SEQ ID NO: 1 that are not replaced.

[261] Herein and throughout this document, the structure of formula (XVI) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. Herein and throughout this document, the structure of formula (XVII) encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof. In some embodiments, the IL-10 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

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

[263] In some embodiments of the IL-10 conjugate. described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is from 1 to 20, or from 1 to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or from 1 to 2. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is 1. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is 2. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is 3. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is 4. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is 5. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is 6. In some embodiments of the conjugate IL-10 described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is 7. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is 8. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is 9. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is 10. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is 11. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is 12. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is 13. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is 14. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is 15. In some embodiments of the conjugate IL-10 described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is 16. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is 17. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is 18. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is 19. In some embodiments of the IL-10 conjugate described herein, m in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is equal to 20.

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

[265] In some embodiments of the IL-10 conjugate described herein, in the compounds of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII), m is an integer from 1 to 6, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-10 conjugate described herein, in the compounds of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII), m is an integer from 2 to 6, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-10 conjugate described herein, in the compounds of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII), m is an integer from 2 to 4, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136 and 1137. In some embodiments of the IL-10 conjugate described herein, in the compounds of Formula (XVI) or Formula (XVII) or a mixture of Formula (XVI) and Formula (XVII), m is 1 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136 and 1137. In some embodiments of the IL-10 conjugate described herein, in the compounds of Formula (XVI) or Formula (XVII) or a mixture of Formula (XVI) and Formula (XVII), m is 2 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-10 conjugate described herein, in the compounds of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII), m is 3 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-10 conjugate described herein, in the compounds of formula (XVI) or formula (XVII) or mixtures of formula (XVI) and formula (XVII), m is 4 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-10 conjugate described herein, in compounds of formula (XVI) or formula (XVII) or mixtures of formula (XVI) and formula (XVII), m is 5 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136 and 1137. In some embodiments of the IL-10 conjugate described herein, in the compounds of Formula (XVI) or Formula (XVII) or a mixture of Formula (XVI) and Formula (XVII), m is 6 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136 and 1137. In some embodiments of the IL-10 conjugate described herein, in the compounds of Formula (XVI) or Formula (XVII) or a mixture of Formula (XVI) and Formula (XVII), m is 7 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-10 conjugate described herein, in the compounds of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII), m is 8 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-10 conjugate described herein, in the compounds of formula (XVI) or formula (XVII) or mixtures of formula (XVI) and formula (XVII), m is 9 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the IL-10 conjugate described herein, in compounds of formula (XVI) or formula (XVII) or mixtures of formula (XVI) and formula (XVII), m is 10 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136 and 1137. In some embodiments of the IL-10 conjugate described herein, in the compounds of Formula (XVI) or Formula (XVII) or a mixture of Formula (XVI) and Formula (XVII), m is 11 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136 and 1137. In some embodiments of the IL-10 conjugate described herein, in the compounds of Formula (XVI) or Formula (XVII) or a mixture of Formula (XVI) and Formula (XVII), m is 12 and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136 and 1137. In some embodiments of the IL-10 conjugate described herein, in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII), m is 2 and n is an integer selected from 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136 and 1137.

[266] In some embodiments of the IL-10 conjugate described herein, n in the compounds of Formula (XVI) or Formula (XVII) or a mixture of Formula (XVI) and Formula (XVII) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545 and 4546.

[267] In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XVI) or formula (XVII) or a mixture of formula (XVI and formula (XVII) in the amino acid sequence of the IL-10 conjugate is selected from N82, K88, A89, K99, K125, N126, N129, and K130. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XVI) or formula (XVII) or a mixture of formula (XVI and formula (XVII) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position N82. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XVI) or formula (XVII) or a mixture of formula (XVI and formula (XVII) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position K88. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XVI) or formula (XVII) or a mixture of formula (XVI and formula (XVII) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position A89. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XVI) or formula (XVII) or a mixture of formula (XVI and formula (XVII) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position K99. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XVI) or formula (XVII) or a mixture of formula (XVI and formula (XVII) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position K125. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XVI) or formula (XVII) or a mixture of formula (XVI and formula (XVII) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position N126. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XVI) or formula (XVII) or a mixture of formula (XVI and formula (XVII) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position N129. In some embodiments of the IL-10 conjugate described herein, the position of the structure of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) in the amino acid sequence of the IL-10 conjugate of SEQ ID NO: 1 is position K130.

[268] In some embodiments of the IL-10 conjugate described herein, the ratio of the amount of the structure of formula (XVI) to the amount of the structure of formula (XVII) contained in the total amount of the IL-10 conjugate is about 1:1. In some embodiments of the IL-10 conjugate described herein, the ratio of the amount of the structure of formula (XVI) to the amount of the structure of formula (XVII) contained in the total amount of the IL-10 conjugate is greater than 1:1. In some embodiments of the IL-10 conjugate described herein, the ratio of the amount of the structure of formula (XVI) to the amount of the structure of formula (XVII) contained in the total amount of the IL-10 conjugate is less than 1:1.

[269] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is selected from N82, K88, A89, K99, K125, N126, N129, and K130, and wherein n is an integer from 100 to about 1150, or from about 100 to about 1100, or from about 100 to about 1000, or from about 100 to about 900, or from about 100 to about 750, or from about 100 to about 700, or from about 100 to about 600, or from about 100 to about 575, or from about 100 to about 500, or from about 100 to about 450, or from about 100 to about 350, or from about 100 to about 275, or from about 100 to about 230, or from about 150 to about 475, or from about 150 to about 340, or from about 113 to about 340, or from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 340 to about 795, or from about 341 to about 682, or from about 568 to about 909, or from about 227 to about 1500, or from about 225 to about 2280, or from about 460 to about 2160, or from about 460 to about 2050, or from about 341 to about 1820, or from about 341 to about 1710, or from about 341 to about 1250, or from about 225 to about 1250, or from about 341 to about 1250, or from about 341 to about 1136, or from about 341 to about 1023, or from about 341 to about 910, or from about 341 to about 796, or from about 341 to about 682, or from about 341 to about 568, or from about 114 to about 1000, or from about 114 to about 950, or from about 114 to about 910, or from about 114 to about 800, or from about 114 to about 690, or from about 114 to about 575. In some embodiments of the IL-10 conjugate described herein, n in the compounds of Formula (XVI) or Formula (XVII) or a mixture of Formula (XVI) and Formula (XVII) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545 and 4546.

[270] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is selected from N82, K88, A89, K99, K125, N126, N129, and K130, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described herein, n in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137 and 1249.

[271] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is selected from N82, K88, A89, K99, K125, N126, N129, and K130, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described herein, n in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) represents an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910.

[272] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is selected from N82, K88, A89, K99, K125, N126, N129, and K130, and wherein n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described herein, n in the compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some embodiments, n is from about 500 to about 1000. In some embodiments, n is from about 550 to about 800. In some embodiments, n is about 681.

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

[274] In some embodiments, described herein are IL-10 conjugates comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (XVI) or formula (XVII), or a mixture of formula (XVI) and formula (XVII), wherein the amino acid residue in SEQ ID NO: 1 that is replaced is selected from N82, K88, A89, K99, K125, N126, N129, and K130, and wherein m is an integer from 1 to 6, and n is an integer from about 450 to about 800, or from about 454 to about 796, or from about 454 to about 682, or from about 568 to about 909. In some embodiments of the IL-10 conjugate described herein, in compounds of formula (XVI) or formula (XVII) or a mixture of formula (XVI) and formula (XVII) m is 2 and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909 and 910.

[275] In some embodiments, described herein are IL-10 conjugates that are modified at an amino acid position. In some cases, the modification is to a naturally occurring amino acid. In some cases, the modification is to a non-naturally occurring amino acid. In some cases, the modification is a substitution with a non-naturally occurring amino acid that is also conjugated. In some cases, the modification is a substitution with a non-naturally occurring amino acid and conjugation to amino acid residues that are not a non-naturally occurring amino acid. In some embodiments, the modification of the IL-10 conjugate comprises modifying and conjugating a parent IL-10 comprising the sequences of SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, the parent IL-10 is wild-type IL-10. In some embodiments, the IL-10 conjugates comprise an optional methionine at the N-terminus, shown as (M) in SEQ ID NOs: 1 and 3-73. In some embodiments, the IL-10 conjugates comprise a methionine at the N-terminus of the wild-type IL-10 sequence or the parental IL-10 sequence, followed by a serine. In some cases, the IL-10 conjugates comprise a serine at the N-terminus of the wild-type IL-10 sequence or the parental IL-10 sequence. In some embodiments, the IL-10 conjugates comprise a methionine replacing and substituting for the serine at the N-terminus of the wild-type IL-10 sequence or the parental IL-10 sequence. In some embodiments, the IL-10 conjugates comprise a methionine at the N-terminus followed by a serine shown as (M) in SEQ ID NO: 1. In some cases, the IL-10 conjugates comprise a serine at the N-terminus of SEQ ID NO: 1. In some embodiments, the IL-10 conjugates comprise a methionine replacing and substituting for the serine at the N-terminus shown as (M) in SEQ ID NO: 1.

[276] In some cases, the document describes an isolated IL-10 conjugate that includes at least one non-natural amino acid. In some cases, the IL-10 conjugate is an isolated and purified mammalian IL-10, such as a rodent IL-10 protein or a human IL-10 protein. In some cases, the IL-10 conjugate is a human IL-10 protein. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 19. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 19. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 19. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 20. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 20. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 20. In some cases, the conjugate IL-10 has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 21. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 21. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 21. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 22. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 22. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 22. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 23. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 23. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 23. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 24. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 24. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 24. In further cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 25. In further instances, the IL-10 conjugate comprises the sequence of SEQ ID NO: 25. In further instances, the IL-10 conjugate consists of the sequence of SEQ ID NO: 25. In some instances, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 26. In some instances, the IL-10 conjugate comprises the sequence of SEQ ID NO: 26. In some instances, the IL-10 conjugate consists of the sequence of SEQ ID NO: 26. In further instances, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 27. In further instances, the IL-10 conjugate comprises the sequence of SEQ ID NO: 27. In further instances, the IL-10 conjugate consists of the sequence of SEQ ID NO: 27. In some instances, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 28. In some instances, the IL-10 conjugate comprises the sequence of SEQ ID NO: 28. In some instances, the IL-10 conjugate consists of the sequence of SEQ ID NO: 28. In further instances, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 29. In further instances, the IL-10 conjugate comprises the sequence of SEQ ID NO: 29. In further instances, the IL-10 conjugate consists of the sequence of SEQ ID NO: 29. In some instances, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 30. In some instances, the IL-10 conjugate comprises the sequence of SEQ ID NO: 30. In some instances, the IL-10 conjugate consists of the sequence of SEQ ID NO: 30. In some instances, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 31. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 31. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 31. In further cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 32. In further cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 32. In further cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 32. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 33. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 33. In further cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34. In further cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 34. In further cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 34. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 35. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 35. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 35. In further cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 36. In further cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 36. In further cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 36. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 37. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 37. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 37. In further cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 38. In further cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 38. In further cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 38. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 39. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 39. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 39. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 40. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 40. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 40. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 41. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 41. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 41. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 42. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 42. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 42. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 43. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 43. In some cases, the IL-10 conjugate consists of from the sequence of SEQ ID NO: 43. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 44. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 44. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 44. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 45. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 45. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 45. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 46. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 46. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 46. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 47. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 47. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 47. In some cases the IL-10 conjugate has approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 48. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 48. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 48. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 49. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 49. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 49. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 50. In some cases, the conjugate IL-10 comprises the sequence of SEQ ID NO: 50. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 50. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 51. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 51. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 51. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 52. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 52. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 52. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 53. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 53. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 53. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 54. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 54. in some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 54. In further cases, the IL-10 conjugate comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 55. In further cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 55. In further cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 55. In some cases, the IL-10 conjugate comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 56. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 56. In some cases, the conjugate IL-10 consists of the sequence of SEQ ID NO: 56. In further instances, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 57. In further instances, the IL-10 conjugate comprises the sequence of SEQ ID NO: 57. In further instances, the IL-10 conjugate consists of the sequence of SEQ ID NO: 57. In some instances, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 58. In some instances, the IL-10 conjugate comprises the sequence of SEQ ID NO: 58. In some instances, the IL-10 conjugate consists of the sequence of SEQ ID NO: 58. In further instances, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 59. In further instances, the IL-10 conjugate comprises the sequence of SEQ ID NO: 59. In further instances, the IL-10 conjugate consists of the sequence of SEQ ID NO: 59. In some instances, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 60. In some instances, the IL-10 conjugate comprises the sequence of SEQ ID NO: 60. In some instances, the IL-10 conjugate consists of the sequence of SEQ ID NO: 60. in some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 61. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 61. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 61. In further cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 62. In further cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 62. In further cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 62. In some cases, the conjugate IL-10 is characterized by about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 63. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 63. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 63. In further cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 64. In further cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 64. In further cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 64. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 65. In some cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 65. In some cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 65. In further cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 66. In further cases, the IL-10 conjugate comprises the sequence of SEQ ID NO: 66. In further cases, the IL-10 conjugate consists of the sequence of SEQ ID NO: 66. In some cases, the IL-10 conjugate has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 67-73. In further cases, the IL-10 conjugate comprises a sequence with any one of SEQ ID NOs: 67-73. In further cases, the IL-10 conjugate consists of a sequence with any one of SEQ ID NOs: 67-73.

[277] In some embodiments, the at least one non-natural amino acid is located proximal to the N-terminus (e.g., proximal to the N-terminal residue). For example, the at least one non-natural amino acid is optionally located within the first 10, 20, 30, 40, or 50 residues from the N-terminus. In some cases, the at least one non-natural amino acid is located at the N-terminus (i.e., the at least one non-natural amino acid is the N-terminal residue of the IL-10 polypeptide).

[278] In other embodiments, the at least one unnatural amino acid is located proximal to the C-terminus (e.g., proximal to the C-terminal residue). For example, the at least one unnatural amino acid is optionally located within the first 10, 20, 30, 40, or 50 residues from the C-terminus. In some cases, the at least one unnatural amino acid is located at the C-terminus (i.e., the at least one unnatural amino acid is the C-terminal residue of the IL-10 polypeptide).

[279] In some cases, the IL-10 conjugate contains one conjugated moiety linked to a non-natural amino acid.

[280] In some cases, the IL-10 conjugate contains an IL-10 monomer that is capable of activating the IL-10R signaling pathway. In other cases, the IL-10 conjugate contains an IL-10 dimer that is functionally active.

[281] In some cases, the IL-10 conjugate comprises two or more conjugated moieties, wherein each of the two or more conjugated moieties is linked to a different unnatural amino acid. In some cases, the two or more conjugated moieties are conjugated to the same IL-10 polypeptide (e.g., presented as either a functionally active IL-10 monomer or a functionally active IL-10 dimer). In other cases, each of the two or more conjugated moieties is conjugated to a different IL-10 polypeptide within an IL-10 dimer. In additional cases, the IL-10 conjugate comprises three, four, five, six or more conjugated moieties, wherein each of the conjugated moieties is linked to a different unnatural amino acid. In such cases, the two IL-10 polypeptides within the dimer are characterized by an uneven distribution of conjugated fragments, for example, one IL-10 polypeptide has one conjugated fragment, while the other IL-10 polypeptide has two or more conjugated fragments.

[282] In some cases, the IL-10 conjugate comprises two or more conjugated moieties. In some cases, each of the two or more conjugated moieties is linked to a non-natural amino acid at the same residue position within the corresponding IL-10 monomer. In other cases, each of the two or more conjugated moieties is linked to a non-natural amino acid located at a different residue position within the IL-10 dimer.

[283] In some cases, the location of the conjugated moiety does not substantially affect the dimerization of the IL-10 polypeptide.

[284] In some cases, the location of the conjugated fragment also does not significantly affect the binding of the IL-10 dimer to the IL-10R.

[285] In some embodiments, the location of the conjugated moiety impairs IL-10R signaling by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, or less. In some cases, the location of the conjugated moiety impairs IL-10R signaling by less than 90%. In some cases, the location of the conjugated moiety impairs IL-10R signaling by less than 80%. In some cases, the location of the conjugated moiety impairs IL-10R signaling by less than 70%. In some cases, the location of the conjugated moiety impairs IL-10R signaling by less than 60%. In some cases, the location of the conjugated moiety interferes with IL-10R signaling by less than 50%. In some cases, the location of the conjugated moiety interferes with IL-10R signaling by less than 40%. In some cases, the location of the conjugated moiety interferes with IL-10R signaling by less than 30%. In some cases, the location of the conjugated moiety interferes with IL-10R signaling by less than 20%. In some cases, the location of the conjugated moiety interferes with IL-10R signaling by less than 10%. In some cases, the location of the conjugated moiety interferes with IL-10R signaling by less than 5%. In some cases, the location of the conjugated moiety interferes with IL-10R signaling by less than 2%. In some cases, the location of the conjugated moiety interferes with IL-10R signaling by less than 1%. In some cases, the location of the conjugated fragment does not significantly interfere with L-10R-mediated signaling.

[286] In additional cases, the location of the conjugated moiety does not interfere with IL-10R signaling.

[287] In some cases, the IL-10 conjugate is characterized by an increased plasma half-life. In some cases, the increased plasma half-life is compared to the plasma half-life of the wild-type IL-10 conjugate or wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 hours, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 21 days, 28 days, 30 days, or longer than the plasma half-life of the wild-type IL-10 conjugate or wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 90 minutes or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 2 hours or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 3 hours or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 4 hours or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 5 hours or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 6 hours or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 10 hours or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 12 hours or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 18 hours or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 24 hours or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 36 hours or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 48 hours or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 3 days or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 4 days or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 5 days or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 6 days or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 7 days or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 10 days or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 12 days or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 14 days or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 21 days or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 28 days or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some cases, the extended plasma half-life of the IL-10 conjugate is at least 30 days or longer than the plasma half-life of the wild-type IL-10 conjugate or the wild-type IL-10 protein.

[288] In some embodiments, also described herein is an IL-10/IL-10R complex comprising a modified IL-10 dimer comprising at least one unnatural amino acid and an IL-10R, wherein the modified IL-10 dimer has an increased plasma half-life compared to the plasma half-life of the wild-type IL-10 protein. In some cases, the modified IL-10 dimer further comprises a conjugation moiety covalently attached to the at least one unnatural amino acid.

[289] In some embodiments, the IL-10 conjugate has a plasma half-life that allows for proliferation and/or expansion of tumor infiltrating lymphocytes (TILs), T cells, B cells, natural killer cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, basophils, or CD4+ or CD8+ T cells.

[290] In some embodiments, the IL-10 conjugate is administered to a subject. In some embodiments, the IL-10 conjugate administered to the subject has reduced toxicity compared to the toxicity of a wild-type IL-10 protein administered to a second subject. In some embodiments, the IL-10 conjugate has reduced toxicity that is reduced by at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 100-fold, or more compared to a wild-type IL-10 dimer. In some cases, the reduced toxicity is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more compared to wild-type IL-10 protein.

[291] In some embodiments, the IL-10 conjugate is administered to a subject. In some embodiments, the IL-10 conjugate administered to the subject does not cause grade 3 or 4 adverse events. In some embodiments, the IL-10 conjugate administered to the subject has a reduced incidence or severity of grade 3 or 4 adverse events compared to the incidence or severity of grade 3 or 4 adverse events caused by wild-type IL-10 protein administered to a second subject. Illustrative grade 3 or 4 adverse events include anemia, leukopenia, thrombocytopenia, ALT increased, anorexia, arthralgia, back pain, chills, diarrhea, dyslipidemia, fatigue, fever, influenza-like symptoms, hypoalbuminemia, lipase increased, injection site reaction, myalgia, nausea, night sweats, pruritis, rash, rash erythematous, rash maculopapular, transaminitis, vomiting, and weakness.

[292] In some embodiments, the IL-10 conjugate reduces the incidence of grade 3 or 4 adverse events in a subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% compared to a second subject administered a wild-type IL-10 protein. In some cases, the IL-10 conjugate reduces the severity of grade 3 or 4 adverse events in a subject by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% compared to a second subject administered a wild-type IL-10 protein.

[293] In some embodiments, an IL-10 conjugate as described herein has a reduced affinity for the IL-10R compared to the affinity of a wild-type IL-10 conjugate or wild-type IL-10 protein for the IL-10R. In some embodiments, the affinity of the IL-10 conjugate for the IL-10R compared to the affinity of a wild-type IL-10 conjugate or wild-type IL-10 protein for the IL-10R is reduced by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or greater than 99%. In some cases, the reduction in affinity is about 10%. In some cases, the reduction in affinity is about 20%. In some cases, the decrease in affinity is approximately 30%. In some cases, the decrease in affinity is approximately 40%. In some cases, the decrease in affinity is approximately 50%. In some cases, the decrease in affinity is approximately 60%. In some cases, the decrease in affinity is approximately 70%. In some cases, the decrease in affinity is approximately 80%. In some cases, the decrease in affinity is approximately 90%. In some cases, the decrease in affinity is approximately 95%. In some cases, the decrease in affinity is approximately 99%. In some cases, the decrease in affinity is approximately 100%.

[294] In some embodiments, the decrease in affinity of the IL-10 conjugate compared to the wild-type IL-10 conjugate or wild-type IL-10 protein is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more. In some cases, the decrease in affinity is about 1-fold. In some cases, the decrease in affinity is about 2-fold. In some cases, the decrease in affinity is about 3-fold. In some cases, the decrease in affinity is approximately 4-fold. In some cases, the decrease in affinity is approximately 5-fold. In some cases, the decrease in affinity is approximately 6-fold. In some cases, the decrease in affinity is approximately 7-fold. In some cases, the decrease in affinity is approximately 8-fold. In some cases, the decrease in affinity is approximately 9-fold. In some cases, the decrease in affinity is approximately 10-fold. In some cases, the decrease in affinity is approximately 30-fold. In some cases, the decrease in affinity is approximately 50-fold. In some cases, the decrease in affinity is approximately 100-fold. In some cases, the decrease in affinity is approximately 200-fold. In some cases, the decrease in affinity is approximately 300-fold. In some cases, the decrease in affinity is approximately 400-fold. In some cases, the decrease in affinity is approximately 500-fold. In some cases, the decrease in affinity is approximately 1000-fold. In some cases, the decrease in affinity is greater than 1000-fold.

[295] In some cases, the IL-10 conjugate does not interact with IL-10R. In some cases, the IL-10 conjugate has approximately the same affinity for IL-10R as the affinity of wild-type IL-10 for IL-10R.

[296] In some embodiments, an IL-10 conjugate as described herein has an increased affinity for the IL-10R compared to the affinity of a wild-type IL-10 conjugate or wild-type IL-10 protein for the IL-10R. In some embodiments, the affinity of the IL-10 conjugate for the IL-10R compared to the affinity of a wild-type IL-10 conjugate or wild-type IL-10 protein for the IL-10R is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or greater than 99%. In some cases, the increase in affinity is about 10%. In some cases, the increase in affinity is about 20%. In some cases, the increase in affinity is about 30%. In some cases, the increase in affinity is approximately 40%. In some cases, the increase in affinity is approximately 50%. In some cases, the increase in affinity is approximately 60%. In some cases, the increase in affinity is approximately 70%. In some cases, the increase in affinity is approximately 80%. In some cases, the increase in affinity is approximately 90%. In some cases, the increase in affinity is approximately 95%. In some cases, the increase in affinity is approximately 99%. In some cases, the increase in affinity is approximately 100%. In some embodiments, the increase in affinity of the IL-10 conjugate compared to the wild-type IL-10 conjugate or wild-type IL-10 protein is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more. In some cases, the increase in affinity is about 1-fold. In some cases, the increase in affinity is about 2-fold. In some cases, the increase in affinity is about 3-fold. In some cases, the increase in affinity is about 4-fold. In some cases, the increase in affinity is approximately 5-fold. In some cases, the increase in affinity is approximately 6-fold. In some cases, the increase in affinity is approximately 7-fold. In some cases, the increase in affinity is approximately 8-fold. In some cases, the increase in affinity is approximately 9-fold. In some cases, the increase in affinity is approximately 10-fold. In some cases, the increase in affinity is approximately 30-fold. In some cases, the increase in affinity is approximately 50-fold. In some cases, the increase in affinity is approximately 100-fold. In some cases, the increase in affinity is approximately 200-fold. In some cases, the increase in affinity is approximately 300-fold. In some cases, the increase in affinity is approximately 400-fold. In some cases, the increase in affinity is approximately 500-fold. In some cases, the increase in affinity is approximately 1000-fold. In some cases, the increase in affinity is more than 1000-fold.

[297] In some cases, the IL-10 mediated signaling efficacy of the IL-10R is measured by the EC50. In some embodiments, the EC50 of the IL-10 conjugate is reduced compared to the EC50 of the wild-type IL-10 conjugate or the wild-type IL-10 protein. In some embodiments, the reduction in the EC50 of the IL-10 conjugate is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or greater than 99%. In some cases, the EC50 of the IL-10 conjugate is reduced by about 10%. In some cases, the EC50 of the IL-10 conjugate is reduced by about 20%. In some cases, the EC50 of the IL-10 conjugate is reduced by about 30%. In some cases, the EC50 of the IL-10 conjugate is reduced by approximately 40%. In some cases, the EC50 of the IL-10 conjugate is reduced by approximately 50%. In some cases, the EC50 of the IL-10 conjugate is reduced by approximately 60%. In some cases, the EC50 of the IL-10 conjugate is reduced by approximately 70%. In some cases, the EC50 of the IL-10 conjugate is reduced by approximately 80%. In some cases, the EC50 of the IL-10 conjugate is reduced by approximately 90%. In some cases, the EC50 of the IL-10 conjugate is reduced by approximately 95%. In some cases, the EC50 of the IL-10 conjugate is reduced by approximately 99%. In some cases, the EC50 of the IL-10 conjugate is reduced by approximately 100%.

[298] In some embodiments, the reduction in the EC50 of the IL-10 conjugate compared to the wild-type IL-10 conjugate or the wild-type IL-10 protein is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more. In some cases, the EC50 of the IL-10 conjugate is reduced by about 1-fold. In some cases, the EC50 of the IL-10 conjugate is reduced by about 2-fold. In some cases, the EC50 of the IL-10 conjugate is reduced by about 3-fold. In some cases, the EC50 of the IL-10 conjugate is reduced by about 4-fold. In some cases, the EC50 of the IL-10 conjugate is reduced by about 5-fold. In some cases, the EC50 of the IL-10 conjugate is reduced by about 6-fold. In some cases, the EC50 of the IL-10 conjugate is reduced by about 7-fold. In some cases, the EC50 of the IL-10 conjugate is reduced by about 8-fold. In some cases, the EC50 of the IL-10 conjugate is reduced by about 9-fold. In some cases, the EC50 of the IL-10 conjugate is reduced by about 10-fold. In some cases, the EC50 of the IL-10 conjugate is reduced by about 30-fold. In some cases, the EC50 of the IL-10 conjugate is reduced by about 50-fold. In some cases, the EC50 of the IL-10 conjugate is reduced by about 100-fold. In some cases, the EC50 of the IL-10 conjugate is reduced by about 200-fold. In some cases, the EC50 of the IL-10 conjugate is reduced by about 300-fold. In some cases, the EC50 of the IL-10 conjugate is reduced by about 400-fold. In some cases, the EC50 of the IL-10 conjugate is reduced by about 500-fold. In some cases, the EC50 of the IL-10 conjugate is reduced by about 1000-fold. In some cases, the EC50 of the IL-10 conjugate is reduced by more than 1000-fold.

[299] In some cases, the EC50 of the IL-10 conjugate is approximately the same as the EC50 of wild-type IL-10 protein.

[300] In some cases, the IL-10 conjugate described herein has an increased EC50 compared to the EC50 of the wild-type IL-10 conjugate or the wild-type IL-10 protein in activating the signaling pathway via the IL-10R. In some embodiments, the increase in the EC50 of the IL-10 conjugate is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or greater than 99%. In some cases, the EC50 of the IL-10 conjugate is increased by about 10%. In some cases, the EC50 of the IL-10 conjugate is increased by about 20%. In some cases, the EC50 of the IL-10 conjugate is increased by about 30%. In some cases, the EC50 of the IL-10 conjugate is increased by approximately 40%. In some cases, the EC50 of the IL-10 conjugate is increased by approximately 50%. In some cases, the EC50 of the IL-10 conjugate is increased by approximately 60%. In some cases, the EC50 of the IL-10 conjugate is increased by approximately 70%. In some cases, the EC50 of the IL-10 conjugate is increased by approximately 80%. In some cases, the EC50 of the IL-10 conjugate is increased by approximately 90%. In some cases, the EC50 of the IL-10 conjugate is increased by approximately 95%. In some cases, the EC50 of the IL-10 conjugate is increased by approximately 99%. In some cases, the EC50 of the IL-10 conjugate is increased by approximately 100%.

[301] In some embodiments, the increase in the EC50 of the IL-10 conjugate compared to the EC50 of the wild-type IL-10 conjugate or wild-type IL-10 protein is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more. In some cases, the EC50 of the IL-10 conjugate is increased by about 1-fold. In some cases, the EC50 of the IL-10 conjugate is increased by about 2-fold. In some cases, the EC50 of the IL-10 conjugate is increased by about 3-fold. In some cases, the EC50 of the IL-10 conjugate is increased by about 4-fold. In some cases, the EC50 of the IL-10 conjugate is increased by about 5-fold. In some cases, the EC50 of the IL-10 conjugate is increased by about 6-fold. In some cases, the EC50 of the IL-10 conjugate is increased by about 7-fold. In some cases, the EC50 of the IL-10 conjugate is increased by about 8-fold. In some cases, the EC50 of the IL-10 conjugate is increased by about 9-fold. In some cases, the EC50 of the IL-10 conjugate is increased by about 10-fold. In some cases, the EC50 of the IL-10 conjugate is increased by about 30-fold. In some cases, the EC50 of the IL-10 conjugate is increased by about 50-fold. In some cases, the EC50 of the IL-10 conjugate is increased by about 100-fold. In some cases, the EC50 of the IL-10 conjugate is increased by about 200-fold. In some cases, the EC50 of the IL-10 conjugate is increased by about 300-fold. In some cases, the EC50 of the IL-10 conjugate is increased by about 400-fold. In some cases, the EC50 of the IL-10 conjugate is increased by about 500-fold. In some cases, the EC50 of the IL-10 conjugate is increased by about 1000-fold. In some cases, the EC50 of the IL-10 conjugate is increased by more than 1000-fold.

[302] In some cases, the IL-10R signaling potency mediated by IL-10 is measured by ED50. In some embodiments, an IL-10 conjugate as described herein has a reduced ED50 compared to the ED50 of a wild-type IL-10 conjugate or a wild-type IL-10 protein. In some embodiments, the reduction in the ED50 of the IL-10 conjugate is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or greater than 99%. In some cases, the ED50 of the IL-10 conjugate is reduced by about 10%. In some cases, the ED50 of the IL-10 conjugate is reduced by about 20%. In some cases, the ED50 of the IL-10 conjugate is reduced by approximately 30%. In some cases, the ED50 of the IL-10 conjugate is reduced by approximately 40%. In some cases, the ED50 of the IL-10 conjugate is reduced by approximately 50%. In some cases, the ED50 of the IL-10 conjugate is reduced by approximately 60%. In some cases, the ED50 of the IL-10 conjugate is reduced by approximately 70%. In some cases, the ED50 of the IL-10 conjugate is reduced by approximately 80%. In some cases, the ED50 of the IL-10 conjugate is reduced by approximately 90%. In some cases, the ED50 of the IL-10 conjugate is reduced by approximately 95%. In some cases, the ED50 of the IL-10 conjugate is reduced by approximately 99%. In some cases, the ED50 of the IL-10 conjugate is reduced by approximately 100%.

[303] In some embodiments, the reduction in the ED50 of the IL-10 conjugate compared to the ED50 of the wild-type IL-10 conjugate or wild-type IL-10 protein is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more. In some cases, the ED50 of the IL-10 conjugate is reduced by about 1-fold. In some cases, the ED50 of the IL-10 conjugate is reduced by about 2-fold. In some cases, the ED50 of the IL-10 conjugate is reduced by about 3-fold. In some cases, the ED50 of the IL-10 conjugate is reduced by about 4-fold. In some cases, the ED50 of the IL-10 conjugate is reduced by about 5-fold. In some cases, the ED50 of the IL-10 conjugate is reduced by about 6-fold. In some cases, the ED50 of the IL-10 conjugate is reduced by about 7-fold. In some cases, the ED50 of the IL-10 conjugate is reduced by about 8-fold. In some cases, the ED50 of the IL-10 conjugate is reduced by about 9-fold. In some cases, the ED50 of the IL-10 conjugate is reduced by about 10-fold. In some cases, the ED50 of the IL-10 conjugate is reduced by about 30-fold. In some cases, the ED50 of the IL-10 conjugate is reduced by about 50-fold. In some cases, the ED50 of the IL-10 conjugate is reduced by about 100-fold. In some cases, the ED50 of the IL-10 conjugate is reduced by about 200-fold. In some cases, the ED50 of the IL-10 conjugate is reduced by about 300-fold. In some cases, the ED50 of the IL-10 conjugate is reduced by about 400-fold. In some cases, the ED50 of the IL-10 conjugate is reduced by about 500-fold. In some cases, the ED50 of the IL-10 conjugate is reduced by about 1000-fold. In some cases, the ED50 of the IL-10 conjugate is reduced by more than 1000-fold.

[304] In some cases, the ED50 of the IL-10 conjugate is approximately the same as the ED50 of the wild-type IL-10 protein.

[305] In some cases, an IL-10 conjugate as described herein has an increased ED50 compared to the ED50 of a wild-type IL-10 conjugate or a wild-type IL-10 protein. In some embodiments, the increase in the ED50 of the IL-10 conjugate is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%, or greater than 99%. In some cases, the ED50 of the IL-10 conjugate is increased by about 10%. In some cases, the ED50 of the IL-10 conjugate is increased by about 20%. In some cases, the ED50 of the IL-10 conjugate is increased by about 30%. In some cases, the ED50 of the IL-10 conjugate is increased by about 40%. In some cases, the ED50 of the IL-10 conjugate is increased by approximately 50%. In some cases, the ED50 of the IL-10 conjugate is increased by approximately 60%. In some cases, the ED50 of the IL-10 conjugate is increased by approximately 70%. In some cases, the ED50 of the IL-10 conjugate is increased by approximately 80%. In some cases, the ED50 of the IL-10 conjugate is increased by approximately 90%. In some cases, the ED50 of the IL-10 conjugate is increased by approximately 95%. In some cases, the ED50 of the IL-10 conjugate is increased by approximately 99%. In some cases, the ED50 of the IL-10 conjugate is increased by approximately 100%.

[306] In some embodiments, the increase in the ED50 of the IL-10 conjugate compared to the ED50 of the wild-type IL-10 conjugate or wild-type IL-10 protein is about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 30-fold, 50-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold, or more. In some cases, the ED50 of the IL-10 conjugate is increased by about 1-fold. In some cases, the ED50 of the IL-10 conjugate is increased by about 2-fold. In some cases, the ED50 of the IL-10 conjugate is increased by about 3-fold. In some cases, the ED50 of the IL-10 conjugate is increased by about 4-fold. In some cases, the ED50 of the IL-10 conjugate is increased by about 5-fold. In some cases, the ED50 of the IL-10 conjugate is increased by about 6-fold. In some cases, the ED50 of the IL-10 conjugate is increased by about 7-fold. In some cases, the ED50 of the IL-10 conjugate is increased by about 8-fold. In some cases, the ED50 of the IL-10 conjugate is increased by about 9-fold. In some cases, the ED50 of the IL-10 conjugate is increased by about 10-fold. In some cases, the ED50 of the IL-10 conjugate is increased by about 30-fold. In some cases, the ED50 of the IL-10 conjugate is increased by about 50-fold. In some cases, the ED50 of the IL-10 conjugate is increased by about 100-fold. In some cases, the ED50 of the IL-10 conjugate is increased by about 200-fold. In some cases, the ED50 of the IL-10 conjugate is increased by about 300-fold. In some cases, the ED50 of the IL-10 conjugate is increased by about 400-fold. In some cases, the ED50 of the IL-10 conjugate is increased by about 500-fold. In some cases, the ED50 of the IL-10 conjugate is increased by about 1000-fold. In some cases, the ED50 of the IL-10 conjugate is increased by more than 1000-fold.

Natural and unnatural amino acids

[307] In some embodiments, described herein is an amino acid residue within a modified IL-10 polypeptide or IL-10 conjugate that is mutated to lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine prior to linking to (or reacting with) a conjugation moiety. For example, a side chain of lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, or tyrosine can link to a conjugation moiety described herein. In some cases, the amino acid residue is mutated to cysteine, lysine, or histidine. In some cases, the amino acid residue is mutated to cysteine. In some cases, the amino acid residue is mutated to lysine. In some cases, the amino acid residue is mutated to histidine. In some cases, the amino acid residue is mutated to tyrosine. In some cases, the amino acid residue is mutated to tryptophan. In some cases, the amino acid residue is proximal to the N- or C-terminus, at the N- or C-terminus, or at an internal residue position. In some cases, the amino acid residue is an N- or C-terminal residue and the mutation results in a substitution of cysteine or lysine. In some cases, the amino acid residue is proximal to the N- or C-terminal residue (e.g., within 50, 40, 30, 20, or 10 residues of the N- or C-terminal residue) and the mutation results in a substitution of cysteine or lysine.

[308] In some cases, the amino acid residue is added to the N- or C-terminal residue, i.e., the IL-10 polypeptide contains an additional amino acid residue at either the N- or C-terminus, and the additional amino acid residue is a cysteine or a lysine. In some cases, the additional amino acid residue is a cysteine. In some cases, the additional amino acid is conjugated to the conjugation moiety.

[309] In some embodiments, an amino acid residue described herein (e.g., within an IL-10 polypeptide) is mutated to a non-natural amino acid. In some embodiments, the non-natural amino acid is not conjugated to a conjugation moiety. In some embodiments, an IL-10 polypeptide described herein comprises a non-natural amino acid, wherein IL-10 is conjugated to the protein, wherein the attachment point is not a non-natural amino acid.

[310] In some embodiments, an amino acid residue described herein (e.g., within an IL-10 polypeptide) is mutated to a non-natural amino acid prior to binding to a conjugation moiety. In some cases, the mutation to a non-natural amino acid prevents or minimizes the immune system's response to the self-antigen. As used herein, the term "non-natural amino acid" refers to an amino acid other than the 20 amino acids that naturally occur in a protein. Non-limiting examples of unnatural amino acids include p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, p-methoxyphenylalanine, O-methyl-L-tyrosine, p-propargyloxyphenylalanine, p-propargylphenylalanine, L-3-(2-naphthyl)alanine, 3-methylphenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p -borophenylalanine, O -propargyltyrosine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, selenocysteine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, N6-[(2-azidoethoxy)carbonyl]-L-lysine (AzK), N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine; unnatural analogue of the amino acid tyrosine; unnatural analogue of the amino acid glutamine; unnatural analogue of the amino acid phenylalanine; unnatural analogue of the amino acid serine; unnatural analogue of the amino acid threonine; an amino acid substituted with alkyl, aryl, acyl, azido, cyano, halogen, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocycle, enone, imine, aldehyde, hydroxylamine, keto or amino, or a combination thereof; an amino acid with a photoactivatable crosslinker; a spin-labeled amino acid; a fluorescent amino acid; a metal-binding amino acid; a metal-containing amino acid; a radioactive amino acid; a photoshielded and/or photoisomerizable amino acid; an amino acid containing biotin or a biotin analog; a keto-containing amino acid; an amino acid containing polyethyleneglycol or a polyether; an amino acid substituted with a heavy atom; a chemically cleavable or photocleavable amino acid; an amino acid with an extended side chain; an amino acid containing a toxic group; an amino acid substituted with a sugar; an amino acid containing a carbon-linked sugar; a redox-active amino acid; an a-hydroxy-containing acid; an amino thioic acid; an α,α-disubstituted amino acid; a β-amino acid; a cyclic amino acid other than proline or histidine, and an aromatic amino acid other than phenylalanine, tyrosine, or tryptophan.

[311] Other examples of unnatural amino acids include N6-((2-azidoethoxy)carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, and N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.

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

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

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

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

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

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

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

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

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

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

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

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

[324] In some embodiments, the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid is N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, the at least one unnatural amino acid is N6-(propargylethoxy)-L-lysine (PraK). In some embodiments, the at least one unnatural amino acid is N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid is N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid is N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid is N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid is N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid is N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.

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

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

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

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

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

[330] In some cases, the unnatural amino acid is a derivative of lysine where the N atom at the epsilon position is replaced by an oxygen atom.

[331] In some cases, the unnatural amino acid is a lysine derivative, which is a non-naturally occurring lysine that has been post-translationally modified.

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

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

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

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

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

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

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

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

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

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

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

[343] In some embodiments, the unnatural amino acid is incorporated into the IL-10 polypeptide via a repurposed amber, opal, or ochre stop codon.

[344] In some embodiments, the unnatural amino acid is incorporated into the IL-10 polypeptide via a 4-base codon.

[345] In some embodiments, the unnatural amino acid is incorporated into the IL-10 polypeptide via a repurposed, rarely used sense codon.

[346] In some embodiments, the non-natural amino acid is incorporated into the IL-10 polypeptide via a synthetic codon containing a non-natural nucleic acid.

Orthogonal pair of synthetase and tRNA

[347] In some cases, the unnatural amino acid is incorporated into the IL-10 polypeptide by a naturally occurring synthetase. In some embodiments, the unnatural amino acid is incorporated into the cytokine by an organism that is auxotrophic for one or more amino acids. In some embodiments, synthetases corresponding to the auxotrophic amino acid are capable of loading the corresponding tRNA with the unnatural amino acid. In some embodiments, the unnatural amino acid is selenocysteine or a derivative thereof. In some embodiments, the unnatural amino acid is selenomethionine or a derivative thereof. In some embodiments, the unnatural amino acid is an aromatic amino acid, wherein the aromatic amino acid includes an aryl halide, such as iodide. In embodiments, the unnatural amino acid is structurally similar to the auxotrophic amino acid.

Conjugated fragments

[348] In certain embodiments, conjugation moieties are disclosed herein that are linked to an IL-10 polypeptide described herein. In some cases, the conjugation moiety is a molecule that disrupts the interaction of IL-10 with its receptor. In some cases, the conjugation moiety is any molecule that, when bound to IL-10, allows the IL-10 conjugate to modulate an immune response. In some cases, the conjugation moiety is linked to IL-10 via a covalent bond. In some cases, the IL-10 described herein is attached to the conjugation moiety via a triazole group. In some cases, the IL-10 described herein is attached to the conjugation moiety via a dihydropyridazine or pyridazine group. In some cases, the conjugation moiety comprises a water-soluble polymer. In other cases, the conjugation moiety comprises a protein or a binding moiety thereof. In additional instances, the conjugation moiety comprises a peptide. In additional instances, the conjugation moiety comprises a nucleic acid. In additional instances, the conjugation moiety comprises a small molecule. In additional instances, the conjugation moiety comprises a bioconjugate (e.g., a TLR agonist such as a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 agonist; or a synthetic ligand such as Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, poly I:C, poly A:U, AGP, MPL A, RC-529, MDF2β, CFA, or flagellin). In some instances, the conjugation moiety increases serum half-life and/or improves stability. In some cases, the conjugation moiety reduces the interaction of the cytokine with one or more domains or subunits of the cytokine receptor. In additional cases, the conjugation moiety blocks the interaction of IL-10 with one or more domains or subunits of IL-10 with its cognate receptor(s). In some embodiments, the IL-10 conjugates described herein comprise multiple conjugation moieties. In some embodiments, the conjugation moiety is attached to a non-natural or natural amino acid in an IL-10 polypeptide. In some embodiments, the IL-10 conjugate comprises a conjugation moiety attached to a natural amino acid. In some embodiments, the IL-10 conjugate is attached to a non-natural amino acid in a cytokine peptide. In some embodiments, the conjugation moiety is attached to the N- or C-terminal amino acid of the IL-10 polypeptide. Various combinations of sites are disclosed herein, such as a first conjugation moiety being attached to a non-natural or natural amino acid in an IL-10 polypeptide and a second conjugation moiety being attached to an N- or C-terminal amino acid of the IL-10 polypeptide. In some embodiments, one conjugation moiety is attached to multiple residues of the IL-10 polypeptide (e.g., in a loop). In some embodiments, the conjugation moiety is attached to both the N-terminal and C-terminal amino acids of the IL-10 polypeptide.

Water-soluble polymers

[349] In some embodiments, the conjugation moiety described herein is a water-soluble polymer. In some cases, the water-soluble polymer is non-peptide, non-toxic, and biocompatible. As used herein, a substance is considered biocompatible if the beneficial effects associated with use of the substance alone or with another substance (e.g., an active agent such as an IL-10 fragment) in combination with living tissue (e.g., when administered to a patient) outweigh any detrimental effects, as judged by a clinician, such as a physician, toxicologist, or clinical development specialist. Additionally, in some cases, the water-soluble polymer is non-immunogenic. In some cases, a substance is considered non-immunogenic if the intended use of the substance in vivo does not elicit an adverse immune response (e.g., antibody formation), or, if an immune response occurs, the response is not considered clinically significant or important, as judged by a clinician, such as a physician, toxicologist, or clinical development specialist.

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

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

[352] In some embodiments, W in any of the IL-10 conjugates described herein, such as any IL-10 conjugates comprising Formula (II), Formula (III), Formula (IV), or Formula (V), is a linear or branched PEG group. In some embodiments, W is a linear PEG group. In some embodiments, W is a branched PEG group. In some embodiments, W is a methoxy-PEG group. In some embodiments, the methoxy-PEG group is linear or branched. In some embodiments, the methoxy-PEG group is linear. In some embodiments, the methoxy-PEG group is branched.

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

[354] Exemplary values for the weight average molecular weight of the water-soluble polymer include values of about 100 Daltons, about 200 Daltons, about 300 Daltons, about 400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons, about 900 Daltons, about 1000 Daltons, about 1500 Daltons, about 2000 Daltons, about 2200 Daltons, about 2500 Daltons, about 3000 Daltons, about 4000 Daltons, about 4400 Daltons, about 4500 Daltons, about 5000 Daltons, about 5500 daltons, approximately 6000 daltons, approximately 7000 daltons, approximately 7500 daltons, approximately 8000 daltons, approximately 9000 daltons, approximately 10000 daltons, approximately 11000 daltons, approximately 12000 daltons, approximately 13000 daltons, approximately 14000 daltons, approximately 15000 daltons, approximately 20000 daltons, approximately 22500 daltons, approximately 25000 daltons, approximately 30000 daltons, approximately 35000 daltons, approximately 40000 daltons, approximately 45000 daltons, approximately 50000 daltons, approximately 55000 daltons, approximately 60,000 daltons, about 65,000 daltons, about 70,000 daltons, and about 75,000 daltons. Branched versions of the water-soluble polymer (e.g., a branched water-soluble polymer with a molecular weight of 40,000 daltons composed of two polymers with a molecular weight of 20,000 daltons) having a combined molecular weight of any of the above can also be used. In one or more embodiments, the conjugate will not contain any moieties that are PEG attached, directly or indirectly, to PEG having a weight average molecular weight of less than about 6,000 daltons.

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

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

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

[358] In some embodiments, the IL-10 polypeptide described herein is conjugated to a water-soluble polymer selected from poly(alkylene glycols) such as polyethylene glycol ("PEG"), poly(propylene glycol) ("PPG"), copolymers of ethylene glycol and propylene glycol, and the like, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), polymethylacrylamide (PDAAm), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polyphosphazene, polyoxazolines ("POZ"), poly(N-acryloylmorpholine), and combinations thereof. In some cases, the IL-10 polypeptide is conjugated to PEG (e.g., pegylated). In some cases, the IL-10 polypeptide is conjugated to PPG. In some cases, the IL-10 polypeptide is conjugated to POZ. In some cases, the IL-10 polypeptide is conjugated to PVP.

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

[360] In some cases, the IL-10 polypeptide described herein is conjugated to a PG, such as HPG, LPG, medium functional PG, linear hyperbranched block copolymer PG, or side chain functional PG.

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

[362] In some cases, the IL-10 polypeptide described herein is conjugated with an alternative to degradable synthetic PEG, such as, for example, POEGM; backbone-modified PEG derivatives created by polymerization of telechelic or double-end functionalized PEG-based macromonomers; P(EO- co -MEO), cyclic ketene acetals such as BMDO, MDO, and MPDL copolymerized with OEGMA; or PCL-g-PEO.

[363] In some embodiments, the water-soluble polymer comprises poly(zwitterions). Exemplary poly(zwitterions) include, but are not limited to, poly(sulfobetaine methacrylate) (PSBMA), poly(carboxybetaine methacrylate) (PCBMA), and poly(2-methacryloyloxyethylphosphorylcholine) (PMPC). In some cases, the IL-10 polypeptide is conjugated to a poly(zwitterion), such as PSBMA, PCBMA, or PMPC.

[364] In some embodiments, the water-soluble polymer comprises a polycarbonate. Exemplary polycarbonates include, but are not limited to, pentafluorophenyl 5-methyl-2-oxo-1,3-dioxane-5-carboxylate (MTC-OC ₆ F ₅ ). In some cases, the IL-10 polypeptide described herein is conjugated to a polycarbonate, such as MTC-OC ₆ F ₅ .

[365] In some embodiments, the water-soluble polymer comprises a polymer hybrid, such as, for example, a polycarbonate/PEG polymer hybrid, a peptide/protein-polymer conjugate, or a hydroxyl-containing and/or zwitterionic derivatized polymer (e.g., a hydroxyl-containing and/or zwitterionic derivatized PEG polymer). In some cases, the IL-10 polypeptide described herein is conjugated to a polymer hybrid, such as a polycarbonate/PEG polymer hybrid, a peptide/protein-polymer conjugate, or a hydroxyl-containing and/or zwitterionic derivatized polymer (e.g., a hydroxyl-containing and/or zwitterionic derivatized PEG polymer).

[366] In some cases, the water-soluble polymer comprises a polysaccharide. Exemplary polysaccharides include, but are not limited to, dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES). In some cases, the IL-10 polypeptide is conjugated to a polysaccharide. In some cases, the IL-10 polypeptide is conjugated to dextran. In some cases, the IL-10 polypeptide is conjugated to PSA. In some cases, the IL-10 polypeptide is conjugated to HA. In some cases, the IL-10 polypeptide is conjugated to amylose. In some cases, the IL-10 polypeptide is conjugated to heparin. In some cases, the IL-10 polypeptide is conjugated to HS. In some cases, the IL-10 polypeptide is conjugated to dextrin. In some cases, the IL-10 polypeptide is conjugated to HES.

[367] In some cases, the water-soluble polymer includes a glycan. Illustrative classes of glycans include N-linked glycans, O-linked glycans, glycolipids, O-GlcNAc, and glycosaminoglycans. In some cases, the IL-10 polypeptide is conjugated to a glycan. In some cases, the IL-10 polypeptide is conjugated toN-linked glycans. In some cases, the IL-10 polypeptide is conjugated toO-linked glycans. In some cases, the IL-10 polypeptide is conjugated to glycolipids. In some cases, the IL-10 polypeptide is conjugated to O-GlcNAc. In some cases, the IL-10 polypeptide is conjugated to glycosaminoglycans.

[368] In some embodiments, the water-soluble polymer comprises a polyoxazoline polymer. The polyoxazoline polymer is a linear synthetic polymer and, like PEG, has low polydispersity. In some cases, the polyoxazoline polymer is a polydisperse polyoxazoline polymer having an average molecular weight. In some cases, the average molecular weight of the polyoxazoline polymer polymer is, for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10000, 12000, 20000, 35000, 40000, 50000, 60000, 100000, 200000, 300000, 400000, or 500000 Da. In some cases, the polyoxazoline polymer comprises poly(2-methyl-2-oxazoline) (PMOZ), poly(2-ethyl-2-oxazoline) (PEOZ), or poly(2-propyl-2-oxazoline) (PPOZ). In some cases, the IL-10 polypeptide is conjugated to the polyoxazoline polymer. In some cases, the IL-10 polypeptide is conjugated to PMOZ. In some cases, the IL-10 polypeptide is conjugated to PEOZ. In some cases, the IL-10 polypeptide is conjugated to PPOZ.

[369] In some cases, the water-soluble polymer comprises a polyacrylic acid-based polymer. In some cases, the IL-10 polypeptide is conjugated to a polyacrylic acid-based polymer.

[370] In some cases, the water-soluble polymer comprises a polyamine. The polyamine is an organic polymer containing two or more primary amine groups. In some embodiments, the polyamine comprises a branched polyamine, a linear polyamine, or a cyclic polyamine. In some cases, the polyamine is a low molecular weight linear polyamine. Exemplary polyamines include putrescine, cadaverine, spermidine, spermine, ethylenediamine, 1,3-diaminopropane, hexamethylenediamine, tetraethylmethylenediamine, and piperazine. In some cases, the IL-10 polypeptide is conjugated to the polyamine. In some cases, the IL-10 polypeptide is conjugated to putrescine, cadaverine, spermidine, spermine, ethylenediamine, 1,3-diaminopropane, hexamethylenediamine, tetraethylmethylenediamine, or piperazine.

[371] In some cases, the water-soluble polymer is described in U.S. Patent Nos. 7,744,861, 8,273,833, and 7,803,777. In some cases, the IL-10 polypeptide is conjugated to a linker described in U.S. Patent Nos. 7,744,861, 8,273,833, or 7,803,777.

Lipids

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

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

[374] In some embodiments, the IL-10 lipid conjugate enhances serum stability and/or serum half-life.

Squirrels

[375] In some embodiments, the conjugated moiety described herein is a protein or a binding fragment thereof. Exemplary proteins include albumin, transferrin, or transthyretin. In some cases, the protein or binding fragment thereof comprises an antibody or binding fragments thereof. In some cases, the IL-10 conjugate comprises a protein or binding fragment thereof. In some cases, the IL-10 conjugate comprising the protein or binding fragment thereof is characterized by an increased serum half-life and/or increased stability. In some cases, the IL-10 conjugate comprising the protein or binding fragment thereof is characterized by a reduced interaction of IL-10 with one or more IL-10R subunits. In additional cases, the protein or binding fragment thereof blocks the interaction of IL-10 with one or more IL-10R subunits.

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

[377] In some cases, the IL-10 polypeptide is conjugated to albumin. In some cases, the IL-10 polypeptide is conjugated to human serum albumin (HSA). In additional cases, the IL-10 polypeptide is conjugated to a functional fragment of albumin.

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

[379] In some cases, the IL-10 polypeptide is conjugated to transferrin. In some cases, the IL-10 polypeptide is conjugated to human transferrin. In additional cases, the IL-10 polypeptide is conjugated to a functional fragment of transferrin.

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

[381] In some cases, the IL-10 polypeptide is conjugated to transthyretin (via one of its ends or via an internal hinge region). In some cases, the IL-10 polypeptide is conjugated to a functional fragment of transthyretin.

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

[383] In some cases, the conjugated fragment comprises an scFv, bis-scFv, (scFv) ₂ , dsFv, or sdAb. In some cases, the conjugated fragment comprises an scFv. In some cases, the conjugated fragment comprises a bis-scFv. In some cases, the conjugated fragment comprises (scFv) ₂ . In some cases, the conjugated fragment comprises a dsFv. In some cases, the conjugated fragment comprises an sdAb.

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

[385] In some cases, the IL-10 polypeptide is conjugated to an antibody or binding fragments thereof. In some cases, the IL-10 polypeptide is conjugated to a humanized antibody or a binding fragment thereof, a murine antibody or a binding fragment thereof, a chimeric antibody or a binding fragment thereof, a monoclonal antibody or a binding fragment thereof, a monovalent Fab', a bivalent Fab ₂ , F(ab)' ₃ fragments, a single-chain variable fragment (scFv), a bis-scFv, (scFv) ₂ , a diabody, a minibody, a nanobody, a tribody, a tetrabody, a human antibody, a disulfide-stabilized Fv protein (dsFv), a single-domain antibody (sdAb), an Ig NAR, a camel antibody or a binding fragment thereof, a bispecific antibody or a binding fragment thereof, or a chemically modified derivative thereof. In additional cases, the IL-10 polypeptide is conjugated to the Fc portion of an antibody. In additional cases, the IL-10 polypeptide is conjugated to the Fc portion of an IgG (e.g., IgG ₁ , IgG ₃ , or IgG ₄ ).

[386] In some embodiments, the IL-10 polypeptide is conjugated to a water-soluble polymer (e.g., PEG) and an antibody or binding fragment thereof. In some cases, the antibody or binding fragments thereof comprises a humanized antibody or binding fragment thereof, a murine antibody or binding fragment thereof, a chimeric antibody or binding fragment thereof, a monoclonal antibody or binding fragment thereof, a monovalent Fab', a bivalent Fab ₂ , F(ab)' ₃ fragments, a single chain variable fragment (scFv), a bis-scFv, (scFv) ₂ , a diabody, a minibody, a nanobody, a triantibody, a tetraantibody, a human antibody, a disulfide-stabilized Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a camel antibody or binding fragment thereof, a bispecific antibody or binding fragment thereof, or a chemically modified derivative thereof. In some cases, the antibody or binding fragments thereof comprises an scFv, bis-scFv, (scFv) ₂ , dsFv, or sdAb. In some cases, the antibody or binding fragments thereof comprises an scFv. In some cases, the antibody or binding fragment thereof directs the IL-10 conjugate to a target cell of interest, and the water-soluble polymer enhances its stability and/or serum half-life.

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

[388] In some embodiments, one or more conjugates of an IL-10 polypeptide and a water-soluble polymer (e.g., PEG) are directly linked to the antibody or binding fragments thereof. In other cases, the IL-10 conjugate is indirectly linked to the antibody or binding fragments thereof via a linker. Exemplary linkers include homobifunctional linkers, heterobifunctional linkers, maleimide-based linkers, traceless linkers, self-cleaving linkers, spacers, and the like.

[389] In some embodiments, the antibody or binding fragments thereof are directly or indirectly linked to a portion of the IL-10 polypeptide in a conjugate of the IL-10 polypeptide and a water-soluble polymer (e.g., PEG). In such cases, the conjugation site of the antibody to the IL-10 polypeptide is a site that will not interfere with binding of the IL-10 polypeptide to the IL-10R. In additional cases, the conjugation site of the antibody to the IL-10 polypeptide is a site that partially blocks binding of the IL-10 polypeptide to the IL-10R. In other embodiments, the antibody or binding fragments thereof are directly or indirectly linked to a portion of the water-soluble polymer in a conjugate of the IL-10 polypeptide and a water-soluble polymer (e.g., PEG).

Peptides

[390] In some embodiments, the conjugated moiety described herein is a peptide. In some cases, the peptide is a non-structured peptide. In some cases, the IL-10 polypeptide is conjugated to a peptide. In some cases, the IL-10 conjugate comprising the peptide is characterized by an increased serum half-life and/or increased stability. In some cases, the IL-10 conjugate comprising the peptide is characterized by reduced interaction of IL-10 with one or more IL-10R subunits. In additional cases, the peptide blocks the interaction of IL-10 with one or more IL-10R subunits.

[391] In some cases, the conjugated moiety is an XTEN™ peptide (Amunix Operating Inc.) and the modification is referred to as ixtenylation. Xtenylation is the genetic fusion of a nucleic acid encoding a polypeptide of interest to a nucleic acid encoding an XTEN™ peptide (Amunix Operating Inc.), a long, unstructured, hydrophilic peptide containing varying percentages of six amino acids: Ala, Glu, Gly, Ser, and Thr. In some cases, the XTEN™ peptide is selected based on properties such as expression, genetic stability, solubility, resistance to aggregation, increased half-life, increased potency, and/or increased in vitro activity when combined with the polypeptide of interest. In some cases, an IL-10 polypeptide is conjugated to an XTEN peptide.

[392] In some cases, the conjugated moiety is a glycine-rich amino acid homopolymer (HAP), and the modification is called gagylation. Gagylation is the genetic fusion of a nucleic acid encoding a polypeptide of interest to a nucleic acid encoding a glycine-rich amino acid homopolymer (HAP). In some cases, the HAP polymer comprises a repeat motif of (Gly ₄ Ser) _n (SEQ ID NO: 67) and is sometimes about 50, 100, 150, 200, 250, 300, or more in length. In some cases, the IL-10 polypeptide is conjugated to HAP.

[393] In some embodiments, the conjugated moiety is a PAS polypeptide, and the modification is referred to as fusion. Fusion is the genetic fusion of a nucleic acid encoding a polypeptide of interest to a nucleic acid encoding a PAS polypeptide. The PAS polypeptide is a hydrophilic, uncharged polypeptide consisting of Pro, Ala, and Ser residues. In some cases, the PAS polypeptide is at least about 100, 200, 300, 400, 500, or 600 amino acids in length. In some cases, the IL-10 polypeptide is conjugated to the PAS polypeptide.

[394] In some embodiments, the conjugated moiety is an elastin-like polypeptide (ELP), and the modification is referred to as epilitation. Eppilitation is a genetic fusion of a nucleic acid encoding a polypeptide of interest to a nucleic acid encoding an elastin-like polypeptide (ELP). ELP comprises a VPGxG repeat motif (SEQ ID NO: 77), wherein x is any amino acid except proline. In some cases, an IL-10 polypeptide is conjugated to ELP.

[395] In some embodiments, the conjugated moiety is a CTP peptide. The CTP peptide comprises a 30 or 31 amino acid residue peptide (FQSSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ (SEQ ID NO: 78) or FQDSSSS*KAPPPS*LPSPS*RLPGPS*DTPILPQ (SEQ ID NO: 79)), wherein S* represents O-glycosylation sites (OPKO). In some cases, the CTP peptide is genetically fused to an IL-10 polypeptide. In some cases, the IL-10 polypeptide is conjugated to the CTP peptide.

[396] In some embodiments, the IL-10 polypeptide is modified by glutamylation. Glutamylation (or polyglutamylation) is a reversible post-translational modification of glutamate in which the γ-carboxyl group of glutamate forms a peptide-like bond with the amino group of free glutamate, in which the α-carboxyl group is converted to a polyglutamate chain.

[397] In some embodiments, the IL-10 polypeptide is modified with a gelatin-like protein (GLK) polymer. In some cases, the GLK polymer comprises multiple Gly-Xaa-Yaa repeats, wherein Xaa and Yaa predominantly comprise proline and 4-hydroxyproline, respectively. In some cases, the GLK-based polymer further comprises Pro, Gly, Glu, Gln, Asn, Ser, and Lys amino acid residues. In some cases, the GLK polymer is about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150 residues, or more in length.

Additional conjugated fragments

[398] In some cases, the conjugated moiety comprises an extracellular biomarker. In some cases, the extracellular biomarker is a tumor antigen. In some cases, an exemplary extracellular biomarker comprises CD19, PSMA, B7-H3, B7-H6, CD70, CEA, CSPG4, EGFRvIII, EphA3, EpCAM, EGFR, ErbB2 (HER2), FAP, FRα, GD2, GD3, Lewis-Y, mesothelin, Muc1, Muc16, ROR1, TAG72, VEGFR2, CD11, Gr-1, CD204, CD16, CD49b, CD3, CD4, CD8, and B220. In some cases, the conjugated moiety is linked or conjugated to IL-10. In some cases, the conjugated fragment is genetically fused, for example, to the N-terminus or C-terminus of IL-10.

[399] In some cases, the conjugated moiety provides a molecule with a post-translational modification. In some cases, examples of post-translational modification include myristoylation, palmitoylation, isoprenylation (or prenylation) (e.g., farnesylation or geranylgeranylation), glypylation, acylation (e.g., O-acylation, N-acylation, S-acylation), alkylation (e.g., with additional alkyl groups such as methyl or ethyl groups), amidation, glycosylation, hydroxylation, iodination, nucleotide addition, oxidation, phosphorylation, succinylation, sulfation, glycosylation, carbamylation, glutamylation, or deamidation. In some cases, IL-10 is modified by post-translational modification such as myristoylation, palmitoylation, isoprenylation (or prenylation) (e.g., farnesylation or geranylgeranylation), glypylation, acylation (e.g., O-acylation, N-acylation, S-acylation), alkylation (e.g., addition of alkyl groups such as methyl or ethyl groups), amidation, glycosylation, hydroxylation, iodination, addition of nucleotides, oxidation, phosphorylation, succinylation, sulfation, glycation, carbamylation, glutamylation, or deamidation.

Conjugation

Linkers

[400] In some embodiments, useful functional reactive groups for conjugating or linking a conjugable moiety to an IL-10 polypeptide described herein include, for example, null linkers or higher order linkers. In some cases, a non-natural amino acid incorporated into an interleukin described herein comprises a functional reactive group. In some cases, a linker comprises a functional reactive group that reacts with a non-natural amino acid incorporated into an interleukin described herein. In some cases, a conjugable moiety comprises a functional reactive group that reacts with a non-natural amino acid incorporated into an interleukin described herein. In some cases, a conjugable moiety comprises a functional reactive group that reacts with a linker (optionally pre-attached to a cytokine peptide) described herein. In some embodiments, the linker comprises a reactive group that reacts with a naturally occurring amino acid in the IL-10 polypeptide described herein. In some cases, higher order linkers include bifunctional linkers, such as homobifunctional linkers or heterobifunctional linkers. Illustrative homobifunctional linkers include, but are not limited to, Lomant's reagent, which is dithiobis(succinimidyl propionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl propionate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), ethylene glycobis(succinimidyl succinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl 3,3'-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), aryl halide-containing compound (DFDNB), such as, for example, 1,5-difluoro-2,4-dinitrobenzene or 1,3-difluoro-4,6-dinitrobenzene, 4,4'-difluoro-3,3'-dinitrophenyl sulfone (DFDNPS), bis-[β-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid acid, N,N'-ethylene-bis(iodoacetamide) or N,N'-hexamethylene-bis(iodoacetamide).

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

[402] In some cases, the reactive functional group provides a nucleophilic group that reacts with an electrophilic group present on the binding moiety (e.g., on the conjugation moiety or on IL-10). Exemplary electrophilic groups include carbonyl groups, such as an aldehyde, ketone, carbonyl group of a carboxylic acid, ester, amide, enone, acyl halide, or acid anhydride. In some embodiments, the reactive functional group is an aldehyde group. Exemplary nucleophilic groups include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In some embodiments, a non-natural amino acid incorporated into an interleukin described herein comprises an electrophilic group.

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

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

[405] In some embodiments, the linker comprises a self-cleaving linker moiety. In some embodiments, the self-cleaving linker moiety comprises p -aminobenzyl alcohol (PAB), p -aminobenzyloxycarbonyl (PABC), or derivatives or analogs thereof. In some embodiments, the linker comprises a dipeptide linker moiety and a self-cleaving linker moiety. In some embodiments, the self-cleaving linker moiety is as described in U.S. Patent No. 9,089,614 and WIPO Application No. WO2015038426, the disclosures of each of which are incorporated herein by reference.

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

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

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

Conjugation Chemistry

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

[410] In some embodiments described herein, the conjugation reaction described herein comprises a 1,3-dipolar cycloaddition reaction. In some embodiments, the 1,3-dipolar cycloaddition reaction comprises a click reaction of an azide and a phosphine. In some embodiments, the conjugation reaction is copper catalyzed. In some embodiments, the conjugation reaction described herein results in a cytokine peptide comprising a linker or conjugation moiety attached via a triazole. In some embodiments, the conjugation reaction described herein comprises a reaction of an azide with a strained olefin. In some embodiments, the conjugation reaction described herein comprises a reaction of an azide with a strained alkyne. In some embodiments, the conjugation reaction described herein involves a reaction of an azide with a cycloalkyne, such as DBCO.

[411] In some embodiments described herein, the conjugation reaction described herein comprises the reaction shown in Scheme 1.

wherein X is a position in an IL-10 conjugate comprising a non-natural amino acid, such as in any of SEQ ID NOs: 3-10. In some embodiments, the conjugated moiety provides a water-soluble polymer. In some embodiments, the reactive group provides an alkyne or azide.

[412] In some embodiments described herein, the conjugation reaction described herein comprises the reaction shown in Scheme 2.

where X is a position in an IL-10 conjugate comprising a non-natural amino acid such as any of SEQ ID NOs: 3-10.

[413] In some embodiments described herein, the conjugation reaction described herein comprises the reaction shown in Scheme 3.

where X is a position in an IL-10 conjugate comprising a non-natural amino acid such as any of SEQ ID NOs: 3-10.

[414] In some embodiments described herein, the conjugation reaction described herein comprises the reaction shown in Scheme 4.

where X is a position in an IL-10 conjugate comprising a non-natural amino acid such as in any of SEQ ID NOs: 3-10.

[415] In some embodiments described herein, the conjugation reaction described herein involves a cycloaddition reaction between an azide moiety, such as one contained in a protein containing an amino acid residue derived from N6 -((2-azidoethoxy)carbonyl)-L-lysine (AzK), and a strained cycloalkyne, such as one derived from DBCO, which is a chemical moiety containing a dibenzocyclooctyne group. PEG moieties containing a DBCO moiety are commercially available or can be prepared by methods known to those of ordinary skill in the art. Exemplary reactions are shown in Schemes 5a-b and 6a-b.

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

[417] In one aspect, provided herein is a method for producing an IL-10 conjugate as described herein, comprising

carrying out a reaction of an IL-10 polypeptide containing an unnatural amino acid of the formula ,

wherein the IL-10 polypeptide comprises the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 polypeptide is replaced with a non-natural amino acid, position X-1 indicates the point of attachment to the previous amino acid residue, position X+1 indicates the point of attachment to the next amino acid residue, and position X indicates the amino acid position at which the non-natural amino acid substitution occurs, with mPEG-DBCO of the formula

where n is such that mPEG-DBCO comprises PEG having a molecular weight of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, or 60 kDa, thereby producing an IL-10 conjugate.

[418] In a further aspect, provided herein is a method for producing an IL-10 conjugate as described herein, comprising

carrying out a reaction of an IL-10 polypeptide containing an unnatural amino acid of the formula , wherein the IL-10 polypeptide comprises the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 polypeptide is replaced with a non-natural amino acid, position X-1 indicates the point of attachment to the previous amino acid residue, position X+1 indicates the point of attachment to the next amino acid residue, and position X indicates the amino acid position at which the non-natural amino acid substitution occurs, with mPEG-DBCO of the formula

where n is such that mPEG-DBCO comprises PEG having a molecular weight of about 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, or 60 kDa, thereby producing an IL-10 conjugate.

Obtaining IL-10 polypeptide

[419] In some cases, the IL-10 conjugates described herein, comprising either a naturally occurring amino acid substitution mutation or a non-naturally occurring amino acid substitution mutation, are recombinantly produced or chemically synthesized. In some cases, the IL-10 conjugates described herein are recombinantly produced, such as in a host cell system or a cell-free system.

[420] In some cases, the IL-10 conjugates are produced recombinantly using a host cell system. In some cases, the host cell is a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell, or a plant cell), a prokaryotic cell (e.g., a gram-positive bacterium or a gram-negative bacterium). In some cases, the eukaryotic host cell is a mammalian host cell. In some cases, the mammalian host cell is a stable cell line or a cell line that contains integrated genetic material of interest into its own genome and has the ability to express a product of the genetic material after many generations of cell division. In other cases, the mammalian host cell is a transient cell line or a cell line that does not contain integrated genetic material of interest into its own genome and does not have the ability to express a product of the genetic material after many generations of cell division.

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

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

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

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

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

[426] In some cases, suitable polynucleic acid molecules or vectors for producing the IL-10 polypeptide described herein include any suitable vectors derived from either a eukaryotic or prokaryotic source. Exemplary polynucleic acid molecules or vectors include vectors derived from a bacterial (e.g., E. coli ), insect, yeast (e.g., Pichia pastoris, K. phaffii ), algae, or mammalian source. Bacterial vectors include, for example, pACYC177, pASK75, the pBAD vector series, the pBADM vector series, the pET vector series, the pETM vector series, the pGEX vector series, pHAT, pHAT2, pMal-c2, pMal-p2, the pQE vector series, pRSET A, pRSET B, pRSET C, the pTrcHis2 series, pZA31-Luc, pZE21-MCS-1, pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC or pTAC-MAT-2.

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

[428] Yeast vectors include, for example, ^Gateway® pDEST™ 14 vector, ^Gateway® pDEST™ 15 vector, ^Gateway® pDEST™ 17 vector, ^Gateway® pDEST™ 24 vector, ^Gateway® pYES-DEST52 vector, ^Gateway® pBAD-DEST49 delivery vector, pAO815 Pichia vector, Pichia pastoris ( K. phaffii ) pFLD1 vector, Pichia pastoris ( K. phaffii ) pGAPZ A, B, and C vector, pPIC3.5K Pichia vector, pPIC6 A, B, and C Pichia vector, pPIC9K Pichia vector, pTEF1/Zeo, pYES2 yeast vector, pYES2/CT yeast vector, pYES2/NT yeast vector A, B and C or yeast vector pYES3/CT.

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

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

[431] In some cases, a cell-free system is used to produce a cytokine polypeptide (e.g., IL-10) described herein. In some cases, the cell-free system comprises a mixture of cytoplasmic and/or nuclear components from a cell and is suitable for in vitro nucleic acid synthesis. In some cases, the cell-free system uses prokaryotic cell components. In other cases, the cell-free system uses eukaryotic cell components. The nucleic acid synthesis is achieved in a cell-free system, such as a Drosophila cell, Xenopus egg, archaea, or HeLa cell based system. Exemplary cell-free systems include the E. coli S30 extract system, the E. coli T7 S30 system, or the PURExpress®, XpressCF, and XpressCF+ system.

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

[433] In some embodiments, a cytokine polypeptide (e.g., IL-10) is produced in a circularly permutated form using either a host expression system or a cell-free system.

Production of IL-10 polypeptide containing unnatural amino acid

[434] The present invention may utilize an orthogonal or expanded genetic code in which one or more specific codons present in the nucleic acid sequence of a cytokine polypeptide (e.g., IL-10) are designed to encode a non-natural amino acid so that it can be genetically inserted into the cytokine (e.g., IL-10) using an orthogonal tRNA synthetase/tRNA pair. The orthogonal tRNA synthetase/tRNA pair is capable of loading a tRNA with a non-natural amino acid and is capable of inserting the non-natural amino acid into a polypeptide chain in response to a codon.

[435] In some embodiments, a polynucleotide is produced comprising the sequence of SEQ ID NO: 76, wherein the codon is replaced with a codon that encodes a non-natural amino acid. In some embodiments, a polynucleotide is produced comprising a sequence having at least 85% identity to SEQ ID NO: 76, wherein the polynucleotide comprises a codon that encodes a non-natural amino acid, wherein optionally the T residues are replaced with U residues. The polynucleotide may encode any of the IL-10 sequences comprising a non-natural amino acid described herein. In some embodiments, the sequence has at least 90%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 76. The polynucleotide may be DNA, such as a plasmid, an expression vector, or an integrated expression construct. The polynucleotide may be RNA, such as mRNA.

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

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

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

[439] In some cases, non-natural nucleic acids are used, resulting in the incorporation of one or more non-natural amino acids into the cytokine (eg, IL-10). Illustrative non-naturally occurring nucleic acids include, but are not limited to, uracil-5-yl, hypoxanthine-9-yl (I), 2-aminoadenine-9-yl, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl- and other alkyl derivatives of adenine and guanine, 2-propyl- and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo-, in particular 5-bromo-, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, as well as 3-deazaguanine and 3-deazaadenine. Certain unnatural nucleic acids such as 5-substituted pyrimidines, 6-azapyrimidines and N-2-substituted purines, N-6-substituted purines, O-6-substituted purines, 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, 5-methylcytosine, those that enhance the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, enlarged nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines and N-2-, N-6- and O-6-substituted purines including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl-, other alkyl derivatives of adenine and guanine, 2-propyl- and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl-(-C≡C-CH₃-)uracil, 5-propynylcytosine-, other alkynyl derivatives of pyrimidine nucleic acids, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo-, 8-amino-, 8-thiol-, 8-thioalkyl-, 8-hydroxyl- and other 8-substituted adenines and guanines, 5-halo-, in particular 5-bromo-, 5-trifluoromethyl-, other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines, phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps, phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine(2H-pyrimido[4,5-b]indol-2-one), pyridoindolcytidine(H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one), those in which the purine or pyrimidine base is replaced by other heterocycles, 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil and 5-iodouracil, 2-aminoadenine, 6-thioguanine, 2-thiothymine, 4-thiothymine, 5-propynyluracil, 4-thiouracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine, and those described in U.S. Patent Nos. 3,687,808; 4,845,205; 4,910,300; 4,948,882; 5093232; 5130302; 5134066; 5175273; 5367066; 5432272; 5457187; 5459255; 5484908; 5502177; 5525711; 5552540; 5587469; 5594121; 5596091; 5614617; 5645985; 5681941; 5750692; 5763588; 5830653 and 6005096; WO 99/62923; Kandimalla et al., (2001) Bioorg. Med. Chem. 9:807-813; The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, J.I., Ed., John Wiley & Sons, 1990, 858- 859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research and Applications, Crooke and Lebleu Eds., CRC Press, 1993, 273-288, the disclosures of each of which are incorporated herein by reference. Additional modifications of the bases can be found in, for example, U.S. Patent No. 3,687,808; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke and Lebleu ed., CRC Press, 1993, the disclosures of each of which are incorporated herein by reference.

[440] Non-naturally occurring nucleic acids containing various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and the nucleic acids in some cases include one or more heterocyclic bases other than the basic five base components of naturally occurring nucleic acids. For example, a heterocyclic base includes in some cases uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo[2.3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2.3-d]pyrimidin-3-yl groups, wherein purines are attached to the sugar moiety of the nucleic acid through position 9, pyrimidines through position 1, pyrrolopyrimidines through position 7, and pyrazolopyrimidines through position 1.

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

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

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

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

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

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

[447] Other modifications at the 2'-position include, but are not limited to, lower _C1 - _C10 alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, _SCH3 , OCN, Cl, Br, CN, _CF3 , _OCF3 , _SOCH3 , SO2CH3, _ONO2 , _NO2 , _N3 , _NH2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, _{polyalkylamino} , substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for _improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents with similar properties. Similar modifications can also be made at other positions in the sugar, particularly at the 3' position of the sugar in the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and at the 5' position of the 5' terminal nucleotide. Modified sugars also include sugars that provide modifications at the bridging oxygen atom of the ring, such as CH ₂ and S. Sugar analog nucleotides can also contain sugar mimetics, such as cyclobutyl moieties in place of the pentofuranosyl sugar. There are numerous United States patents that teach the preparation of such modified sugar structures and that detail and specify the range of base modifications, such as U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5466786; 5514785; 5519134; 5567811; 5576427; 5591722; 5597909; 5610300; 5627053; 5639873; 5646265; 5658873; 5670633; 4845205; 5130302; 5134066; 5175273; 5367066; 5432272; 5457187; 5459255; 5484908; 5502177; 5525711; 5552540; 5587469; 5594121, 5596091; 5614617; 5681941 and 5700920, the disclosures of each of which are incorporated herein by reference in their entireties.

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

[449] In certain embodiments, the nucleic acids described herein include one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acid comprises a bridge between the 4' and 2' atoms of the ribosyl ring. In certain embodiments, the nucleic acids provided herein include one or more bicyclic nucleic acids, wherein the bridge comprises a 4'-2' bicyclic nucleic acid. Examples of such 4'-2' bicyclic nucleic acids include, but are not limited to, one of the formulas: 4'-(CH ₂ )-O-2'(LNA);4'-(CH ₂ )-S-2';4'-(CH ₂ ) ₂ -O-2'(ENA);4'-CH(CH ₃ )-O-2' and 4'-CH(CH ₂ OCH ₃ )-O-2' and their analogs (see U.S. Patent No. 7,399,845); 4'-C(CH ₃ )(CH ₃ )-O-2' and its analogs (see WO2009/006478, WO2008/150729, US2004/0171570, U.S. Patent No. 7,427,672, Chattopadhyaya et al., J. Org. Chem., 209, 74, 118-134 and WO2008/154401). See also, for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Oram et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Patents 4,849,513; 5,015,733; 5,118,800; 5,118,802; 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; 6,525,191; 6,670,461; and 7,399,845; International Publication Nos. WO2004/106356, WO1994/14226, WO2005/021570, WO2007/090071, and WO2007/134181; U.S. Patent Publication Nos. US2004/0171570, US2007/0287831, and US2008/0039618; U.S. Provisional Patent Application Nos. 60/989574, 61/026995, 61/026998, 61/056564, 61/086231, 61/097787, and 61/099844; and International Application Nos. PCT/US2008/064591, PCT US2008/066154, PCT US2008/068922 and PCT/DK98/00393; the disclosures of each of which are incorporated herein by reference.

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

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

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

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

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

[455] Substituents for the phosphate include, for example, short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatom or heterocyclic internucleoside linkages. These include those having morpholine linkages (formed in part from the sugar moiety of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimine and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones and others having mixed parts of N, O, S and CH components₂Numerous United States patents disclose the preparation and use of these types of phosphate substitutions and include, without limitation, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439. It should also be understood that when replacing a nucleotide, both the sugar and phosphate moieties of the nucleotide can be replaced, for example, by an amide-type (aminoethylglycine) linkage (PNA). United States Patent Nos. 5,539,082; 5,714,331; and 5,719,262 describe the preparation and use of a PNA molecule, each of which is incorporated herein by reference. See also Nielsen et al., Science, 1991, 254, 1497-1500. It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance, for example, cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, for example, hexyl-S-tritylthiol (Manoharan et al., Ann. KY. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, for example dodecanediol or undecyl residues (Saison-Behmoaras et al., EM5OJ, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipid, for example di-hexadecyl-rac-glycerol or triethylammonium 1-di-O-hexadecyl-rac-glycero-S-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973) or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochem. Biophys. Acta, 1995, 1264, 229-237) or an octadecylamine or hexylaminocarbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). Numerous United States patents describe the preparation of such conjugates and include, but are not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5414077; 5486603; 5512439; 5578718; 5608046; 4587044; 4605735; 4667025; 4762779; 4789737; 4824941; 4835263; 4876335; 4904582; 4958013; 5082830; 5112963; 5214136; 5082830; 5112963; 5214136; 5245022; 5254469; 5258506; 5262536; 5272250; 5292873; 5317098; 5371241, 5391723; 5416203, 5451463; 5510475; 5512667; 5514785; 5565552; 5567810; 5574142; 5585481; 5587371; 5595726; 5597696; 5599923; 5599928 and 5688941. The disclosure of each of these literature sources is incorporated herein by reference.

[456] In some cases, the non-natural nucleic acids further form non-natural base pairs. Exemplary non-natural nucleotides capable of forming a non-natural DNA or RNA base pair (UBP) in vivo include, but are not limited to, TAT1, dTAT1, 5FM, d5FM, TPT3, dTPT3, 5SICS, d5SICS, NaM, dNaM, CNMO, dCNMO, and combinations thereof. In some embodiments, the non-natural nucleotides include

Illustrative non-natural base pairs include (d)TPT3-(d)NaM; (d)5SICS-(d)NaM; (d)CNMO-(d)TAT1; (d)NaM-(d)TAT1; (d)CNMO-(d)TPT3; and (d)5FM-(d)TAT1.

[457] Other examples of unnatural nucleotides capable of forming unnatural UBPs that can be used to prepare the IL-10 conjugates disclosed herein can be found in Dien et al., J Am Chem Soc., 2018, 140:16115-16123; Feldman et al., J Am Chem Soc, 2017, 139:11427-11433; Ledbetter et al., J Am Chem Soc., 2018, 140:758-765; Dhami et al., Nucleic Acids Res. 2014, 42:10235-10244; Malyshev et al., Nature, 2014, 509:385-388; Betz et al., J Am Chem Soc., 2013, 135:18637-18643; Lavergne et al., J Am Chem Soc. 2013, 135:5408-5419; and Malyshev et al. Proc Natl Acad Sci USA, 2012, 109:12005-12010; the disclosures of each of which are incorporated herein by reference. In some embodiments, the non-naturally occurring nucleotides comprise

.

[458] In some embodiments, non-natural nucleotides that can be used to produce the IL-10 conjugates disclosed herein can be derived from a compound of the formula,

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

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

[459] In some embodiments, each X is a carbon atom. In some embodiments, at least one X is a carbon atom. In some embodiments, one X is a carbon atom. In some embodiments, at least two X are carbon atoms. In some embodiments, two X are carbon atoms. In some embodiments, at least one X is a nitrogen atom. In some embodiments, one X is a nitrogen atom. In some embodiments, at least two X are nitrogen atoms. In some embodiments, two X are nitrogen atoms.

[460] In some embodiments, Y is a sulfur atom. In some embodiments, Y is an oxygen atom. In some embodiments, Y is a selenium atom. In some embodiments, Y is a secondary amine.

[461] In some embodiments, E is a sulfur atom. In some embodiments, E is an oxygen atom. In some embodiments, E is a selenium atom.

[462] In some embodiments, R ₂ is present when X is a carbon atom. In some embodiments, R ² is absent when X is a nitrogen atom. In some embodiments, each R ₂ , when present, is a hydrogen atom. In some embodiments, R ₂ is alkyl, such as methyl, ethyl, or propyl. In some embodiments, R ₂ is alkenyl, such as -CH ₂ =CH ₂ . In some embodiments, R ₂ is alkynyl, such as ethynyl. In some embodiments, R ₂ is methoxy. In some embodiments, R ₂ is methanethiol. In some embodiments, R ₂ is methaneseleno. In some embodiments, R ₂ is a halogen, such as chloro, bromo, or fluoro. In some embodiments, R ₂ is cyano. In some embodiments, R ₂ is azide.

[463] In some embodiments, E is sulfur, Y is sulfur, and each X is independently a carbon or nitrogen atom. In some embodiments, E is a sulfur atom, Y is a sulfur atom, and each X is a carbon atom.

[464] In some embodiments, non-natural nucleotides that can be used to produce the IL-10 conjugates disclosed herein can be derived from

In some embodiments, non-natural nucleotides that can be used to produce the IL-10 conjugates disclosed herein include , , , , , , , , , , And or their salts.

[465] In some embodiments, the unnatural base pair generates a unnatural amino acid as described in Dumas et al., “Designing logical codon reassignment - Expanding the chemistry in biology,” Chemical Science , 6 : 50-69 (2015), the disclosure of which is incorporated herein by reference.

[466] In some embodiments, a non-natural amino acid is introduced into a cytokine (e.g., an IL polypeptide) via a synthetic codon containing a non-natural nucleic acid. In some cases, a non-natural amino acid is introduced into a cytokine via an orthogonal modified synthetase/tRNA pair. Such orthogonal pairs comprise a non-natural synthetase that is capable of loading a non-natural tRNA with the non-natural amino acid while minimizing the loading of a) other endogenous amino acids onto the non-natural tRNA and b) non-natural amino acids onto other endogenous tRNAs. Such orthogonal pairs comprise tRNAs that are capable of carrying the non-natural synthetase while avoiding the loading of a) other endogenous amino acids via endogenous synthetases. In some embodiments, such pairs have been identified in various organisms, such as bacteria, yeast, archaea, or humans. In some embodiments, the orthogonal synthetase/tRNA pair comprises components from a single organism. In some embodiments, the orthogonal synthetase/tRNA pair comprises components from two different organisms. In some embodiments, the orthogonal synthetase/tRNA pair comprises components that, prior to modification, facilitate the translation of two different amino acids. In some embodiments, the orthogonal synthetase is a modified alanine synthetase. In some embodiments, the orthogonal synthetase is a modified arginine synthetase. In some embodiments, the orthogonal synthetase is a modified asparagine synthetase. In some embodiments, the orthogonal synthetase is a modified aspartic acid synthetase. In some embodiments, the orthogonal synthetase is a modified cysteine synthetase. In some embodiments, the orthogonal synthetase is a modified glutamine synthetase. In some embodiments, the orthogonal synthetase is a modified glutamic acid synthetase. In some embodiments, the orthogonal synthetase is a modified alanine-glycine. In some embodiments, the orthogonal synthetase is a modified histidine synthetase. In some embodiments, the orthogonal synthetase is a modified leucine synthetase. In some embodiments, the orthogonal synthetase is a modified isoleucine synthetase. In some embodiments, the orthogonal synthetase is a modified lysine synthetase. In some embodiments, the orthogonal synthetase is a modified methionine synthetase. In some embodiments, the orthogonal synthetase is a modified phenylalanine synthetase. In some embodiments, the orthogonal synthetase is a modified proline synthetase. In some embodiments, the orthogonal synthetase is a modified serine synthetase. In some embodiments, the orthogonal synthetase is a modified threonine synthetase. In some embodiments, the orthogonal synthetase is a modified tryptophan synthetase. In some embodiments, the orthogonal synthetase is a modified tyrosine synthetase. In some embodiments, the orthogonal synthetase is a modified valine synthetase. In some embodiments, the orthogonal synthetase is a modified phosphoserine synthetase. In some embodiments, the orthogonal tRNA is a modified alanine tRNA. In some embodiments, the orthogonal tRNA is a modified arginine tRNA. In some embodiments, the orthogonal tRNA is a modified asparagine tRNA. In some embodiments, the orthogonal tRNA is a modified aspartic acid tRNA. In some embodiments, the orthogonal tRNA is a modified cysteine tRNA. In some embodiments, the orthogonal tRNA is a modified glutamine tRNA. In some embodiments, the orthogonal tRNA is a modified glutamic acid tRNA. In some embodiments, the orthogonal tRNA is a modified alanine-glycine. In some embodiments, the orthogonal tRNA is a modified histidine tRNA. In some embodiments, the orthogonal tRNA is a modified leucine tRNA. In some embodiments, the orthogonal tRNA is a modified isoleucine tRNA. In some embodiments, the orthogonal tRNA is a modified lysine tRNA. In some embodiments, the orthogonal tRNA is a modified methionine tRNA. In some embodiments, the orthogonal tRNA is a modified phenylalanine tRNA. In some embodiments, the orthogonal tRNA is a modified proline tRNA. In some embodiments, the orthogonal tRNA is a modified serine tRNA. In some embodiments, the orthogonal tRNA is a modified threonine tRNA. In some embodiments, the orthogonal tRNA is a modified tryptophan tRNA. In some embodiments, the orthogonal tRNA is a modified tyrosine tRNA. In some embodiments, the orthogonal tRNA is a modified valine tRNA. In some embodiments, the orthogonal tRNA is a modified phosphoserine tRNA.

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

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

[469] In some cases, an unnatural amino acid is inserted into a cytokine (e.g., an IL polypeptide) via a pyrrolyl-tRNA pair. In some cases, the PylRS is derived from an archaebacterium, such as a methanogenic archaebacterium. In some cases, the PylRS is derived from Methanosarcina barkeri , Methanosarcina mazei , or Methanosarcina acetivorans . Illustrative UAAs that can be inserted via a pyrrolyl-tRNA pair include, but are not limited to, amide- and carbamate-substituted lysines such as 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid, N -ε- _D -prolyl- _L -lysine, and N -ε-cyclopentyloxycarbonyl _-L -lysine; N -ε-acryloyl- _L -lysine; N -ε-[(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl] _-L- lysine and N -ε-(1-methylcyclopro-2-ene-carboxamido)lysine. In some embodiments, the IL-10 conjugates disclosed herein can be produced by using M. mazei tRNA that is selectively loaded with a non-natural amino acid, such as N 6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), using M. barkeri pyrrolizyl-tRNA synthetase ( Mb PylRS). Other methods are known to those skilled in the art, such as those disclosed in Zhang et al., Nature 2017, 551(7682): 644-647, the disclosure of which is incorporated herein by reference.

[470] In some cases, a non-natural amino acid is incorporated into a cytokine described herein (e.g., an IL polypeptide) using a synthetase disclosed in US 9988619 and US 9938516, the disclosures of each of which are incorporated herein by reference.

[471] The host cell into which the constructs or vectors disclosed herein are introduced is cultured or maintained in a suitable medium such that tRNA, tRNA synthetase, and the protein of interest are produced. The medium also contains unnatural amino acid(s) such that the unnatural amino acid(s) is(are) incorporated into the protein of interest. In some embodiments, a nucleoside triphosphate transporter (NTT) from a bacterium, plant, or algae is also present in the host cell. In some embodiments, the IL-10 conjugates disclosed herein are produced using a host cell that expresses NTT. In some embodiments, the nucleotide nucleoside triphosphate transporter used in the host cell can be selected from TpNTT1, TpNTT2, TpNTT3, TpNTT4, TpNTT5, TpNTT6, TpNTT7, TpNTT8 (T. pseudonana), PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, PtNTT6 (P. tricornutum), GsNTT (Galdieria sulphuraria), AtNTT1, AtNTT2 (Arabidopsis thaliana), CtNTT1, CtNTT2 (Chlamydia trachomatis), PamNTT1, PamNTT2 (Protochlamydia amoebophila), CcNTT (Caedibacter caryophilus), RpNTT1 (Rickettsia prowazekii). In some embodiments, NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6. In some embodiments, NTT is PtNTT1. In some embodiments, NTT is PtNTT2. In some embodiments, NTT is PtNTT3. In some embodiments, NTT is PtNTT4. In some embodiments, NTT is PtNTT5. In some embodiments, NTT is PtNTT6. Other NTTs that may be used are disclosed in Zhang et al., Nature 2017, 551(7682): 644-647; Malyshev et al. Nature 2014 (509(7500)), 385-388; and Zhang et al. Proc Natl Acad Sci USA, 2017, 114:1317-1322; the disclosure of each of which is incorporated herein by reference.

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

[473] In some embodiments, methods for producing the IL-10 conjugates disclosed herein are provided in a cellular system that expresses NTT and a tRNA synthetase. In some embodiments described herein, NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6, and the tRNA synthetase is selected from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , and M. mazei . In some embodiments, NTT is PtNTT1 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei . In some embodiments, NTT is PtNTT2 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei . In some embodiments, NTT is PtNTT3 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei . In some embodiments, NTT is PtNTT3 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei . In some embodiments, NTT is PtNTT4 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei . In some embodiments, NTT is PtNTT5 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei . In some embodiments, NTT is PtNTT6 and the tRNA synthetase is derived from Methanococcus jannaschii , E. coli TyrRS ( Ec-Tyr )/ B. stearothermophilus , or M. mazei .

[474] In some embodiments, an NTT as used herein is an NTT that is truncated at the N-terminus, at the C-terminus, or at both the N- and C-terminus. In some embodiments, the truncated NTT is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to an untruncated NTT. In some cases, an NTT as used herein is PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, or PtNTT6. In some cases, a PtNTT as used herein is truncated at the N-terminus, at the C-terminus, or at both the N- and C-terminus. In some embodiments, the truncated PtNTT is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to the untruncated PtNTT. In some cases, the NTT used herein is a truncated PtNTT2, wherein the truncated PtNTT2 has an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identical to the amino acid sequence of the untruncated PtNTT2. An example of non-truncated PtNTT2 (NCBI accession number EEC49227.1, GI:217409295) has the amino acid sequence SEQ ID NO: 74:

1 mrpyptiali svflsaatri satsshqasa lpvkkgthvp

41 dspklsklyi maktksvsss fdpprggstv apttplatgg

81 alrkvrqavf piygnqevtk flligsikff iilaltltrd

121 tkdtlivtqc gaeaiaflki ygvlpaataf ialyskmsna

161 mgkkmlfyst cipfftffgl fdvfiypnae rlhpsleavq

201 ailpggaasg gmavlakiat hwtsalfyvm aeiyssvsvg

241 llfwqfandv vnvdqakrfy plfaqmsgla pvlagqyvvr

281 faskavnfea smhrltaavt fagimicify qlsssyvert

321 esakpaadne qsikpkkkkp kmsmvesgkf lassqylrli

361 amlvlgygls infteimwks lvkkqypdpl dyqrfmgnfs

401 savglstciv iffgvhvirl lgwkvgalat pgimailalp

441 ffacillgld sparleiavi fgtiqsllsk tskyalfdpt

481 tqmayipldd eskvkgkaai dvlgsrigks ggsliqqglv

521 fvfgniinaa pvvgvvyysv lvawmsaagr lsglfqaqte

561 mdkadkmeak tnkek

[475] In some embodiments, the IL-10 conjugates disclosed herein can be produced in a cell, such asE. colicontaining (a) a nucleotide triphosphate transporter PtNTT2 (including a truncated variant in which the first 65 amino acid residues of the full-length protein are deleted), (b) a plasmid containing a double-stranded oligonucleotide that encodes an IL-10 variant having the desired amino acid sequence and that contains a non-natural base pair comprising a first non-natural nucleotide and a second non-natural nucleotide to provide a codon at the desired position into which the non-natural amino acid will be inserted, such asN6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine or N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine, (c) a plasmid encoding tRNA, obtained fromM. mazei, and which contains a non-natural nucleotide to provide a recognizable anticodon (for the IL-10 variant codon) in place of its native sequence, and (d) a plasmid encoding the derivedM. barkeripyrrolizyl-tRNA synthetase (MbPylRS), which may be the same plasmid that encodes the tRNA or a different plasmid. Furthermore, in some embodiments, the cell is supplemented with deoxyribotriphosphates containing one or more unnatural bases. Furthermore, in some embodiments, the cell is supplemented with ribotriphosphates containing one or more unnatural bases. In some embodiments, the cell is supplemented with one or more unnatural amino acids, such asN6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-(propargylethoxy)-L-lysine (PraK), N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine or N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)- L-lysine or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, a double-stranded oligonucleotide that encodes the amino acid sequence of a desired IL-10 variant comprises an AXC codon, such as at position 67, 70, 74, 75, 79, 82, 88, 89, 99, 125, 126, 129, 130 or 132 a sequence that encodes a protein having SEQ ID NO: 1, wherein X is a non-natural nucleotide, such as those disclosed herein, such as NaM. In some embodiments, the cell further comprises a plasmid, which may be a protein expression plasmid or another plasmid that encodes the orthogonal tRNA gene fromM. mazei, which comprises a corresponding AXC anticodon GYT in place of its native sequence, wherein Y is a non-natural nucleotide disclosed herein, such as TPT3, that is complementary to and may be the same as or different from a non-natural nucleotide in the codon. In some embodiments, the non-natural nucleotide in the codon is different from the non-natural nucleotide and is complementary to the non-natural nucleotide in the anticodon. In some embodiments, the non-natural nucleotide in the codon is the same as the non-natural nucleotide in the anticodon. In some embodiments, the first and second non-natural nucleotides comprising a non-natural base pair in the double-stranded oligonucleotide can be derived from,,,, And . In some embodiments, the first and second non-natural nucleotides comprising a non-natural base pair in the double-stranded oligonucleotide can be obtained from And In some embodiments, the triphosphates of the first and second non-natural nucleotides comprise, And or salts thereof. In some embodiments, the triphosphates of the first and second non-natural nucleotides comprise And or salts thereof. In some embodiments, mRNA derived from a double-stranded oligonucleotide comprising a first non-natural nucleotide and a second non-natural nucleotide may comprise a codon comprising a non-natural nucleotide derived from, And . In some embodiments, the tRNA M. mazeimay contain an anticodon containing an unnatural nucleotide that recognizes a codon containing an unnatural nucleotide in mRNA. Anticodon in tRNAM. mazeimay contain an unnatural nucleotide derived from,,,, And . In some embodiments, the mRNA comprises a non-natural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from. In some embodiments, the tRNA comprises a non-natural nucleotide derived from. In some embodiments, the tRNA comprises a non-natural nucleotide derived from. In some embodiments, the tRNA comprises a non-natural nucleotide derived from. In some embodiments, the tRNA comprises a non-natural nucleotide derived from. In some embodiments, the tRNA comprises a non-natural nucleotide derived from. In some embodiments, the tRNA comprises a non-natural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from, and tRNA contains an unnatural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from, and tRNA contains an unnatural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from, and tRNA contains an unnatural nucleotide derived from. In some embodiments, the mRNA comprises a non-natural nucleotide derived from, and tRNA contains an unnatural nucleotide derived from. The host cell is cultured in a medium containing appropriate nutrients and supplemented with (a) deoxyribonucleoside triphosphates containing one or more unnatural bases that are necessary for the replication of the plasmid(s) encoding the cytokine gene carrying the codon, (b) ribonucleoside triphosphates containing one or more unnatural bases necessary for the transcription of (i) an mRNA corresponding to the cytokine coding sequence and containing a codon containing one or more unnatural bases, and (ii) a tRNA containing an anticodon containing one or more unnatural bases, and (c) unnatural amino acid(s) to be inserted into the polypeptide sequence of the cytokine of interest. The host cells are then maintained under conditions that allow expression of the protein of interest.

[476] The resulting protein comprising one or more unnatural amino acids, such as AzK, N6-(propargylethoxy)-L-lysine (PraK), N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine, which is expressed, can be purified by methods known to those skilled in the art and then reacted with an alkyne, such as DBCO, containing a PEG chain having the desired average molecular weight disclosed herein, under conditions known to those skilled in the art, to produce the IL-10 conjugates disclosed herein. Other methods are known to those skilled in the art, such as those disclosed in Zhang et al., Nature 2017, 551(7682): 644-647; WO 2015157555; WO 2015021432; WO 2016115168; WO 2017106767; WO 2017223528; WO 2019014262; WO 2019014267; WO 2019028419; and WO2019/028425, the disclosures of each of which are incorporated herein by reference.

[477] Alternatively, a cytokine polypeptide (e.g., IL-10) comprising unnatural amino acid(s) is produced by introducing into a host cell the nucleic acid constructs described herein comprising a tRNA and an aminoacyl-tRNA synthetase and comprising a nucleic acid sequence of interest with one or more orthogonal (stop) codons in frame. The host cell is cultured in a medium containing appropriate nutrients and supplemented with (a) deoxyribonucleoside triphosphates containing one or more unnatural bases required for replication of the plasmid(s) encoding the cytokine gene(s) carrying the novel codon and anticodon, (b) ribonucleoside triphosphates required for transcription of the mRNA corresponding to (i) the cytokine sequence containing the codon and (ii) the orthogonal tRNA containing the anticodon, and (c) unnatural amino acid(s). The host cells are then maintained under conditions that allow expression of the protein of interest. The unnatural amino acid(s) is(are) incorporated into the polypeptide chain in response to the unnatural codon. For example, one or more unnatural amino acids are incorporated into a cytokine polypeptide (e.g., IL-10). Alternatively, two or more unnatural amino acids may be inserted into a cytokine polypeptide (e.g., IL-10) at two or more sites on the protein.

[478] Once the cytokine (e.g., IL-10) with the incorporated non-natural amino acid(s) has been produced in the host cell, it can be recovered therefrom by a variety of techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption. The cytokine polypeptide (e.g., IL-10) can be purified by standard techniques known in the art, such as preparative ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, or any other suitable technique known to those skilled in the art.

[479] In some embodiments, the IL-10 conjugates disclosed herein can be produced in a cell, such asE. colicontaining (a) a nucleotide triphosphate transporter PtNTT2 (including a truncated variant in which the first 65 amino acid residues of the full-length protein are deleted), (b) a plasmid containing a double-stranded oligonucleotide that encodes an IL-10 variant having the desired amino acid sequence and that contains a non-natural base pair comprising a first non-natural nucleotide and a second non-natural nucleotide to ensure that there is a codon at the desired position into which the non-natural amino acid will be inserted, such asN6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), (c) plasmid encoding tRNA, obtained fromM. mazei, and which contains a non-natural nucleotide to provide a recognizable anticodon (for the IL-10 variant codon) in place of its native sequence, and (d) a plasmid encoding pyrrolizyl-tRNA synthetase derived fromM. barkeri(MbPylRS), which may be the same plasmid that encodes the tRNA or a different plasmid. Furthermore, in some embodiments, the cell is supplemented with deoxyribotriphosphates containing one or more unnatural bases. Furthermore, in some embodiments, the cell is supplemented with ribotriphosphates containing one or more unnatural bases. Furthermore, in some embodiments, the cells are supplemented with one or more unnatural amino acids, such asN6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, a double-stranded oligonucleotide that encodes an amino acid sequence of a desired IL-10 variant comprises an AXC codon, such as at position N82, K88, A89, K99, K125, N126, N129, or K130 of a sequence that encodes a protein having SEQ ID NO: 1, wherein X is a non-natural nucleotide.

[480] In some embodiments, the cell further comprises a plasmid, which may be a protein expression plasmid or another plasmid, that encodes an orthogonal tRNA gene from M. mazei that comprises an AXC-corresponding GYT anticodon in place of its native sequence, wherein Y is a non-natural nucleotide that is complementary to and may be the same as or different from a non-natural nucleotide in the codon. In some embodiments, the non-natural nucleotide in the codon is different from the non-natural nucleotide and is complementary to the non-natural nucleotide in the anticodon. In some embodiments, the non-natural nucleotide in the codon is the same as the non-natural nucleotide in the anticodon. In some embodiments, non-natural nucleotides comprising a non-natural base pair in a double-stranded oligonucleotide can be derived from

, , , , And . In some embodiments, the first and second non-natural nucleotides comprising a non-natural base pair in the double-stranded oligonucleotide can be obtained from And . In some embodiments, the first and second non-natural nucleotides comprising a non-natural base pair in the double-stranded oligonucleotide can be obtained from And In some embodiments, the triphosphates of the first and second non-natural nucleotides comprise , , , , And or salts thereof. In some embodiments, the triphosphates of the first and second non-natural nucleotides comprise And or salts thereof. In some embodiments, the triphosphates of the first and second non-natural nucleotides comprise And or salts thereof. In some embodiments, mRNA derived from a double-stranded oligonucleotide comprising a first non-natural nucleotide and a second non-natural nucleotide may comprise a codon comprising a non-natural nucleotide derived from , , , , And . In some embodiments, the M. mazei tRNA may comprise an anticodon comprising a non-natural nucleotide that recognizes a codon comprising a non-natural nucleotide of the mRNA. The anticodon in the M. mazei tRNA may comprise a non-natural nucleotide derived from , , , , And . In some embodiments, the mRNA comprises a non-natural nucleotide derived from . In some embodiments, the mRNA comprises a non-natural nucleotide derived from . In some embodiments, the mRNA comprises a non-natural nucleotide derived from . In some embodiments, the mRNA comprises a non-natural nucleotide derived from . In some embodiments, the mRNA comprises a non-natural nucleotide derived from . In some embodiments, the mRNA comprises a non-natural nucleotide derived from . In some embodiments, the tRNA comprises a non-natural nucleotide derived from . In some embodiments, the tRNA comprises a non-natural nucleotide derived from . In some embodiments, the tRNA comprises a non-natural nucleotide derived from . In some embodiments, the tRNA comprises a non-natural nucleotide derived from . In some embodiments, the tRNA comprises a non-natural nucleotide derived from . In some embodiments, the tRNA comprises a non-natural nucleotide derived from . In some embodiments, the mRNA comprises a non-natural nucleotide derived from , and tRNA contains an unnatural nucleotide derived from . In some embodiments, the mRNA comprises a non-natural nucleotide derived from , and tRNA contains an unnatural nucleotide derived from . In some embodiments, the mRNA comprises a non-natural nucleotide derived from , and tRNA contains an unnatural nucleotide derived from . In some embodiments, the mRNA comprises a non-natural nucleotide derived from , and tRNA contains an unnatural nucleotide derived from . The host cell is cultured in a medium containing appropriate nutrients and supplemented with (a) deoxyribonucleoside triphosphates containing one or more unnatural bases that are necessary for the replication of the plasmid(s) encoding the cytokine gene carrying the codon, (b) ribonucleoside triphosphates containing one or more unnatural bases necessary for the transcription of (i) an mRNA corresponding to the cytokine coding sequence and containing a codon containing one or more unnatural bases, and (ii) a tRNA containing an anticodon containing one or more unnatural bases, and (c) unnatural amino acid(s) to be inserted into the polypeptide sequence of the cytokine of interest. The host cells are then maintained under conditions that allow expression of the protein of interest.

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

Table 2. List of non-limiting examples of codons and anticodons containing X and Y. Codon (mRNA) Anticodon (tRNA) UUX YAA or XAA UGX YCA or XCA CGX YCG or XCG AGX YCU or XCU GAX YUC or XUC CAX YUG or XUG GXU AYC CXU AYG GXG CYC AXG CYU GXC GYC AXC GYU GXA UYC CXC GYG UXC GYA AUX YAU or XAU CUX XAG or YAG GUX XAC or YAC UAX XUA or YUA GGX XCC or YCC

[482] The resulting protein containing one or more unnatural amino acids, such as Azk, that is expressed can be purified by methods known to those skilled in the art and then reacted with an alkyne, such as DBCO, containing a PEG chain of the desired average molecular weight, as disclosed herein, under conditions known to those skilled in the art to produce the IL-10 conjugates disclosed herein. Other methods are known to those skilled in the art, such as those disclosed in Zhang et al., Nature 2017, 551(7682): 644-647; WO 2015157555; WO 2015021432; WO 2016115168; WO 2017106767; WO 2017223528; WO2019014262; WO2019014267; WO2019028419; and WO2019/028425; the disclosures of each of which are incorporated herein by reference.

[483] Alternatively, a cytokine polypeptide (e.g., IL-10) comprising unnatural amino acid(s) is produced by introducing into a host cell the nucleic acid constructs described herein comprising a tRNA and aminoacyl-tRNA synthetase and comprising a nucleic acid sequence of interest with one or more orthogonal (stop) codons in frame. The host cell is cultured in a medium containing appropriate nutrients and supplemented with (a) deoxyribonucleoside triphosphates containing one or more unnatural bases required for replication of the plasmid(s) encoding the cytokine gene(s) carrying the novel codon and anticodon, (b) ribonucleoside triphosphates required for transcription of the mRNA corresponding to (i) the cytokine sequence containing the codon and (ii) the orthogonal tRNA containing the anticodon, and (c) unnatural amino acid(s). The host cells are then maintained under conditions that allow expression of the protein of interest. The unnatural amino acid(s) is(are) incorporated into the polypeptide chain in response to the unnatural codon. For example, one or more unnatural amino acids are incorporated into a cytokine polypeptide (e.g., IL-10). Alternatively, two or more unnatural amino acids may be inserted into a cytokine polypeptide (e.g., IL-10) at two or more sites on the protein.

[484] Once the cytokine (e.g., IL-10) with the incorporated non-natural amino acid(s) has been produced in the host cell, it can be recovered from the host cell by various techniques known in the art, including enzymatic, chemical and/or osmotic lysis and physical disruption. The cytokine polypeptide (e.g., IL-10) can be purified by standard techniques known in the art, such as preparative ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, or any other suitable technique known to those skilled in the art.

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

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

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

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

Methods of application

[489] In some embodiments, described herein is a method of treating a cancer in a subject comprising administering to a subject in need thereof an effective amount of any of the modified IL-10 polypeptide or IL-10 conjugates described herein. In some cases, the cancer is a solid tumor or a hematological malignancy. In some cases, the solid tumor is a metastatic cancer. In some cases, the cancer is a relapsed or refractory cancer from previous treatment. In some embodiments, the cancer to be treated as described herein is selected from renal cell carcinoma, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, squamous cell carcinoma, pancreatic cancer, and prostate cancer. In some embodiments, the cancer to be treated as described herein is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin's lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability cancer, microsatellite stable cancer, microsatellite stable colorectal cancer, gastric cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), melanoma, small cell lung cancer (SCLC), esophageal cancer, glioblastoma, mesothelioma, breast cancer, triple negative breast cancer, prostate cancer, bladder cancer, ovarian cancer, tumors with moderate to low mutational burden, squamous cell cutaneous squamous cell carcinoma (CSCC), cutaneous squamous cell carcinoma (SCSC), tumors with low to absent PD-L1 expression, tumors systemically disseminating to the liver and CNS beyond their primary anatomical site of origin, and diffuse large B-cell lymphoma.

[490] In some embodiments, the cancer to be treated as described herein is a hematological malignancy. In some cases, the hematological malignancy comprises leukemia, lymphoma, or myeloma. In some embodiments, the hematological malignancy is a T-cell malignancy. In some embodiments, the hematological malignancy is a B-cell malignancy. In some embodiments, the hematological malignancy is a metastatic hematological malignancy. In some embodiments, the hematological malignancy is a relapsed hematological malignancy. In some embodiments, the hematological malignancy is a refractory hematological malignancy. In some cases, the cancer to be treated with any of the modified IL-10 polypeptides or IL-10 conjugates is a cancer selected from chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, high-grade B-cell non-Burkitt's lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-cell lymphoblastic lymphoma, prolymphocytic B-cell leukemia, lymphoplasmacytic lymphomas, splenic marginal zone lymphomas, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

Proliferative diseases or conditions

[491] In some embodiments, described herein is a method of treating a proliferative disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the modified IL-10 polypeptides or IL-10 conjugates described herein. In some embodiments, the proliferative disease or condition is cancer. In some cases, the cancer is a solid tumor. Exemplary solid tumors include, but are not limited to, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer. In some cases, the solid tumor is metastatic cancer. In some cases, the solid tumor is relapsed or refractory cancer from prior treatment.

[492] In some cases, any of the modified IL-10 polypeptides or IL-10 conjugates described herein is administered to a subject in need thereof to treat a solid tumor. In such cases, the subject has bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer (or renal cell carcinoma), lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer. In some cases, the IL-10 conjugate is administered to the subject to treat bladder cancer. In some cases, the IL-10 conjugate is administered to the subject to treat breast cancer. In some cases, the IL-10 conjugate is administered to the subject to treat colorectal cancer. In some cases, the IL-10 conjugate is administered to the subject to treat esophageal cancer. In some cases, the IL-10 conjugate is administered to a subject to treat head and neck cancer. In some cases, the IL-10 conjugate is administered to a subject to treat kidney cancer (or renal cell carcinoma or RCC). In some cases, the IL-10 conjugate is administered to a subject to treat lung cancer. In some cases, the IL-10 conjugate is administered to a subject to treat melanoma. In some cases, the IL-10 conjugate is administered to a subject to treat ovarian cancer. In some cases, the IL-10 conjugate is administered to a subject to treat pancreatic cancer. In some cases, the IL-10 conjugate is administered to a subject to treat prostate cancer. In some cases, the cancer is metastatic cancer. In other cases, the cancer is recurrent cancer. In additional cases, the cancer is refractory cancer.

[493] In some embodiments, the cancer is an untreated cancer. In such cases, the untreated cancer includes a cancer that has not been treated with a therapy. In some cases, the untreated cancer is a solid tumor, such as bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer (or RCC), lung cancer, melanoma, ovarian cancer, pancreatic cancer, or prostate cancer. In some embodiments, described herein is a method of treating an untreated solid tumor in a subject in need thereof, comprising administering to the subject an IL-10 conjugate described herein.

[494] In some embodiments, the cancer is a hematological malignancy. In some cases, an IL-10 conjugate described herein is administered to a subject in need thereof to treat a hematological malignancy. In some cases, the hematological malignancy comprises leukemia, lymphoma, or myeloma. In some cases, the hematological malignancy is a T-cell malignancy. In other cases, the hematological malignancy is a B-cell malignancy. In some cases, the hematological malignancy is a metastatic hematological malignancy. In other cases, the hematological malignancy is a relapsed hematological malignancy. In further cases, the hematological malignancy is a refractory hematological malignancy. In some cases, the subject has a T-cell malignancy. In some cases, the subject has a B-cell malignancy. In some cases, the subject has chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, high-grade non-Burkitt's B-cell lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-cell lymphoblastic lymphoma, prolymphocytic B-cell leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis. In some cases, the IL-10 conjugate is administered to a subject to treat CLL. In some cases, the IL-10 conjugate is administered to a subject to treat SLL. In some cases, the IL-10 conjugate is administered to a subject to treat FL. In some cases, the IL-10 conjugate is administered to a subject to treat DLBCL. In some cases, the IL-10 conjugate is administered to a subject to treat MCL. In some cases, the IL-10 conjugate is administered to a subject to treat Waldenstrom's macroglobulinemia. In some cases, the IL-10 conjugate is administered to a subject to treat multiple myeloma. In some cases, the IL-10 conjugate is administered to a subject to treat Burkitt's lymphoma.

Complementary Therapies

[495] In some embodiments, the subject is further administered an additional therapeutic agent. In some cases, the additional therapeutic agent is administered concurrently with the IL-10 conjugate and/or co-formulated. In other cases, the additional therapeutic agent and the IL-10 conjugate are administered sequentially, such as the IL-10 conjugate is administered prior to the additional therapeutic agent or the IL-10 conjugate is administered subsequent to the additional therapeutic agent. In some cases, the one or more additional agents are one or more immune checkpoint inhibitors selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, CTLA-4 inhibitors, OX40 agonists, and 4-1BB agonists. In some cases, the one or more immune checkpoint inhibitors are selected from PD-1 inhibitors. Illustrative PD-1 inhibitors include pembrolizumab, nivolumab, cemiplimab, lambrolizumab, AMP-224, sintilimab, toripalimab, camrelizumab, tislelizumab, dostarlimab (GSK), PDR001 (Novartis), MGA012 (Macrogenics/Incyte), GLS-010 (Arcus/Wuxi), AGEN2024 (Agenus), cetrelimab (Janssen), ABBV-181 (Abbvie), AMG-404 (Amgen), BI-754091 (Boehringer Ingelheim), CC-90006 (Celgene), JTX-4014 (Jounce), PF-06801591 (Pfizer), and genolimzumab (Apollomics/Genor BioPharma). In some cases, one or more immune checkpoint inhibitors are selected from PD-L1 inhibitors. Exemplary PD-L1 inhibitors include atezolizumab, avelumab, durvalumab, ASC22 (Alphamab/Ascletis), CX-072 (Cytomx), CS1001 (Cstone), cosibelimab (Checkpoint Therapeutics), INCB86550 (Incyte), and TG-1501 (TG Therapeutics). In some cases, one or more immune checkpoint inhibitors are selected from CTLA-4 inhibitors. In some embodiments, the CTLA-4 inhibitors are selected from tremelimumab, ipilimumab, and AGEN-1884 (Agenus). In some cases, one or more additional agents include folinic acid, 5-fluorouracil, and oxaliplatin for the treatment of pancreatic cancer and pancreatic ductal adenocarcinoma (PDAC).

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

[497] In some cases, the additional therapeutic agent comprises a first-line therapy. As used herein, the term "first-line therapy" includes the initial treatment of a subject with cancer. In some cases, the cancer is a primary or locally advanced cancer. In other cases, the cancer is a metastatic or recurrent cancer. In some cases, the first-line therapy comprises chemotherapy. In other cases, the first-line treatment comprises immunotherapy, targeted therapy, or radiation therapy. One skilled in the art will readily appreciate that different types of first-line treatment may be applicable to different types of cancer.

[498] In some cases, the IL-10 conjugate is administered with an additional therapeutic agent selected from an alkylating agent such as altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxalaplatin, temozolomide, or thiotepa; an antimetabolite such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, or pemetrexed; an anthracycline such as daunorubicin, doxorubicin, epirubicin, or idarubicin; a topoisomerase I inhibitor such as topotecan or irinotecan (CPT-11); a topoisomerase II inhibitor such as etoposide (VP-16), teniposide, or mitoxantrone; a mitotic inhibitor such as docetaxel, estramustine, ixabepilone, paclitaxel, vinblastine, vincristine, or vinorelbine; or a corticosteroid such as prednisone, methylprednisolone, or dexamethasone.

[499] In some cases, the IL-10 conjugate described herein is administered with an inhibitor of the enzyme poly-ADP-ribose polymerase (PARP). Illustrative PARP inhibitors include, but are not limited to, olaparib (AZD-2281, Lynparza®, from Astra Zeneca), rucaparib (PF-01367338, Rubraca®, from Clovis Oncology), niraparib (MK-4827, Zejula®, from Tesaro), talazoparib (BMN-673, from BioMarin Pharmaceutical Inc.), veliparib (ABT-888, from AbbVie), CK-102 (formerly CEP 9722, from Teva Pharmaceutical Industries Ltd.), E7016 (from Eisai), iniparib (BSI 201, from Sanofi), and pamiparib (BGB-290, from BeiGene). In some cases, the IL-10 conjugate is administered in combination with a PARP inhibitor such as olaparib, rucaparib, niraparib, talazoparib, veliparib, CK-102, E7016, iniparib, or pamiparib.

[500] In some embodiments, the IL-10 conjugate described herein is administered with a tyrosine kinase inhibitor (TKI). Exemplary TKIs include, but are not limited to, afatinib, alectinib, axitinib, bosutinib, cabozantinib, ceritinib, cobimetinib, crizotinib, dabrafenib, dasatinib, erlotinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, nilotinib, nintedanib, osimertinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib, tofacitinib, and vandetanib.

[501] In some cases, the IL-10 conjugate described herein is administered with an immune checkpoint inhibitor. Exemplary immune checkpoint inhibitors include PD-L1 inhibitors such as durvalumab (Imfinzi) from AstraZeneca, atezolizumab (MPDL3280A) from Genentech, avelumab from EMD Serono/Pfizer, CX-072 from CytomX Therapeutics, FAZ053 from Novartis Pharmaceuticals, KN035 from 3D Medicine/Alphamab, LY3300054 from Eli Lilly, or M7824 (an anti-PD-L1 antibody/TGF-beta trap) from EMD Serono; PD-L2 inhibitors such as GlaxoSmithKline's AMP-224 (amplimmune) and rHIgM12B7; PD-1 inhibitors such as nivolumab (Opdivo) from Bristol-Myers Squibb, pembrolizumab (Keytruda) from Merck, AGEN 2034 from Agenus, BGB-A317 from BeiGene, Bl-754091 from Boehringer-Ingelheim Pharmaceuticals, CBT-501 (genolimzumab) from CBT Pharmaceuticals, INCSHR1210 from Incyte, JNJ-63723283 from Janssen Research & Development, MEDI0680 from MedImmune, MGA 012 from MacroGenics, PDR001 from Novartis Pharmaceuticals, PF-06801591 from Pfizer, REGN2810 (SAR439684) from Regeneron Pharmaceuticals/Sanofi, or TSR-042 from TESARO; CTLA-4 inhibitors such as ipilimumab (also known as Yervoy®, MDX-010, BMS-734016, and MDX-101) from Bristol Meyers Squibb, tremelimumab (CP-675206, ticilimumab) from Pfizer, or AGEN 1884 from Agenus; LAG3 inhibitors such as BMS-986016 from Bristol-Myers Squibb, IMP701 from Novartis Pharmaceuticals, LAG525 from Novartis Pharmaceuticals, or REGN3767 from Regeneron Pharmaceuticals; B7-H3 inhibitors such as enoblitusumab (MGA271) from MacroGenics; KIR inhibitors such as lirilumab (IPH2101; BMS-986015) from Innate Pharma; CD137 inhibitors such as urelumab (BMS-663513, Bristol-Myers Squibb), PF-05082566 (anti-4-1BB antibody, PF-2566, Pfizer), or XmAb-5592 (Xencor); PS inhibitors such as bavituximab; and inhibitors such as an antibody or fragments thereof (e.g., a monoclonal antibody, human, humanized, or chimeric antibody), RNAi molecules, or small molecules against TIM3, CD52, CD30, CD20, CD33, CD27, OX40, GITR, ICOS, BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2, or SLAM.

[502] In some embodiments, the PD-1 inhibitor is pembrolizumab, nivolumab, or cemiplimab. In some embodiments, the PD-1 inhibitor is pembrolizumab. In some embodiments, the PD-1 inhibitor is nivolumab. In some embodiments, the PD-1 inhibitor is cemiplimab.

[503] In some embodiments, the PD-L1 inhibitor is atezolizumab. In some embodiments, the PD-L1 inhibitor is avelumab. In some embodiments, the PD-L1 inhibitor is durvalumab.

[504] In some embodiments, the CTLA-4 inhibitors are selected from tremelimumab, ipilimumab, and AGEN-1884 (Agenus). In some embodiments, the CTLA-4 inhibitor is tremelimumab. In some embodiments, the CTLA-4 inhibitor is ipilimumab. In some cases, the IL-10 conjugate is administered in combination with pembrolizumab, nivolumab, tremelimumab, or ipilimumab.

[505] In some cases, the IL-10 conjugate described herein is administered with an antibody such as alemtuzumab, trastuzumab, ibritumomab tiuxetan, brentuximab vedotin, ado-trastuzumab emtansine, or blinatumomab.

[506] In some cases, the IL-10 conjugate is administered with an additional therapeutic agent selected from an additional cytokine. In some cases, the additional cytokine enhances and/or synergizes with respect to the expansion and/or proliferation of effector T cells. In some cases, the additional cytokine comprises IL-1β, IL-2, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, or TNFα. In some cases, the additional cytokine is IL-7. In some cases, the additional cytokine is IL-15. In some cases, the additional cytokine is IL-21. In some cases, the additional cytokine is TNFα.

[507] In some cases, the IL-10 conjugate is administered with an additional therapeutic agent selected from a receptor agonist. In some cases, the receptor agonist comprises a Toll-like receptor (TLR) ligand. In some cases, the TLR ligand comprises TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9. In some cases, the TLR ligand comprises a synthetic ligand, such as, for example, Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, poly I:C, poly A:U, AGP, MPL A, RC-529, MDF2β, CFA, or flagellin. In some cases, the IL-10 conjugate is administered with one or more TLR agonists selected from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In some cases, the IL-10 conjugate is administered with one or more TLR agonists selected from Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, poly I:C, poly A:U, AGP, MPL A, RC-529, MDF2β, CFA, and flagellin.

[508] In some embodiments, the IL-10 conjugate is used in combination with adoptive T cell transfer (ACT) therapy. In one embodiment, ACT comprises identifying autologous T lymphocytes in a subject, e.g., with anti-tumor activity, expanding the autologous T lymphocytes in vitro , and then reinfusing the expanded T lymphocytes into the subject. In another embodiment, ACT comprises using allogeneic T lymphocytes, e.g., with anti-tumor activity, expanding the T lymphocytes in vitro, and then infusing the expanded allogeneic T lymphocytes into a subject in need thereof. In some cases, the IL-10 conjugate described herein is used in combination with autologous T lymphocytes as a component of ACT therapy. In other instances, the IL-10 conjugate described herein is used in combination with allogeneic T lymphocytes as a component of ACT therapy. In some instances, the IL-10 conjugate is administered concurrently with ACT therapy to a subject in need thereof. In other instances, the IL-10 conjugate is administered sequentially with ACT therapy to a subject in need thereof.

[509] In some embodiments, the IL-10 conjugate is used to ex vivo activate and/or expand autologous and/or allogeneic T cells upon transfer. In such cases, the IL-10 conjugate is used to activate and/or expand a sample containing autologous and/or allogeneic T cells, and the IL-10 conjugate is optionally removed from the sample prior to administration of the sample to a subject in need thereof.

[510] In some embodiments, the IL-10 conjugate is administered with a vaccine. In some cases, the IL-10 conjugate is used in combination with an oncolytic virus. In such cases, the IL-10 conjugate acts as a stimulatory agent to modulate the immune response. In some cases, the IL-10 conjugate is used with an oncolytic virus as a component of adjuvant therapy. Exemplary oncolytic viruses include T-Vec (Amgen), G47Δ (Todo et al.), JX-594 (Sillajen), CG0070 (Cold Genesys), and reolysin (Oncolytics Biotech). In some cases, the IL-10 conjugate is used in combination with an oncolytic virus, such as T-Vec, G47Δ, JX-594, CG0070, or reolysin.

[511] In some embodiments, the IL-10 conjugate is administered in combination with radiation therapy.

Treatment methods for other diseases

[512] In some embodiments, described herein is a method of treating a fibrotic disorder in a subject by administering any of the modified IL-10 polypeptides or IL-10 conjugates described herein. In some cases, the fibrotic disorder may include liver fibrosis, idiopathic pulmonary fibrosis, and periportal fibrosis. In some embodiments, described herein is a method of treating non-alcoholic steatohepatitis (NASH) in a subject by administering any of the modified IL-10 polypeptides or IL-10 conjugates described herein. In some embodiments, described herein is a method of treating non-alcoholic fatty liver disease (NAFLD) in a subject by administering any of the modified IL-10 polypeptides or IL-10 conjugates described herein.

Pharmaceutical compositions and formulations

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

[514] In some embodiments, pharmaceutical formulations include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, liposomal dispersions, aerosols, immediate release formulations, controlled release formulations, delayed release formulations, extended release formulations, pulsatile release formulations, and mixed immediate and controlled release formulations.

[515] In some embodiments, the pharmaceutical formulations include a carrier or carrier materials selected based on compatibility with the composition disclosed herein and the release profile characteristics of the desired dosage form. See, e.g., Remington: The Science and Practice of Pharmacy , Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995), Hoover, John E., Remington 's Pharmaceutical Sciences , Mack Publishing Co., Easton, Pennsylvania 1975, Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms , Marcel Decker, New York, NY, 1980, and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999); the disclosures of each of which are incorporated herein by reference.

[516] In some cases, the pharmaceutical composition is formulated as an immunoliposome that comprises a plurality of IL-10 conjugates directly or indirectly bound to a lipid bilayer of the liposomes. Exemplary lipids include, but are not limited to, fatty acids; phospholipids; sterols such as cholesterols; sphingolipids such as sphingomyelin; glycosphingolipids such as gangliosides, globosides, and cerebrosides; surfactant amines such as stearyl, oleyl, and linoleylamines. In some cases, the lipid comprises a cationic lipid. In some cases, the lipid comprises a phospholipid. Illustrative phospholipids include, but are not limited to, phosphatidic acid ("PA"), phosphatidylcholine ("PC"), phosphatidylglycerol ("PG"), phosphatidylethanolamine ("PE"), phosphatidylinositol ("PI"), and phosphatidylserine ("PS"), sphingomyelin (including brain sphingomyelin), lecithin, lysolecithin, lysophosphatidylethanolamine, cerebrosides, diarachidoylphosphatidylcholine ("DAPC"), didecanoyl-L-alpha-phosphatidylcholine ("DDPC"), dielaidoylphosphatidylcholine ("DEPC"), dilauroylphosphatidylcholine ("DLPC"), dilinoleoylphosphatidylcholine, dimyristoylphosphatidylcholine ("DMPC"), dioleoylphosphatidylcholine ("DOPC"), dipalmitoylphosphatidylcholine ("DPPC"), Distearoylphosphatidylcholine ("DSPC"), 1-palmitoyl-2-oleoylphosphatidylcholine ("POPC"), diarachidoylphosphatidylglycerol ("DAPG"), didecanoyl-L-alpha-phosphatidylglycerol ("DDPG"), dielaidoylphosphatidylglycerol ("DEPG"), dilauroylphosphatidylglycerol ("DLPG"), dilinoleoylphosphatidylglycerol, dimyristoylphosphatidylglycerol ("DMPG"), dioleoylphosphatidylglycerol ("DOPG"), dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol ("DSPG"), 1-palmitoyl-2-oleoylphosphatidylglycerol ("POPG"), diarachidoylphosphatidylethanolamine ("DAPE"), didecanoyl-L-alpha-phosphatidylethanolamine ("DDPE"), dielaidoylphosphatidylethanolamine ("DEPE"), dilauroylphosphatidylethanolamine ("DLPE"), dilinoleoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine ("DMPE"), dioleoylphosphatidylethanolamine ("DOPE"), dipalmitoylphosphatidylethanolamine ("DPPE"), distearoylphosphatidylethanolamine ("DSPE"), 1-palmitoyl-2-oleoylphosphatidylethanolamine ("POPE"), diarachidoylphosphatidylinositol ("DAPI"), didecanoyl-L-alpha-phosphatidylinositol ("DDPI"), dielaidoylphosphatidylinositol ("DEPI"), dilauroylphosphatidylinositol ("DLPI"), dilinoleoylphosphatidylinositol, dimyristoylphosphatidylinositol ("DMPI"), dioleoylphosphatidylinositol ("DOPI"), Dipalmitoylphosphatidylinositol ("DPPI"), Distearoylphosphatidylinositol ("DSPI"), 1-Palmitoyl-2-oleoylphosphatidylinositol ("POPI"), Diarachidoylphosphatidylserine ("DAPS"), Didecanoyl-L-alpha-phosphatidylserine ("DDPS"), Dielaidoylphosphatidylserine ("DEPS"), Dilauroylphosphatidylserine ("DLPS"), Dilinoleoylphosphatidylserine, Dimyristoylphosphatidylserine ("DMPS"), Dioleoylphosphatidylserine ("DOPS"), Dipalmitoylphosphatidylserine ("DPPS"), Distearoylphosphatidylserine ("DSPS"), 1-Palmitoyl-2-oleoylphosphatidylserine ("POPS"), Diarachidoylsphingomyelin, Didecanoylsphingomyelin, dielaidoylsphingomyelin, dilauroylsphingomyelin, dilinoleoylsphingomyelin, dimyristoylsphingomyelin, sphingomyelin, dioleoylsphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin and 1-palmitoyl-2-oleoylsphingomyelin.

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

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

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

[520] In some cases, pharmaceutical formulations further include a diluent, which is used to stabilize the compounds because they are capable of providing a more stable environment. Salts dissolved in buffer solutions (which are also capable of providing pH control or maintenance) are used as diluents in the art, including, but not limited to, phosphate buffered sodium chloride solution.

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

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

Therapeutic regimens

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

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

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

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

[527] In some embodiments, the methods comprise administering a dose of an IL-10 conjugate to a subject in need thereof at a dose in the range of 1 μg of IL-10 conjugate per kg of subject body weight to about 200 μg of IL-10 conjugate per kg of subject body weight, or from about 2 μg of IL-10 conjugate per kg of subject body weight to about 200 μg of IL-10 conjugate per kg of subject body weight, or from about 4 μg of IL-10 conjugate per kg of subject body weight to about 200 μg of IL-10 conjugate per kg of subject body weight, or from about 6 μg of IL-10 conjugate per kg of subject body weight to about 200 μg of IL-10 conjugate per kg of subject body weight, or from about 8 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 10 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 12 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 14 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 16 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 18 μg of IL-10 conjugate per kg of subject body weight to about 200 μg of IL-10 conjugate per kg of subject body weight, or from about 20 μg of IL-10 conjugate per kg of subject body weight to about 200 μg of IL-10 conjugate per kg of subject body weight, or from about 22 μg of IL-10 conjugate per kg of subject body weight to about 200 μg of IL-10 conjugate per kg of subject body weight, or from about 24 μg of IL-10 conjugate per kg of subject body weight to about 200 μg of IL-10 conjugate per kg of subject body weight, or from about 26 μg of IL-10 conjugate per kg of subject body weight to about 200 μg of IL-10 per kg subject body weight, or from about 28 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 32 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 34 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 36 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 40 μg IL-10 conjugate per kg subject body weight to about 200 μg of IL-10 conjugate per kg of subject body weight, or from about 45 μg of IL-10 conjugate per kg of subject body weight to about 200 μg of IL-10 conjugate per kg of subject body weight, or from about 50 μg of IL-10 conjugate per kg of subject body weight to about 200 μg of IL-10 conjugate per kg of subject body weight, or from about 55 μg of IL-10 conjugate per kg of subject body weight to about 200 μg of IL-10 conjugate per kg of subject body weight, or from about 60 μg of IL-10 conjugate per kg of subject body weight to about 200 μg of IL-10 conjugate per kg of subject body weight, or from about 65 μg of conjugate IL-10 per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 70 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 75 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 80 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 85 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 90 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 95 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 100 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 110 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 120 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 per kg subject body weight, or from about 130 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 140 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 150 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 160 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 170 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 180 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight, or from about 190 μg IL-10 conjugate per kg subject body weight to about 200 μg IL-10 conjugate per kg subject body weight. The above ranges are only suggestive since the number of variables with respect to an individual treatment regimen is large and significant deviations from these recommended values are not uncommon. Such dosages will vary depending on a number of variables, including, but not limited to, the activity of the compound being administered, the disease or condition being treated, the route of administration, the needs of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

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

[529] Once the patient's condition has improved, a maintenance dose is administered as needed. Thereafter, the dose or frequency of administration, or both, may be reduced as symptoms dictate, to a level that maintains improvement in the disease, disorder, or condition.

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

[531] In some embodiments, the dose may be determined at least in part by the incidence or severity of grade 3 or 4 adverse events in the subject. Non-limiting examples of adverse events include hypothermia; shock; bradycardia; ventricular extrasystole; myocardial ischemia; syncope; hemorrhage; atrial arrhythmia; phlebitis; second degree atrioventricular block; endocarditis; pericardial effusion; peripheral gangrene; thrombosis; coronary artery disorder; stomatitis; nausea and vomiting; liver function tests abnormal; gastrointestinal hemorrhage; hematemesis; hemorrhagic diarrhea; gastrointestinal disorder; intestinal perforation; pancreatitis; anemia; leukopenia; leukocytosis; hypocalcemia; alkaline phosphatase increased; increased blood urea nitrogen (BUN); hyperuricemia; increased non-protein nitrogen (NPN); respiratory acidosis; somnolence; agitation; neuropathy; paranoid reaction; seizures; grand mal seizure; delirium; asthma, pulmonary edema; hyperventilation; hypoxia; hemoptysis; hypoventilation; pneumothorax; mydriasis; abnormal pupillary function; impaired renal function; renal failure; acute tubular necrosis; duodenal ulcer; intestinal necrosis; myocarditis; supraventricular tachycardia; permanent or transient blindness due to optic neuritis; transient ischemic attacks; meningitis; cerebral edema; pericarditis; allergic interstitial nephritis; tracheoesophageal fistula; malignant hyperthermia; cardiac arrest; myocardial infarction; pulmonary embolism; stroke; liver or kidney failure; severe depression leading to suicide; pulmonary edema; respiratory arrest; respiratory failure; leukopenia, thrombocytopenia, alanine aminotransferase (ALT) increased, anorexia, arthralgia, back pain, chills, diarrhea, dyslipidemia, fatigue, fever, influenza-like symptoms, hypoalbuminemia, lipase increased, injection site reaction, myalgia, nausea, night sweats, pruritis, rash, rash erythematous, rash maculopapular, transaminitis, vomiting, and weakness.

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

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

Sets/product

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

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

[536] For example, the container(s) comprises one or more IL-10 polypeptides or conjugates disclosed herein, and optionally one or more pharmaceutical excipients described herein to facilitate delivery of the IL-10 polypeptides or conjugates. Such kits further optionally include an identifying description or label, or instructions relating to their use in the methods described herein.

[537] The kit typically includes labels listing the contents and/or instructions for use, and package inserts containing instructions for use. An instruction set is also typically included.

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

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

[540] In some embodiments, the kits comprise articles of manufacture that are useful for developing adoptive cell therapy agents. In some embodiments, the kits comprise one or more of the cytokine polypeptides (e.g., IL-10) or cytokine conjugates (e.g., IL-10) disclosed herein, and optionally one or more pharmaceutical excipients described herein to facilitate delivery of the cytokine polypeptides (e.g., IL-10) or cytokine conjugates (e.g., IL-10). Such kits may optionally include one or more excipients comprising inducers of tumor infiltrating lymphocytes (TILs), T cells, B cells, natural killer cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, basophils, or CD4+ or CD8+ T cells. Such kits further optionally include an identifying description or label, or instructions relating to their use in the methods described herein. In some embodiments, the kits comprise one or more polynucleic acid sequences encoding the IL-10 conjugates disclosed herein, inducers of tumor infiltrating lymphocytes (TILs), T cells, B cells, natural killer cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, basophils, or CD4+ or CD8+ T cells and/or a pharmaceutical composition thereof.

[541] In some embodiments, the kits and articles of manufacture described herein comprise a modified IL-10 polypeptide comprising at least one unnatural amino acid. In some embodiments, the at least one unnatural amino acid is a lysine analog, comprises an aromatic side chain, comprises an azido group, comprises an alkyne group, or comprises an aldehyde or ketone group. In some embodiments, the at least one unnatural amino acid does not comprise an aromatic side chain. In some embodiments, the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), N6-((propargyloxy)-carbonyl)-L-lysine (PraK), BCN-L-lysine, norbornenelysine, TCO-lysine, methyltetrazinelysine, allyloxycarbonyllysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxononanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, meta-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargylphenylalanine, 3-methylphenylalanine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic acid, selenocysteine, N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid comprises N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK) or N6-((propargyloxy)-carbonyl)-L-lysine (PraK). In some embodiments, the at least one unnatural amino acid is N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, the at least one unnatural amino acid is N6-((propargyloxy)-carbonyl)-L-lysine (PraK).

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

[543] In some embodiments, the modified IL-10 polypeptide provides a conjugation moiety. In some embodiments, the conjugation moiety provides a water-soluble polymer, a lipid, a protein, and/or a peptide. In some embodiments, the water-soluble polymer provides polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefin alcohol), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acid), poly(vinyl alcohol), polyphosphazene, polyoxazolines (POZ), poly(N-acryloylmorpholine), or a combination thereof. In some embodiments, the water-soluble polymer provides a PEG molecule

[544] In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-0 polypeptide. In some cases, the conjugation moiety provides a PEG molecule that is associated with a longer in vivo plasma half-life of the modified IL-10 polypeptide than the in vivo plasma half-life of a PEG that is smaller in size than the conjugation moiety. In some cases, the conjugation moiety provides a PEG molecule that is associated with a shorter in vivo plasma half-life of the modified IL-10 polypeptide than the in vivo plasma half-life of a PEG that is larger in size than the conjugation moiety.

[545] In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the efficiency of receptor signaling to the IL-10R signaling pathway. In some embodiments, the molecular weight of the PEG does not affect or has minimal effect on the desired reduced IL-10R binding or maintained IL-10R binding, wherein the reduced IL-10R binding is compared to the binding of wild-type IL-10 protein and IL-10R.

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

[547] For example, the container(s) comprise one or more modified IL-10 polypeptides comprising a mutated amino acid residue E67, Q70, E74, E75, Q79, N82, K88, A89, K99, K125, N126, N129, K130, or Q132 at residue positions corresponding to 67, 70, 74, 75, 79, 82, 88, 89, 99, 125, 126, 129, 130, and 132 set forth in SEQ ID NO: 1. In some embodiments, the modified IL-10 polypeptide comprises a conjugation moiety comprising PEG having a molecular weight of about 2,000-50,000 Da. In some embodiments, the molecular weight is 5,000 Da. In some embodiments, the molecular weight is 10,000 Da. In some embodiments, the molecular weight is 15,000 Da. In some embodiments, the molecular weight is 20,000 Da. In some embodiments, the molecular weight is 25,000 Da. In some embodiments, the molecular weight is 30,000 Da. In some embodiments, the molecular weight is 35,000 Da. In some embodiments, the molecular weight is 40,000 Da. In some embodiments, the molecular weight is 45,000 Da. In some embodiments, the molecular weight is 50,000 Da. In some embodiments, the molecular weight of the PEG determines, at least in part, the in vivo plasma half-life of the modified IL-10 polypeptide. In some cases, PEG is associated with a longer in vivo plasma half-life of the modified IL-10 polypeptide compared to the in vivo plasma half-life of a smaller PEG. In some cases, PEG is associated with a shorter in vivo plasma half-life of the modified IL-10 polypeptide compared to the in vivo plasma half-life of a larger PEG. In some embodiments, the molecular weight of the PEG does not affect or has a minimal effect on the efficiency of receptor signaling from the modified IL-10 polypeptide to the IL-10R signaling pathway. In some embodiments, the molecular weight of the PEG does not affect or has a minimal effect on the desired reduced binding of the modified IL-10 polypeptide to the IL-10R or maintained binding to the IL-10R, wherein the reduced binding to the IL-10R is compared to binding of the wild-type IL-10 protein to the IL-10R.

Illustrative embodiments

[548] The present invention is further described by means of the following embodiments. The features of each embodiment are combined with any of the other embodiments where appropriate and practicable.

[549] Embodiment 1. An IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced by a structure of formula (I),

Where

Z is CH ₂ and Y is ;

Y is CH ₂ , and Z is ;

Z is CH ₂ and Y is ; or

Y is CH ₂ , and Z is ;

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

X has the structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[550] Embodiment 1.1. An IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced by a structure of formula (I),

Where

Z is CH ₂ and Y is ;

Y is CH ₂ , and Z is ;

Z is CH ₂ and Y is ; or

Y is CH ₂ , and Z is ;

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

q is equal to 1, 2, or 3; and

X has the structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[551] Embodiment 2. The IL-10 conjugate of embodiment 1 or 1.1, wherein Z is CH ₂ , and Y is , or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[552] Embodiment 3. The IL-10 conjugate of embodiment 1 or 1.1, wherein Y is CH ₂ , and Z is , or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[553] Embodiment 4. The IL-10 conjugate of Embodiment 1, wherein Z is CH ₂ and Y is , or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[554] Embodiment 5. The IL-10 conjugate of embodiment 1 or 1.1, wherein Y is CH ₂ , and Z is , or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[555] Embodiment 6. The IL-10 conjugate of embodiment 1 or 1.1, wherein the PEG group has an average molecular weight selected from 5 kDa, 10 kDa, 20 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[556] Embodiment 7. The IL-10 conjugate of embodiment 6, wherein the PEG group has an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[557] Embodiment 8. The IL-10 conjugate of embodiment 6, wherein the PEG group has an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[558] Embodiment 9. The IL-10 conjugate of embodiment 1 or 1.1, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is selected from N82, K88, A89, K99, K125, N126, N129, and K130, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is at the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[559] Embodiment 10. The IL-10 conjugate of embodiment 9, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is selected from N82, K88, K99, N126, N129, and K130, wherein the position of the structure of formula (I) in the amino acid sequence of the IL-10 conjugate is at the positions in SEQ ID NO: 1, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[560] Embodiment 11. An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 19-26, wherein [AzK_PEG] has a structure of formula (II), formula (III), or is a mixture of formula (II) and formula (III),

Where

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

X has a structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[561] Embodiment 11.1: An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 19-26, wherein [AzK_PEG] has a structure of formula (II), formula (III), or is a mixture of formula (II) and formula (III),

Where

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

q is equal to 1, 2, or 3; and

X has the structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[562] Embodiment 12. The IL-10 conjugate of embodiment 11 or 11.1, wherein [AzK_PEG] is a mixture of formula (II) and formula (III), or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[563] Embodiment 13. The IL-10 conjugate of embodiment 11 or 11.1, wherein [AzK_PEG] has the structure of formula (II),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[564] Embodiment 14. The IL-10 conjugate of embodiment 13, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 19, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[565] Embodiment 15. The IL-10 conjugate of embodiment 14, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

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

[567] Embodiment 17. The IL-10 conjugate of embodiment 16, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[568] Embodiment 18. The IL-10 conjugate of embodiment 16, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[569] Embodiment 19. The IL-10 conjugate of embodiment 13, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 20, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[570] Embodiment 20. The IL-10 conjugate of embodiment 19, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

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

[572] Embodiment 22. The IL-10 conjugate of embodiment 21, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[573] Embodiment 23. The IL-10 conjugate of embodiment 21, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[574] Embodiment 24. The IL-10 conjugate of embodiment 13, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 21, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[575] Embodiment 25. The IL-10 conjugate of embodiment 24, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

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

[577] Embodiment 27. The IL-10 conjugate of embodiment 26, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[578] Embodiment 28. The IL-10 conjugate of embodiment 26, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[579] Embodiment 29. The IL-10 conjugate of embodiment 13, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 22, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[580] Embodiment 30. The IL-10 conjugate of embodiment 29, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

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

[582] Embodiment 32. The IL-10 conjugate of embodiment 31, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[583] Embodiment 33. The IL-10 conjugate of embodiment 31, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[584] Embodiment 34. The IL-10 conjugate of embodiment 13, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 23, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[585] Embodiment 35. The IL-10 conjugate of embodiment 34, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[586] Embodiment 36. The IL-10 conjugate of embodiment 35, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[587] Embodiment 37. The IL-10 conjugate of embodiment 36, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[588] Embodiment 38. The IL-10 conjugate of embodiment 36, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[589] Embodiment 39. The IL-10 conjugate of embodiment 13, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 24, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[590] Embodiment 40. The IL-10 conjugate of embodiment 39, wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[591] Embodiment 41. The IL-10 conjugate of embodiment 40, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[592] Embodiment 42. The IL-10 conjugate of embodiment 41, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[593] Embodiment 43. The IL-10 conjugate of embodiment 41, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[594] Embodiment 44. The IL-10 conjugate of embodiment 13, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 25, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[595] Embodiment 45. The IL-10 conjugate of embodiment 44, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[596] Embodiment 46. The IL-10 conjugate of embodiment 45, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[597] Embodiment 47. The IL-10 conjugate of embodiment 46, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[598] Embodiment 48. The IL-10 conjugate of embodiment 46, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[599] Embodiment 49. The IL-10 conjugate of embodiment 13, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 26, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[600] Embodiment 50. The IL-10 conjugate of embodiment 49, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[601] Embodiment 51. The IL-10 conjugate of embodiment 50, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[602] Embodiment 52. The IL-10 conjugate of embodiment 51, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[603] Embodiment 53. The IL-10 conjugate of embodiment 51, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[604] Embodiment 54. The IL-10 conjugate of embodiment 11 or 11.1, wherein [AzK_PEG] has the structure of formula (III),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[605] Embodiment 55. The IL-10 conjugate of embodiment 54, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 19, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[606] Embodiment 56. The IL-10 conjugate of embodiment 55, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[607] Embodiment 57. The IL-10 conjugate of embodiment 56, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[608] Embodiment 58. The IL-10 conjugate of embodiment 57, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[609] Embodiment 59. The IL-10 conjugate of embodiment 57, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[610] Embodiment 60. The IL-10 conjugate of embodiment 54, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 20, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[611] Embodiment 61. The IL-10 conjugate of embodiment 60, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[612] Embodiment 62. The IL-10 conjugate of embodiment 61, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[613] Embodiment 63. The IL-10 conjugate of embodiment 62, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[614] Embodiment 64. The IL-10 conjugate of embodiment 62, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[615] Embodiment 65. The IL-10 conjugate of embodiment 54, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 21, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[616] Embodiment 66. The IL-10 conjugate of embodiment 65, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[617] Embodiment 67. The IL-10 conjugate of embodiment 66, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[618] Embodiment 68. The IL-10 conjugate of embodiment 67, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[619] Embodiment 69. The IL-10 conjugate of embodiment 67, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[620] Embodiment 70. The IL-10 conjugate of embodiment 54, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 22, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[621] Embodiment 71. The IL-10 conjugate of embodiment 70, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[622] Embodiment 72. The IL-10 conjugate of embodiment 71, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[623] Embodiment 73. The IL-10 conjugate of embodiment 72, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[624] Embodiment 74. The IL-10 conjugate of embodiment 72, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[625] Embodiment 75. The IL-10 conjugate of embodiment 54, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 23, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[626] Embodiment 76. The IL-10 conjugate of embodiment 75, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[627] Embodiment 77. The IL-10 conjugate of embodiment 76, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[628] Embodiment 78. The IL-10 conjugate of embodiment 77, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[629] Embodiment 79. The IL-10 conjugate of embodiment 77, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[630] Embodiment 80. The IL-10 conjugate of embodiment 54, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 24, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[631] Embodiment 81. The IL-10 conjugate of embodiment 80, wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[632] Embodiment 82. The IL-10 conjugate of embodiment 81, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[633] Embodiment 83. The IL-10 conjugate of embodiment 82, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[634] Embodiment 84. The IL-10 conjugate of embodiment 82, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[635] Embodiment 85. The IL-10 conjugate of embodiment 54, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 25, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[636] Embodiment 86. The IL-10 conjugate of embodiment 85, wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[637] Embodiment 87. The IL-10 conjugate of embodiment 86, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[638] Embodiment 88. The IL-10 conjugate of embodiment 87, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[639] Embodiment 89. The IL-10 conjugate of embodiment 87, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[640] Embodiment 90. The IL-10 conjugate of embodiment 54, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 26, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[641] Embodiment 91. The IL-10 conjugate of embodiment 90, wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[642] Embodiment 92. The IL-10 conjugate of embodiment 91, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[643] Embodiment 93. The IL-10 conjugate of embodiment 92, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[644] Embodiment 94. The IL-10 conjugate of embodiment 92, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[645] Embodiment 95. The IL-10 conjugate of any one of embodiments 1-94, wherein W is a linear or branched PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[646] Embodiment 96. The IL-10 conjugate of any one of embodiments 1-94, wherein W is a linear PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[647] Embodiment 97. The IL-10 conjugate of any one of embodiments 1-94, wherein W is a branched PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[648] Embodiment 98. The IL-10 conjugate of any one of embodiments 1-94, wherein W is a methoxy-PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[649] Embodiment 99. The IL-10 conjugate of embodiment 98, wherein the methoxy-PEG group is linear or branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[650] Embodiment 100. The IL-10 conjugate of embodiment 98, wherein the methoxy-PEG group is linear, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[651] Embodiment 101. The IL-10 conjugate of embodiment 98, wherein the methoxy-PEG group is branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[652] Embodiment 102. An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 27-34, wherein [AzK_PEG20kDa] has a structure of formula (II), formula (III), or is a mixture of formula (II) and formula (III),

Where

W is a PEG group having an average molecular weight of 20 kDa; and

X has the structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[653] Embodiment 102.1: An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 27-34, wherein [AzK_PEG20kDa] has a structure of formula (II), formula (III), or is a mixture of formula (II) and formula (III),

Where

W is a PEG group with an average molecular weight of 20 kDa;

q is equal to 1, 2, or 3; and

X has the structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[654] Embodiment 103. The IL-10 conjugate of embodiment 102 or 102.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 27, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[655] Embodiment 104. The IL-10 conjugate of embodiment 102 or 102.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 28, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[656] Embodiment 105. The IL-10 conjugate of embodiment 102 or 102.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 29, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[657] Embodiment 106. The IL-10 conjugate of embodiment 102 or 102.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 30, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[658] Embodiment 107. The IL-10 conjugate of embodiment 102 or 102.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 31, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[659] Embodiment 108. The IL-10 conjugate of embodiment 102 or 102.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 32, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[660] Embodiment 109. The IL-10 conjugate of embodiment 102 or 102.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 33, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[661] Embodiment 110. The IL-10 conjugate of embodiment 102 or 102.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 34, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[662] Embodiment 111. The IL-10 conjugate of embodiment 102 or 102.1, wherein [AzK_PEG20kDa] has the structure of formula (II),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[663] Embodiment 112. The IL-10 conjugate of embodiment 111, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 27, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[664] Embodiment 113. The IL-10 conjugate of embodiment 111, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 28, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[665] Embodiment 114. The IL-10 conjugate of embodiment 111, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 29, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[666] Embodiment 115. The IL-10 conjugate of embodiment 111, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 30, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[667] Embodiment 116. The IL-10 conjugate of embodiment 111, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 31, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[668] Embodiment 117. The IL-10 conjugate of embodiment 111, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 32, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[669] Embodiment 118. The IL-10 conjugate of embodiment 111, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 33, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[670] Embodiment 119. The IL-10 conjugate of embodiment 111, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 34, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[671] Embodiment 120. The IL-10 conjugate of embodiment 102 or 102.1, wherein [AzK_PEG20kDa] has the structure of formula (III),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[672] Embodiment 121. The IL-10 conjugate of embodiment 120, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 27, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[673] Embodiment 122. The IL-10 conjugate of embodiment 120, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 28, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[674] Embodiment 123. The IL-10 conjugate of embodiment 120, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 29, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[675] Embodiment 124. The IL-10 conjugate of embodiment 120, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 30, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[676] Embodiment 125. The IL-10 conjugate of embodiment 120, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 31, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[677] Embodiment 126. The IL-10 conjugate of embodiment 120, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 32, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[678] Embodiment 127. The IL-10 conjugate of embodiment 120, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 33, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[679] Embodiment 128. The IL-10 conjugate of embodiment 120, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 34, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[680] Embodiment 129. An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 35-42, wherein [AzK_PEG30kDa] has a structure of formula (II), formula (III), or is a mixture of structures of formula (II) and formula (III),

Where

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

X has the structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[681] Embodiment 129.1: An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 35-42, wherein [AzK_PEG30kDa] has a structure of formula (II), formula (III), or is a mixture of structures of formula (II) and formula (III),

Where

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

q is equal to 1, 2, or 3; and

X has the structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[682] Embodiment 130. The IL-10 conjugate of embodiment 129 or 129.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 35, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[683] Embodiment 131. The IL-10 conjugate of embodiment 129 or 129.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 36, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[684] Embodiment 132. The IL-10 conjugate of embodiment 129 or 129.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 37, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[685] Embodiment 133. The IL-10 conjugate of embodiment 129 or 129.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 38, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[686] Embodiment 134. The IL-10 conjugate of embodiment 129 or 129.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 39, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[687] Embodiment 135. The IL-10 conjugate of embodiment 129 or 129.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 40, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[688] Embodiment 136. The IL-10 conjugate of embodiment 129 or 129.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 41, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[689] Embodiment 137. The IL-10 conjugate of embodiment 129 or 129.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 42, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[690] Embodiment 138. The IL-10 conjugate of embodiment 129 or 129.1, wherein [AzK_PEG30kDa] has the structure of formula (II),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[691] Embodiment 139. The IL-10 conjugate of embodiment 138, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 35, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[692] Embodiment 140. The IL-10 conjugate of embodiment 138, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 36, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[693] Embodiment 141. The IL-10 conjugate of embodiment 138, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 37, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[694] Embodiment 142. The IL-10 conjugate of embodiment 138, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 38, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[695] Embodiment 143. The IL-10 conjugate of embodiment 138, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 39, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[696] Embodiment 144. The IL-10 conjugate of embodiment 138, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 40, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[697] Embodiment 145. The IL-10 conjugate of embodiment 138, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 41, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[698] Embodiment 146. The IL-10 conjugate of embodiment 138, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 42, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[699] Embodiment 147. The IL-10 conjugate of embodiment 129 or 129.1, wherein [AzK_PEG30kDa] has the structure of formula (III),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[700] Embodiment 148. The IL-10 conjugate of embodiment 147, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 35, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[701] Embodiment 149. The IL-10 conjugate of embodiment 147, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 36, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[702] Embodiment 150. The IL-10 conjugate of embodiment 147, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 37, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[703] Embodiment 151. The IL-10 conjugate of embodiment 147, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 38, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[704] Embodiment 152. The IL-10 conjugate of embodiment 147, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 39, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[705] Embodiment 153. The IL-10 conjugate of embodiment 147, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 40, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[706] Embodiment 154. The IL-10 conjugate of embodiment 147, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 41, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[707] Embodiment 155. The IL-10 conjugate of embodiment 147, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 42, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[708] Embodiment 156. An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 19-26, wherein [AzK_PEG] is a mixture of structures of formula (II) and formula (III),

Where

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

q is equal to 1, 2, or 3; and

X has the structure

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

[709] Embodiment 156.1: An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 19-26, wherein [AzK_PEG] is a mixture of structures of formula (II) and formula (III),

Where

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

q is equal to 1, 2, or 3; and

X has the structure

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

[710] Embodiment 157. The IL-10 conjugate of embodiment 156 or 156.1, wherein the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) making up the total amount of [AzK_PEG] in the IL-10 conjugate is about 1:1.

[711] Embodiment 158. The IL-10 conjugate of embodiment 156 or 156.1, wherein the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) making up the total amount of [AzK_PEG] in the IL-10 conjugate is greater than 1:1.

[712] Embodiment 159. The IL-10 conjugate of embodiment 156 or 156.1, wherein the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) making up the total amount of [AzK_PEG] in the IL-10 conjugate is less than 1:1.

[713] Embodiment 160. The IL-10 conjugate of any one of embodiments 156-159, wherein W is a linear or branched PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[714] Embodiment 161. The IL-10 conjugate of any one of embodiments 156-159, wherein W is a linear PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[715] Embodiment 162. The IL-10 conjugate of any one of embodiments 156-159, wherein W is a branched PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[716] Embodiment 163. The IL-10 conjugate of any one of embodiments 156-159, wherein W is a methoxy-PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[717] Embodiment 164. The IL-10 conjugate of embodiment 163, wherein the methoxy-PEG group is linear or branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[718] Embodiment 165. The IL-10 conjugate of embodiment 164, wherein the methoxy-PEG group is linear, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[719] Embodiment 166. The IL-10 conjugate of embodiment 164, wherein the methoxy-PEG group is branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[720] Embodiment 167. An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 27-34, wherein [AzK_PEG20kDa] is a mixture of structures of formula (II) and formula (III),

Where

W is a PEG group having an average molecular weight of 20 kDa; and

X has the structure

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

[721] Embodiment 167.1: An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 27-34, wherein [AzK_PEG20kDa] is a mixture of structures of formula (II) and formula (III),

Where

W is a PEG group with an average molecular weight of 20 kDa;

q is equal to 1, 2, or 3; and

X has a structure

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

[722] Embodiment 168. The IL-10 conjugate of embodiment 167 or 167.1, wherein the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) making up the total amount of [AzK_PEG20kDa] in the IL-10 conjugate is about 1:1.

[723] Embodiment 169. The IL-10 conjugate of embodiment 167 or 167.1, wherein the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) making up the total amount of [AzK_PEG20kDa] in the IL-10 conjugate is greater than 1:1.

[724] Embodiment 170. The IL-10 conjugate of embodiment 167 or 167.1, wherein the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) making up the total amount of [AzK_PEG20kDa] in the IL-10 conjugate is less than 1:1.

[725] Embodiment 171. An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 35-42, wherein [AzK_PEG30kDa] is a mixture of structures of formula (II) and formula (III),

Where

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

X has the structure

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

[726] Embodiment 171.1: An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 35-42, wherein [AzK_PEG30kDa] is a mixture of structures of formula (II) and formula (III),

Where

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

q is equal to 1, 2, or 3; and

X has the structure

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

[727] Embodiment 172. The IL-10 conjugate of embodiment 171 or 171.1, wherein the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) making up the total amount of [AzK_PEG30kDa] in the IL-10 conjugate is about 1:1.

[728] Embodiment 173. The IL-10 conjugate of embodiment 171 or 171.1, wherein the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) making up the total amount of [AzK_PEG30kDa] in the IL-10 conjugate is greater than 1:1.

[729] Embodiment 174. The IL-10 conjugate of embodiment 171 or 171.1, wherein the ratio of the amount of the structure of formula (II) to the amount of the structure of formula (III) making up the total amount of [AzK_PEG30kDa] in the IL-10 conjugate is less than 1:1.

[730] Embodiment 175. An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 59-66, wherein [AzK_L1_PEG] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V),

Where

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

X has the structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[731] Embodiment 175.1: An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 59-66, wherein [AzK_L1_PEG] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V),

Where

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

q is equal to 1, 2, or 3; and

X has a structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[732] Embodiment 176. The IL-10 conjugate of embodiment 175 or 175.1, wherein [AzK_L1_PEG] is a mixture of formula (IV) and formula (V), or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[733] Embodiment 177. The IL-10 conjugate of embodiment 175 or 175.1, wherein [AzK_L1_PEG] has the structure of formula (IV),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[734] Embodiment 178. The IL-10 conjugate of embodiment 177, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 59, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[735] Embodiment 179. The IL-10 conjugate of embodiment 178, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[736] Embodiment 180. The IL-10 conjugate of embodiment 179, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[737] Embodiment 181. The IL-10 conjugate of embodiment 180, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[738] Embodiment 182. The IL-10 conjugate of embodiment 180, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[739] Embodiment 183. The IL-10 conjugate of embodiment 177, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 60, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[740] Embodiment 184. The IL-10 conjugate of embodiment 183, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[741] Embodiment 185. The IL-10 conjugate of embodiment 184, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[742] Embodiment 186. The IL-10 conjugate of embodiment 185, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[743] Embodiment 187. The IL-10 conjugate of embodiment 185, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[744] Embodiment 188. The IL-10 conjugate of embodiment 177, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 61, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[745] Embodiment 189. The IL-10 conjugate of embodiment 188, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[746] Embodiment 190. The IL-10 conjugate of embodiment 189, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[747] Embodiment 191. The IL-10 conjugate of embodiment 190, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[748] Embodiment 192. The IL-10 conjugate of embodiment 190, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[749] Embodiment 193. The IL-10 conjugate of embodiment 177, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 62, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[750] Embodiment 194. The IL-10 conjugate of embodiment 193, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[751] Embodiment 195. The IL-10 conjugate of embodiment 194, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[752] Embodiment 196. The IL-10 conjugate of embodiment 195, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[753] Embodiment 197. The IL-10 conjugate of embodiment 195, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[754] Embodiment 198. The IL-10 conjugate of embodiment 177, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 63, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[755] Embodiment 199. The IL-10 conjugate of embodiment 198, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[756] Embodiment 200. The IL-10 conjugate of embodiment 199, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[757] Embodiment 201. The IL-10 conjugate of embodiment 200, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[758] Embodiment 202. The IL-10 conjugate of embodiment 200, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[759] Embodiment 203. The IL-10 conjugate of embodiment 177, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 64, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[760] Embodiment 204. The IL-10 conjugate of embodiment 203, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[761] Embodiment 205. The IL-10 conjugate of embodiment 204, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[762] Embodiment 206. The IL-10 conjugate of embodiment 205, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[763] Embodiment 207. The IL-10 conjugate of embodiment 205, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[764] Embodiment 208. The IL-10 conjugate of embodiment 177, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 65, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[765] Embodiment 209. The IL-10 conjugate of embodiment 208, wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[766] Embodiment 210. The IL-10 conjugate of embodiment 209, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[767] Embodiment 211. The IL-10 conjugate of embodiment 210, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[768] Embodiment 212. The IL-10 conjugate of embodiment 210, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[769] Embodiment 213. The IL-10 conjugate of embodiment 177, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 66, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[770] Embodiment 214. The IL-10 conjugate of embodiment 213, wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[771] Embodiment 215. The IL-10 conjugate of embodiment 214, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[772] Embodiment 216. The IL-10 conjugate of embodiment 215, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[773] Embodiment 217. The IL-10 conjugate of embodiment 215, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[774] Embodiment 218. The IL-10 conjugate of embodiment 175 or 175.1, wherein [AzK_L1_PEG] has the structure of formula (V),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[775] Embodiment 219. The IL-10 conjugate of embodiment 218, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 59, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[776] Embodiment 220. The IL-10 conjugate of embodiment 219, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[777] Embodiment 221. The IL-10 conjugate of embodiment 220, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[778] Embodiment 222. The IL-10 conjugate of embodiment 221, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[779] Embodiment 223. The IL-10 conjugate of embodiment 221, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[780] Embodiment 224. The IL-10 conjugate of embodiment 218, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 60, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[781] Embodiment 225. The IL-10 conjugate of embodiment 224, wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[782] Embodiment 226. The IL-10 conjugate of embodiment 225, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[783] Embodiment 227. The IL-10 conjugate of embodiment 226, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[784] Embodiment 228. The IL-10 conjugate of embodiment 226, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[785] Embodiment 229. The IL-10 conjugate of embodiment 218, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 61, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[786] Embodiment 230. The IL-10 conjugate of embodiment 229, wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[787] Embodiment 231. The IL-10 conjugate of embodiment 230, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[788] Embodiment 232. The IL-10 conjugate of embodiment 231, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[789] Embodiment 233. The IL-10 conjugate of embodiment 231, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[790] Embodiment 234. The IL-10 conjugate of embodiment 218, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 62, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[791] Embodiment 235. The IL-10 conjugate of embodiment 234, wherein W is a PEG moiety having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[792] Embodiment 236. The IL-10 conjugate of embodiment 235, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[793] Embodiment 237. The IL-10 conjugate of embodiment 236, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[794] Embodiment 238. The IL-10 conjugate of embodiment 236, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[795] Embodiment 239. The IL-10 conjugate of embodiment 218, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 63, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[796] Embodiment 240. The IL-10 conjugate of embodiment 239, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[797] Embodiment 241. The IL-10 conjugate of embodiment 240, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[798] Embodiment 242. The IL-10 conjugate of embodiment 241, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[799] Embodiment 243. The IL-10 conjugate of embodiment 241, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[800] Embodiment 244. The IL-10 conjugate of embodiment 218, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 64, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[801] Embodiment 245. The IL-10 conjugate of embodiment 244, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[802] Embodiment 246. The IL-10 conjugate of embodiment 245, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[803] Embodiment 247. The IL-10 conjugate of embodiment 246, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[804] Embodiment 248. The IL-10 conjugate of embodiment 246, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[805] Embodiment 249. The IL-10 conjugate of embodiment 218, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 65, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[806] Embodiment 250. The IL-10 conjugate of embodiment 249, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[807] Embodiment 251. The IL-10 conjugate of embodiment 250, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[808] Embodiment 252. The IL-10 conjugate of embodiment 251, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[809] Embodiment 253. The IL-10 conjugate of embodiment 251, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[810] Embodiment 254. The IL-10 conjugate of embodiment 218, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 66, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[811] Embodiment 255. The IL-10 conjugate of embodiment 254, wherein W is a PEG group having an average molecular weight selected from 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[812] Embodiment 256. The IL-10 conjugate of embodiment 255, wherein W is a PEG group having an average molecular weight selected from 20 kDa and 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[813] Embodiment 257. The IL-10 conjugate of embodiment 256, wherein W is a PEG group having an average molecular weight of 20 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[814] Embodiment 258. The IL-10 conjugate of embodiment 256, wherein W is a PEG group having an average molecular weight of 30 kDa, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[815] Embodiment 259. The IL-10 conjugate of any one of embodiments 175-258, wherein W is a linear or branched PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[816] Embodiment 260. The IL-10 conjugate of any one of embodiments 178-258, wherein W is a linear PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[817] Embodiment 261. The IL-10 conjugate of any one of embodiments 175-258, wherein W is a branched PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[818] Embodiment 262. The IL-10 conjugate of any one of embodiments 175-258, wherein W is a methoxy-PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[819] Embodiment 263. The IL-10 conjugate of embodiment 262, wherein the methoxy-PEG group is linear or branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[820] Embodiment 264. The IL-10 conjugate of embodiment 263, wherein the methoxy-PEG group is linear, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[821] Embodiment 265. The IL-10 conjugate of embodiment 263, wherein the methoxy-PEG group is branched, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[822] Embodiment 266. An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 43-50, wherein [AzK_L1_PEG20kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V),

Where

W is a PEG group having an average molecular weight of 20 kDa; and

X has the structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[823] Embodiment 266.1: An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 43-50, wherein [AzK_L1_PEG20kDa] has a structure of formula (IV), formula (V), or is a mixture of formula (IV) and formula (V),

Where

W is a PEG group with an average molecular weight of 20 kDa;

q is equal to 1, 2, or 3; and

X has the structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[824] Embodiment 267. The IL-10 conjugate of embodiment 266 or 266.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 43, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[825] Embodiment 268. The IL-10 conjugate of embodiment 266 or 266.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 44, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[826] Embodiment 269. The IL-10 conjugate of embodiment 266 or 266.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 45, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[827] Embodiment 270. The IL-10 conjugate of embodiment 266 or 266.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 46, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[828] Embodiment 271. The IL-10 conjugate of embodiment 266 or 266.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 47, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[829] Embodiment 272. The IL-10 conjugate of embodiment 266 or 266.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 48, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[830] Embodiment 273. The IL-10 conjugate of embodiment 266 or 266.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 49, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[831] Embodiment 274. The IL-10 conjugate of embodiment 266 or 266.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 50, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[832] Embodiment 275. The IL-10 conjugate of embodiment 266 or 266.1, wherein [AzK_L1_PEG20kDa] has the structure of formula (IV),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[833] Embodiment 276. The IL-10 conjugate of embodiment 275, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 43, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[834] Embodiment 277. The IL-10 conjugate of embodiment 275, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 44, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[835] Embodiment 278. The IL-10 conjugate of embodiment 275, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 45, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[836] Embodiment 279. The IL-10 conjugate of embodiment 275, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 46, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[837] Embodiment 280. The IL-10 conjugate of embodiment 275, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 47, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[838] Embodiment 281. The IL-10 conjugate of embodiment 275, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 48, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[839] Embodiment 282. The IL-10 conjugate of embodiment 275, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 49, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[840] Embodiment 283. The IL-10 conjugate of embodiment 275, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 50, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[841] Embodiment 284. The IL-10 conjugate of embodiment 266 or 266.1, wherein [AzK_L1_PEG20kDa] has the structure of formula (V),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[842] Embodiment 285. The IL-10 conjugate of embodiment 284, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 43, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[843] Embodiment 286. The IL-10 conjugate of embodiment 284, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 44, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[844] Embodiment 287. The IL-10 conjugate of embodiment 284, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 45, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[845] Embodiment 288. The IL-10 conjugate of embodiment 284, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 46, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[846] Embodiment 289. The IL-10 conjugate of embodiment 284, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 47, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[847] Embodiment 290. The IL-10 conjugate of embodiment 284, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 48, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[848] Embodiment 291. The IL-10 conjugate of embodiment 284, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 49, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[849] Embodiment 292. The IL-10 conjugate of embodiment 284, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 50, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[850] Embodiment 293. An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 51-58, wherein [AzK_L1_PEG30kDa] has a structure of formula (IV), formula (V), or is a mixture of structures of formula (IV) and formula (V),

Where

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

X has the structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[851] Embodiment 293.1: An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 51-58, wherein [AzK_L1_PEG30kDa] has a structure of formula (IV), formula (V), or is a mixture of structures of formula (IV) and formula (V),

Where

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

q is equal to 1, 2, or 3; and

X has the structure

;

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[852] Embodiment 294. The IL-10 conjugate of embodiment 293 or 293.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 51, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[853] Embodiment 295. The IL-10 conjugate of embodiment 293 or 293.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 52, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[854] Embodiment 296. The IL-10 conjugate of embodiment 293 or 293.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 53, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[855] Embodiment 297. The IL-10 conjugate of embodiment 293 or 293.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 54, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[856] Embodiment 298. The IL-10 conjugate of embodiment 293 or 293.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 55, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[857] Embodiment 299. The IL-10 conjugate of embodiment 293 or 293.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 56, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[858] Embodiment 300. The IL-10 conjugate of embodiment 293 or 293.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 57, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[859] Embodiment 301. The IL-10 conjugate of embodiment 293 or 293.1, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 58, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[860] Embodiment 302. The IL-10 conjugate of embodiment 293 or 293.1, wherein [AzK_L1_PEG30kDa] has the structure of formula (IV),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[861] Embodiment 303. The IL-10 conjugate of embodiment 302, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 51, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[862] Embodiment 304. The IL-10 conjugate of embodiment 302, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 52, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[863] Embodiment 305. The IL-10 conjugate of embodiment 302, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 53, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[864] Embodiment 306. The IL-10 conjugate of embodiment 302, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 54, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[865] Embodiment 307. The IL-10 conjugate of embodiment 302, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 55, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[866] Embodiment 308. The IL-10 conjugate of embodiment 302, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 56, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[867] Embodiment 309. The IL-10 conjugate of embodiment 302, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 57, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[868] Embodiment 310. The IL-10 conjugate of embodiment 302, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 58, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[869] Embodiment 311. The IL-10 conjugate of embodiment 293 or 293.1, wherein [AzK_L1_PEG30kDa] has the structure of formula (V),

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[870] Embodiment 312. The IL-10 conjugate of embodiment 311, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 51, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[871] Embodiment 313. The IL-10 conjugate of embodiment 311, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 52, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[872] Embodiment 314. The IL-10 conjugate of embodiment 311, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 53, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[873] Embodiment 315. The IL-10 conjugate of embodiment 311, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 54, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[874] Embodiment 316. The IL-10 conjugate of embodiment 311, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 55, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[875] Embodiment 317. The IL-10 conjugate of embodiment 311, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 56, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[876] Embodiment 318. The IL-10 conjugate of embodiment 311, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 57, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[877] Embodiment 319. The IL-10 conjugate of embodiment 311, wherein the IL-10 conjugate has the amino acid sequence of SEQ ID NO: 58, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[878] Embodiment 320. An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 43-50, wherein [AzK_L1_PEG20kDa] is a mixture of structures of formula (IV) and formula (V),

Where

W is a PEG group having an average molecular weight of 20 kDa; and

X has the structure

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

[879] Embodiment 320.1: An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 43-50, wherein [AzK_L1_PEG20kDa] is a mixture of structures of formula (IV) and formula (V),

Where

W is a PEG group with an average molecular weight of 20 kDa;

q is equal to 1, 2, or 3; and

X has the structure

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

[880] Embodiment 321. The IL-10 conjugate of embodiment 320 or 320.1, wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) making up the total amount of [AzK_L1_PEG20kDa] in the IL-10 conjugate is about 1:1.

[881] Embodiment 322. The IL-10 conjugate of embodiment 320 or 320.1, wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) making up the total amount of [AzK_L1_PEG20kDa] in the IL-10 conjugate is greater than 1:1.

[882] Embodiment 323. The IL-10 conjugate of embodiment 320 or 320.1, wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) making up the total amount of [AzK_L1_PEG20kDa] in the IL-10 conjugate is less than 1:1.

[883] Embodiment 324. An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 51-58, wherein [AzK_L1_PEG30kDa] is a mixture of structures of formula (IV) and formula (V),

Where

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

X has the structure

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

[884] Embodiment 324.1: An IL-10 conjugate comprising the amino acid sequence of any one of SEQ ID NOs: 51-58, wherein [AzK_L1_PEG30kDa] is a mixture of structures of formula (IV) and formula (V),

Where

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

q is equal to 1, 2, or 3; and

X has the structure

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

[885] Embodiment 325. The IL-10 conjugate of embodiment 324 or 324.1, wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) making up the total amount of [AzK_L1_PEG30kDa] in the IL-10 conjugate is about 1:1.

[886] Embodiment 326. The IL-10 conjugate of embodiment 324 or 324.1, wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) making up the total amount of [AzK_L1_PEG30kDa] in the IL-10 conjugate is greater than 1:1.

[887] Embodiment 327. The IL-10 conjugate of embodiment 324 or 324.1, wherein the ratio of the amount of the structure of formula (IV) to the amount of the structure of formula (V) making up the total amount of [AzK_L1_PEG30kDa] in the IL-10 conjugate is less than 1:1.

[888] Embodiment 328. An IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII),

Where

n is an integer such that the molecular weight of the PEG group is from about 5,000 daltons to about 60,000 daltons; and

X has a structure ,

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[889] Embodiment 328.1: An IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII),

Where

q is equal to 1, 2, or 3;

n is an integer such that the molecular weight of the PEG group is from about 5,000 daltons to about 60,000 daltons; and

X has a structure ,

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[890] Embodiment 329. The IL-10 conjugate of embodiment 328 or 328.1, wherein the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is selected from N82, K88, A89, K99, K125, N126, N129, and K130, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[891] Embodiment 330. The IL-10 conjugate of embodiment 329, wherein the position of the structure of formula (VI), formula (VII), or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is selected from N82, K88, K99, N126, N129, and K130, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[892] Embodiment 331. The IL-10 conjugate of any one of embodiments 328-330, wherein the ratio of the amount of the structure of formula (VI) to the amount of the structure of formula (VII) contained in the total amount of the IL-10 conjugate is about 1:1.

[893] Embodiment 332. The IL-10 conjugate of any one of embodiments 328-330, wherein the ratio of the amount of the structure of formula (VI) to the amount of the structure of formula (VII) contained in the total amount of the IL-10 conjugate is greater than 1:1.

[894] Embodiment 333. The IL-10 conjugate of any one of embodiments 328-330, wherein the ratio of the amount of the structure of formula (VI) to the amount of the structure of formula (VII) contained in the total amount of the IL-10 conjugate is less than 1:1.

[895] Embodiment 334. The IL-10 conjugate of any one of embodiments 328-333, wherein n is an integer such that the molecular weight of the PEG group is from about 5,000 Daltons to about 40,000 Daltons.

[896] Embodiment 335. The IL-10 conjugate of embodiment 334, wherein n is an integer such that the molecular weight of the PEG group is from about 5,000 Daltons to about 30,000 Daltons.

[897] Embodiment 336. The IL-10 conjugate of embodiment 334, wherein n is an integer such that the molecular weight of the PEG group is from about 5,000 Daltons to about 25,000 Daltons.

[898] Embodiment 337. The IL-10 conjugate of embodiment 334, wherein n is an integer such that the molecular weight of the PEG group is from about 7,500 Daltons to about 30,000 Daltons.

[899] Embodiment 338. The IL-10 conjugate of embodiment 334, wherein n is an integer such that the molecular weight of the PEG group is from about 10,000 Daltons to about 20,000 Daltons.

[900] Embodiment 339. The IL-10 conjugate of embodiment 328 or 328.1, wherein the position of the structure of formula (VI) or formula (VII) or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is selected from N82, K88, A89, N129, and K130, and wherein n is an integer such that the molecular weight of the PEG group is from about 7,500 Daltons to about 30,000 Daltons.

[901] Embodiment 340. The IL-10 conjugate of embodiment 339, wherein n is an integer such that the molecular weight of the PEG group is from about 10,000 Daltons to about 20,000 Daltons.

[902] Embodiment 341. The IL-10 conjugate of embodiment 340, wherein the position of the structure of formula (VI) or formula (VII) or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is N82.

[903] Embodiment 342. The IL-10 conjugate of embodiment 340, wherein the position of the structure of formula (VI) or formula (VII) or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is K88.

[904] Embodiment 343. The IL-10 conjugate of embodiment 340, wherein the position of the structure of formula (VI) or formula (VII) or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is A89.

[905] Embodiment 344. The IL-10 conjugate of embodiment 340, wherein the position of the structure of formula (VI) or formula (VII) or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is N129.

[906] Embodiment 345. The IL-10 conjugate of embodiment 340, wherein the position of the structure of formula (VI) or formula (VII) or a mixture of formula (VI) and formula (VII) in the amino acid sequence of the IL-10 conjugate is K130.

[907] Embodiment 346. An IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII) or formula (IX) or a mixture of formula (VIII) and formula (IX),

Where

n is an integer such that the molecular weight of the PEG group is from about 5,000 daltons to about 60,000 daltons; and

X has a structure ,

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[908] Embodiment 346.1: An IL-10 conjugate comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 conjugate is replaced with a structure of formula (VIII) or formula (IX) or a mixture of formula (VIII) and formula (IX),

Where

q is equal to 1, 2, or 3;

n is an integer such that the molecular weight of the PEG group is from about 5,000 daltons to about 60,000 daltons; and

X has a structure ,

or a pharmaceutically acceptable salt, solvate or hydrate thereof.

[909] Embodiment 347. The IL-10 conjugate of embodiment 346 or 346.1, wherein the position of the structure of formula (VIII) or formula (IX) or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is selected from N82, K88, A89, K99, K125, N126, N129, and K130, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[910] Embodiment 348. The IL-10 conjugate of embodiment 346 or 346.1, wherein the position of the structure of formula (VIII) or formula (IX), or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is selected from N82, K88, K99, N126, N129, and K130, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.

[911] Embodiment 349. The IL-10 conjugate of any one of embodiments 346-348, wherein the ratio of the amount of the structure of formula (VIII) to the amount of the structure of formula (IX) contained in the total amount of the IL-10 conjugate is about 1:1.

[912] Embodiment 350. The IL-10 conjugate of any one of embodiments 346-348, wherein the ratio of the amount of the structure of formula (VIII) to the amount of the structure of formula (IX) contained in the total amount of the IL-10 conjugate is greater than 1:1.

[913] Embodiment 351. The IL-10 conjugate of any one of embodiments 346-348, wherein the ratio of the amount of the structure of formula (VIII) to the amount of the structure of formula (IX) contained in the total amount of the IL-10 conjugate is less than 1:1.

[914] Embodiment 352. The IL-10 conjugate of any one of embodiments 346-351, wherein n is an integer such that the molecular weight of the PEG group is from about 5,000 Daltons to about 40,000 Daltons.

[915] Embodiment 353. The IL-10 conjugate of embodiment 352, wherein n is an integer such that the molecular weight of the PEG group is from about 5,000 Daltons to about 30,000 Daltons.

[916] Embodiment 354. The IL-10 conjugate of embodiment 352, wherein n is an integer such that the molecular weight of the PEG group is from about 5,000 Daltons to about 25,000 Daltons.

[917] Embodiment 355. The IL-10 conjugate of embodiment 352, wherein n is an integer such that the molecular weight of the PEG group is from about 7,500 Daltons to about 30,000 Daltons.

[918] Embodiment 356. The IL-10 conjugate of embodiment 352, wherein n is an integer such that the molecular weight of the PEG group is from about 10,000 Daltons to about 20,000 Daltons.

[919] Embodiment 357. The IL-10 conjugate of embodiment 346 or 346.1, wherein the position of the structure of formula (VIII) or formula (IX) or a mixture of (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is selected from N82, K88, A89, N129, and K130, and wherein n is an integer such that the molecular weight of the PEG group is from about 7,500 Daltons to about 30,000 Daltons.

[920] Embodiment 358. The IL-10 conjugate of embodiment 357, wherein n is an integer such that the molecular weight of the PEG group is from about 10,000 daltons to about 20,000 daltons.

[921] Embodiment 359. The IL-10 conjugate of embodiment 358, wherein the position of the structure of formula (VIII) or formula (IX) or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is N82.

[922] Embodiment 360. The IL-10 conjugate of embodiment 358, wherein the position of the structure of formula (VIII) or formula (IX) or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is K88.

[923] Embodiment 361. The IL-10 conjugate of embodiment 358, wherein the position of the structure of formula (VIII) or formula (IX) or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is A89.

[924] Embodiment 362. The IL-10 conjugate of embodiment 358, wherein the position of the structure of formula (VIII) or formula (IX) or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is N129.

[925] Embodiment 363. The IL-10 conjugate of embodiment 358, wherein the position of the structure of formula (VIII) or formula (IX) or a mixture of formula (VIII) and formula (IX) in the amino acid sequence of the IL-10 conjugate is K130.

[926] Embodiment 364. A method of treating cancer in a subject comprising administering to the subject in need thereof an effective amount of an IL-10 conjugate of any one of embodiments 1-363.

[927] Embodiment 365. The method of embodiment 364, wherein the cancer is a solid tumor or a hematological malignancy.

[928] Embodiment 366. The method of embodiment 365, wherein the cancer is a solid tumor.

[929] Embodiment 367. The method of embodiment 366, wherein the solid tumor is metastatic cancer.

[930] Embodiment 368. The method of embodiment 366, wherein the solid tumor is relapsed or refractory cancer from prior treatment.

[931] Embodiment 369. The method of any one of embodiments 364-368, wherein the cancer in the subject is selected from renal cell carcinoma, bladder cancer, bone cancer, brain cancer, breast cancer, colorectal cancer, esophageal cancer, eye cancer, head and neck cancer, kidney cancer, lung cancer, melanoma, ovarian cancer, pancreatic cancer, squamous cell carcinoma, pancreatic cancer, and prostate cancer.

[932] Embodiment 370. The method of embodiment 364, wherein the cancer in the subject is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin's lymphoma (cHL), primary mediastinal large B cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability cancer, microsatellite stable cancer, microsatellite stable colorectal cancer, gastric cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), melanoma, small cell lung cancer (SCLC), esophageal cancer, glioblastoma, mesothelioma, breast cancer, triple negative breast cancer, prostate cancer, bladder cancer, ovarian cancer, tumors with mutation moderate to low burden, cutaneous squamous cell carcinoma (CSCC), cutaneous squamous cell carcinoma (SCSC), tumors with low to absent PD-L1 expression, tumors systemically disseminating to the liver and CNS beyond their primary site of anatomical origin, and diffuse large B-cell lymphoma.

[933] Embodiment 371. The method of embodiment 364, wherein the cancer in the subject is a hematological malignancy.

[934] Embodiment 372. The method of embodiment 371, wherein the hematological malignancy comprises leukemia, lymphoma, or myeloma.

[935] Embodiment 373. The method of embodiment 371, wherein the hematological malignancy is a T-cell malignancy.

[936] Embodiment 374. The method of embodiment 371, wherein the hematological malignancy is a B-cell malignancy.

[937] Embodiment 375. The method of embodiment 371, wherein the hematological malignancy is a metastatic malignancy.

[938] Embodiment 376. The method of embodiment 371, wherein the hematological malignancy is a recurrent hematological malignancy.

[939] Embodiment 377. The method of embodiment 371, wherein the hematological malignancy is a refractory malignancy.

[940] Embodiment 378. The method of embodiment 371, wherein the cancer is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, high-grade B-cell non-Burkitt's lymphoma, primary mediastinal B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, progenitor B-cell lymphoblastic lymphoma, prolymphocytic B-cell leukemia, lymphoplasmacytic lymphoma, splenic marginal zone cell lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

[941] Embodiment 379. The method of any one of embodiments 364-378, wherein the method further comprises administering to a subject in need thereof an effective amount of one or more additional agents.

[942] Embodiment 380. The method of embodiment 379, wherein the one or more additional agents is one or more immune checkpoint inhibitors selected from the group consisting of PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, CTLA-4 inhibitors, OX40 agonists, and 4-1BB agonists.

[943] Embodiment 381. The method of embodiment 380, wherein the one or more immune checkpoint inhibitors are selected from PD-1 inhibitors.

[944] Embodiment 382. The method of embodiment 381, wherein the one or more PD-1 inhibitors are selected from pembrolizumab, nivolumab, cemiplimab, lambrolizumab, AMP-224, sintilimab, toripalimab, camrelizumab, tislelizumab, dostarlimab (GSK), PDR001 (Novartis), MGA012 (Macrogenics/Incyte), GLS-010 (Arcus/Wuxi), AGEN2024 (Agenus), cetrelimab (Janssen), ABBV-181 (Abbvie), AMG-404 (Amgen), BI-754091 (Boehringer Ingelheim), CC-90006 (Celgene), JTX-4014 (Jounce), PF-06801591 (Pfizer) and genolimzumab (Apollomics/Genor BioPharma).

[945] Embodiment 383. The method of embodiment 380, wherein the one or more immune checkpoint inhibitors are selected from PD-L1 inhibitors.

[946] Embodiment 384. The method of embodiment 383, wherein the PD-L1 inhibitors are selected from atezolizumab, avelumab, durvalumab, ASC22 (Alphamab/Ascletis), CX-072 (Cytomx), CS1001 (Cstone), cosibelimab (Checkpoint Therapeutics), INCB86550 (Incyte), and TG-1501 (TG Therapeutics).

[947] Embodiment 385. The method of embodiment 380, wherein the one or more immune checkpoint inhibitors are selected from CTLA-4 inhibitors.

[948] Embodiment 386. The method of embodiment 385, wherein the CTLA-4 inhibitors are selected from tremelimumab, ipilimumab, and AGEN-1884 (Agenus).

[949] Embodiment 387. The method of embodiment 379, wherein the one or more additional agents comprise folinic acid, 5-fluorouracil, and oxaliplatin.

[950] Embodiment 388. The method of embodiment 387, wherein the cancer is pancreatic cancer.

[951] Embodiment 389. The method of embodiment 388, wherein the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

[952] Embodiment 390. A method of treating a fibrotic disorder in a subject, comprising administering to the subject in need thereof an effective amount of an IL-10 conjugate of any one of embodiments 1-363.

[953] Embodiment 391. The method of embodiment 390, wherein the fibrotic disorder in the subject is selected from liver fibrosis, idiopathic pulmonary fibrosis, and periportal fibrosis.

[954] Embodiment 392. The method of embodiment 391, wherein the fibrotic disorder in the subject is liver fibrosis.

[955] Embodiment 393. The method of embodiment 391, wherein the fibrotic disorder in the subject is idiopathic pulmonary fibrosis.

[956] Embodiment 394. The method of embodiment 391, wherein the fibrotic disorder in the subject is periportal fibrosis.

[957] Embodiment 395. A method of treating non-alcoholic steatohepatitis (NASH) in a subject, comprising administering to the subject in need thereof an effective amount of an IL-10 conjugate of any one of embodiments 1-363.

[958] Embodiment 396. A method of treating non-alcoholic fatty liver disease (NAFLD) in a subject, comprising administering to the subject in need thereof an effective amount of an IL-10 conjugate of any one of embodiments 1-363.

[959] Embodiment 397. The method of any one of embodiments 364-396, wherein administering an effective amount of IL-10 to a subject in need thereof does not cause a grade 3 or 4 adverse event in the subject.

[960] Embodiment 398. The method of embodiment 397, wherein the grade 3 or 4 adverse event is selected from anemia, leukopenia, thrombocytopenia, elevated ALT, anorexia, arthralgia, back pain, chills, diarrhea, dyslipidemia, fatigue, fever, flu-like symptoms, hypoalbuminemia, elevated lipase, injection site reaction, myalgia, nausea, night sweats, pruritis, rash, erythematous rash, maculopapular rash, transaminitis, vomiting, and weakness.

[961] Embodiment 399. The method of embodiment 398, wherein the grade 3 or 4 adverse event is selected from anemia, leukopenia, thrombocytopenia, erythematous rash, and maculopapular rash.

[962] Embodiment 400. The method of embodiment 399, wherein the grade 3 or 4 adverse event is selected from anemia, thrombocytopenia, erythematous rash, and maculopapular rash.

[963] Embodiment 401. The method of embodiment 400, wherein the grade 3 or 4 adverse event is selected from anemia and thrombocytopenia.

[964] Embodiment 402. The method of embodiment 401, wherein the grade 3 or 4 adverse event is anemia.

[965] Embodiment 403. The method of embodiment 401, wherein the grade 3 or 4 adverse event is thrombocytopenia.

[966] Embodiment 404. The method of any one of embodiments 364-403, wherein administering an effective amount of the IL-10 conjugate to a group of subjects does not cause one or more grade 4 adverse events in more than 1% of the subjects following administration of the IL-10 conjugate to the subjects.

[967] Embodiment 405. The method of any one of embodiments 364-404, wherein the IL-10 conjugate is administered to a subject in need thereof once daily, twice daily, three times daily, once weekly, once every two weeks, once every three weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, or once every 8 weeks.

[968] Embodiment 406. The method of embodiment 405, wherein the IL-10 conjugate is administered to a subject in need thereof once daily, twice daily, three times daily, once weekly, once every two weeks, once every three weeks, or once every 4 weeks.

[969] Embodiment 407. The method of embodiment 406, wherein the IL-10 conjugate is administered to a subject in need thereof once daily, twice daily, once weekly, once every two weeks, once every three weeks, or once every four weeks.

[970] Embodiment 408. The method of embodiment 407, wherein the IL-10 conjugate is administered to a subject in need thereof once daily.

[971] Embodiment 409. The method of embodiment 407, wherein the IL-10 conjugate is administered to a subject in need thereof once per week.

[972] Embodiment 410. The method of embodiment 407, wherein the IL-10 conjugate is administered to a subject in need thereof once every two weeks.

[973] Embodiment 411. The method of embodiment 407, wherein the IL-10 conjugate is administered to a subject in need thereof once every three weeks.

[974] Embodiment 412. The method of embodiment 407, wherein the IL-10 conjugate is administered to a subject in need thereof once every four weeks.

[975] Embodiment 413. The IL-10 conjugate of any one of embodiments 1-363 or the method of any one of embodiments 364-412, wherein the IL-2 conjugate is a pharmaceutically acceptable salt, solvate, or hydrate.

[976] Embodiment 414. An IL-10 conjugate for use in the method of any one of embodiments 364-412.

[977] Embodiment 415. The IL-10 conjugate of any one of embodiments 1-363 or the method of any one of embodiments 364-412, wherein q is 1 in the IL-10 conjugate.

[978] Embodiment 416. The IL-10 conjugate of any one of embodiments 1-363 or the method of any one of embodiments 364-412, wherein q is 2 in the IL-10 conjugate.

[979] Embodiment 417. The IL-10 conjugate of any one of embodiments 1-363 or the method of any one of embodiments 364-412, wherein q is 3 in the IL-10 conjugate.

[980] Embodiment 418. Use of an IL-10 conjugate of any one of embodiments 1-363 or 415-417 for the manufacture of a medicament for treating cancer, fibrotic disorder, NASH, or NAFLD according to the method of any one of embodiments 364-412.

EXAMPLES

[981] These examples are provided for illustrative purposes only and do not limit the scope of the claims presented herein.

EXAMPLE 1

[982] Biochemical interactions of pegylated IL-10 with the human IL-10 receptor

[983] Kinetic and equilibrium dissociation constants for the interaction of a pegylated IL-10 compound with the human IL-10 receptor were measured using surface plasmon resonance (SPR) via Biosensor Tools LLC. For these studies, the extracellular domain of the IL-10R fused to human IgG1 Fc was immobilized on the surface of a protein A-coated CM4 biosensor chip. The surface was probed in duplicate with a two-fold dilution series starting at 2 μM of either native IL-10 (also referred to herein as "native IL-10" or "wild-type IL-10") or IL-10 muteins using a Biacore 2000 or similar SPR instrument. Test samples were injected for 60 s or more to allow association to be measured until a plateau was reached, followed by buffer alone (washout) for 30 s or more to measure dissociation. Response units (RU, Y-axis) were plotted against time (s, X-axis).

[984] Ex-vivo immune response profiling for IL-10 mutein in human primary leukocyte removal system (LRS)-derived PBMC samples

[985] To determine how different receptor specificities of IL-10 muteins affect the activation of primary immune cell subsets, concentration-response profiling of lymphocyte activation was performed in LRS-derived human peripheral blood mononuclear cell (PBMC) samples using multicolor flow cytometry. These studies were performed through PrimityBio LLC (Fremont, CA, USA). Fresh LRS-derived samples were treated with either native IL-10 or IL-10 mutein in a 5-fold dilution series starting at a maximum concentration of 30 μg/mL. After 45 min of incubation, samples were fixed and stained with antibodies to detect the phosphorylated form of the transcription factor STAT3 (pSTAT3), a marker of upstream recruitment and activation of IL-10 receptor signaling complexes, and a panel of surface markers ( Table 3 ) to monitor pSTAT3 formation in subsets of specific T cells and natural killer (NK) cells.

Table 3. Staining panel for flow cytometric analysis of LRS-derived PBMC samples Cell type Marker profile Effector T cells (Teff) CD3+, CD4+, CD8+, CD127+ NK cells CD3-, CD16+ Regulatory T cells (Treg) CD3+, CD4+, CD8-, IL-2Rα+, CD127-

EXAMPLE 2

[986] PEGylated IL-10 compounds were prepared as homogeneous di-PEGylated dimers

[987] Samples of IL-10 conjugates corresponding to SEQ ID NOs: 43, 46, 47, 49, 50, and 59 tagged with the N-terminal [His] as defined above (corresponding to Compound A, Compound B, Compound C, Compound D, Compound E, and Compound F, respectively; for Compound F, in this case and in other cases, the PEG was 10 kDa) were prepared using the methods described herein. Samples of IL-10 conjugates corresponding to SEQ ID NOs: 43 and 49 in which the N-terminal [His] was removed (corresponding to Compound G and H, respectively) were prepared using the methods described herein. Compounds AH contain [AzK_L1_PEG] and as such are characterized by a structure of formula (IV) or formula (V) or formula (XII) or formula (XIII) wherein the substituent q is present and q is 3.

A brief summary of the structural features of compounds A-H is presented in Table 3-A.

Table 3-A. Summary of structural features of compounds A-H. Compound Relevant SEQ ID NO: An amino acid residue substituted by an unnatural amino acid Molecular weight of PEG (kDa) [His] tag present at the N-terminus or immediately following the parent methionine A 43 N82 20 Yes B 46 K99 20 Yes C 47 K125 20 Yes D 49 N129 20 Yes E 50 K130 20 Yes F 59 N82 10 Yes G 43 N82 20 No H 49 N129 20 No

[His] indicates a sequence containing the His tag and TEV recognition site defined above.

[988] Briefly, IL-10 conjugates were expressed as inclusion bodies inE. coliusing the methods disclosed herein, wherein expression plasmids encoding a protein with a desired amino acid sequence were obtained, which contained (a) a non-natural base pair comprising a first non-natural nucleotide and a second non-natural nucleotide to ensure the presence of a codon at the desired position in which the non-natural amino acid was includedN6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), and the corresponding anticodon in tRNA, (b) a plasmid encoding tRNA, obtained from PylM. mazei, which contains an unnatural nucleotide to ensure the presence of the corresponding anticodon in place of its native sequence, (c) a plasmid encoding pyrrolizyl-tRNA synthetase derived fromM. barkeri(MbPylRS), and (d)N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK). A double-stranded oligonucleotide that encodes the amino acid sequence of a desired IL-10 variant comprising an AXC codon, such as at position 82, 88, 89, 99, 125, 126, 129, or 130 of the sequence that encodes the protein of SEQ ID NO: 1, wherein X is a non-natural nucleotide as disclosed herein. In some embodiments, the cell further comprises a plasmid, which may be a protein expression plasmid or another plasmid, that encodes an orthogonal tRNA gene fromM. mazei, which contains a GYT anticodon corresponding to AXC in place of its native sequence, wherein Y is a non-natural nucleotide disclosed herein and may be the same as or different from the non-natural nucleotide in the codon. X and Y are selected from the non-natural nucleotides dTPT3TP, dNaMTP and dCNMOTP disclosed herein. The expressed protein was purified and refolded using standard procedures prior to site-specific PEGylation of the AzK-containing IL-10 product using copper-free DBCO-mediated click chemistry to stably covalently attach mPEG moieties to AzK, which is depicted in Scheme 6b, where q is 3.

[989] In some embodiments, the natural His-TEV-IL-10 has the nucleotide sequence of SEQ ID NO: 75:

ATGCATCATCACCATCATCATGGTAGCAGCGAAAATCTGTATTTTCAGAGCCCTGGTCAGGGCACCCAGAGCGAAAATTCATGTACCCATTTTCCGGGTAATCTGCCGAATATGCTGCGCGATCTGCGTGAT GCATTTAGCCGTGTTAAAACCTTTTTCCAGATGAAAGATCAGCTGGATAATCTGCTGCTGAAAGAAAGCCTGCTGGAAGATTTCAAAGGTTATCTGGGTTGTCAGGCACTGAGCGAAATGATTCAGTTTTATC TGGAAGAAGTTATGCCGCAGGCAGAAAATCAGGATCCGGATATTAAAGCACATGTTAATAGCCTGGGCGAAAATCTGAAAACCCTGCGTCTGCGCCTGCGTCGTTGTCATCGTTTTCTGCCGTGTGAAAACAA AAGCAAAGCAGTTGAACAGGTGAAAAACGCCTTTAACAAACTGCAAGAGAAAGGCATCTATAAAGCCATGAGCGAATTCGACATCTTCATCAACTATATCGAAGCCTACATGACCATGAAAATCCGCAATTAA

[990] In some embodiments, natural IL-10 has the nucleotide sequence of SEQ ID NO: 76:

ATGAGCCCTGGTCAGGGAACCCAATCCGAAAATTCATGTACCCATTTTCCGGGTAATCTGCCGAATATGCTGCGCGATCTGCGTGATGCATTTAGCCGTGTTAAAACCTTTTTCCAGATGA AAGATCAGCTGGATAATCTGCTGCTGAAAGAAAGCCTGCTGGAAGATTTCAAAGGTTATCTGGGTTGTCAGGCACTGAGCGAAATGATTCAGTTTTATCTGGAAGAAGTTATGCCGCAGGCA GAAAATCAGGATCCGGATATTAAAGCACATGTTAATAGCCTGGGCGAAAATCTGAAAACCCTGCGTCTGCGCCTGCGTCGTTGTCATCGTTTTCTGCCGTGTGAAAACAAAAGCAAAGCAGTTGAACAGGTGAAAAACGCCTTTAACAAACTGCAAGAGAAAGGCATCTATAAAGCCATGAGCGAATTCGACATCTTCATCAACTATATCGAAGCCTACATGACCATGAAAATCCGCAATTAA

[991] The conjugates after bioconjugation with the corresponding PEG-containing DBCO reagent were incubated with Laemmli sample buffer under reducing conditions for 5 min at 95°C. After cooling the samples to room temperature, the samples were loaded onto SDS-PAGE gels for electrophoretic separation of proteins. The gel was incubated with water-soluble Coomassie dye or transferred to a nitrocellulose membrane for detection of the corresponding compounds by Western blotting using an antibody to IL-10. Figure 1 shows an illustrative SDS-PAGE and Western blot analysis of compound A under reducing conditions, illustrating homogeneous PEGylation of IL-10 monomers. Molar mass determination of the conjugates was performed using size exclusion chromatography-multiangle light scattering (SEC-MALS) and the results were consistent with compounds AF being di-PEGylated dimers (1:1 protein:PEG ratio). Further analysis of compound dilutions showed no dissociation of the subunits over the concentration range tested. Figure 2 shows an exemplary molecular mass determination of compound A using SEC-MALS. Figure 3 shows an exemplary stability analysis of the dimer of compound A at low concentrations using size exclusion chromatography (SEC).

[992] Removal of the [His] tag in the preparation of Compound A, Compound B, Compound C, Compound D, Compound E, and Compound F can be accomplished using tobacco etch virus (TEV) protease according to methods known in the art. Typically, TEV protease recognizes a linear epitope of the general form E-Xaa-Xaa-Y-Xaa-Q-(G/S), with cleavage occurring between Q and G or Q and S. Cleavage of the protein tag by TEV can be accomplished intracellularly during expression or during purification of the IL-10 conjugates described herein. For example, removal of the [His] tag from the unpegylated precursor of Compound A was accomplished using TEV protease at room temperature overnight. Detection was performed by SDS-PAGE under reducing conditions, followed by Western blot analysis using anti-IL-10 antibodies. Additional methods for [His] tag removal using TEV protease are presented in Raran-Kurussi et al. (2017) Removal of Affinity Tags with TEV Protease. In: Burgess-Brown N. (eds) Heterologous Gene Expression in E. coli. Methods in Molecular Biology, vol 1586. Humana Press, New York, NY; Phan et al. (2002). Structural basis for the substrate specificity of tobacco etch virus protease. J. Biol. Chem. 277: 50564–50572; and Kapust et al. (2000). Controlled intracellular processing of fusion proteins by TEV protease. Protein Expr. Purif. 19: 312–318.

EXAMPLE 3

[993] Biological activity of IL-10 conjugates

[994] The biological activity of Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, Compound G, and Compound H, having the structural features listed in Table 3-A, was determined using two orthogonal assays: the MC/9 Cell Proliferation Assay and the PathHunter® Cytokine Receptor Assay (DiscoverX). MC/9 cells depend on cytokines for growth. MC/9 cell cultures were established in the presence of IL-2, which was removed prior to stimulation with IL-10. The MC/9 Cell Proliferation Assay measured the proliferation of MC/9 cells treated with increasing concentrations of IL-10 and PEGylated compounds following 4 h of IL-2 starvation at 37°C. After 72 h hIL-10 treatment, WST-1 cell proliferation reagent (Sigma, 11644807001) was added and the cells were incubated for an additional 3 h at 37 °C before measuring the sample absorbance at OD450 compared to the background control. Figures 4-7b illustrate the concentration traces of IL-10 conjugates (pg/mL) versus proliferation ( _OD450 ) for compounds A, D, E, F, G, and H, respectively, in the MC/9 proliferation assay. The data show that the N-terminal [His] tag was well tolerated with no significant difference in potency between native IL-10 and native [His]-IL-10. Table 4 shows the potency ( _EC50 ) for the different IL-10 compounds in the MC/9 proliferation assay.

Table 4. Potency of illustrative IL-10 conjugates. Compound EC ₅₀ (ng/ml) Connection A 9.69 Connection B 129 Connection C 440 Compound D 38.2 Compound E 89.8 F connection 2.29 G connection 10.5 H connection 29.8

[995] The biological activity of Compound A, Compound B, Compound C, Compound D, Compound E, and Compound F was also measured using the PathHunter® Cytokine Receptor Assay (DiscoverX/Eurofins), which measures the interaction of two IL-10 receptor chains with cytokines. In this assay, one receptor chain was labeled with a small peptide epitope, ProLink (PK), and the other chain was labeled with an enzyme acceptor (EA). Binding of IL-10 or IL-10 conjugates to the receptor induces dimerization, such that complementation of the PK and EA fragments results in the formation of an active β-galactosidase unit that is detected by chemiluminescence. Figure 8 illustrates the measurement of the biological activity of wild-type IL-10 in the PathHunter® assay. Figure 9 illustrates the measurement of biological activity of Compound A in the PathHunter® assay. Table 5 shows the biological activity of Compound A in the PathHunter® IL-10 R1/R2 Dimerization Assay compared to wild-type IL-10.

Table 5. Biological activity of illustrative IL-10 conjugates. Compound EC ₅₀ (µM) IL-10 wild type 0,0139 Connection A 0.2123

EXAMPLE 4

[996] Profiling of IL-10 conjugates in mouse spleen

[997] This study assessed STAT3 phosphorylation of CD8, NK and B cells in response to stimulation with native [His]-IL-10, Compound A and Compound D. Treatments were performed on C57BL/6 and Balb/c splenocytes. The dose curve consisted of 12 dose points, 3-fold lower than the maximum dose, with a maximum dose of 1 μg/mL for native HisIL-10 and 10 μg/mL for Compound A and Compound D. Mouse spleen splenocytes were prepared by cutting the spleen into small pieces, pressing and washing with PBS through a mesh strainer. The cell suspension was centrifuged and the supernatant was removed by aspiration. 1x RBC Lysis Buffer Working Solution (BioLegend 420301) was used to resuspend the cells. After 4 min at room temperature, the reaction was stopped by adding a 4-8-fold dilution with PBS and passed through a 70 μm mesh strainer. The cells were centrifuged again and washed with complete RPMI splenocyte medium (RPMI, Gibco 22-400-089 with 10% fetal bovine serum and 1% penicillin/streptomycin (P/S)). Finally, the cells were resuspended in complete RPMI medium diluted to 5.5 × 10 ⁶ cells/mL, and 90 μL of cells were added to each well of a 96-well U-bottom plate. The cells were incubated for 20 min or longer at 37°C before stimulation. The cells were stimulated for 45 min at 37°C, followed by fixation with 200 μl pre-warmed Fixation Buffer (BD 554655) and incubation for 10 min in a 37°C water bath. The cells were centrifuged and washed twice with Stain Buffer (BD 554657). The cells were incubated with the different antibodies described in Table 6. Table 7 shows the potency of wild-type [His]-IL-10, Compound A, and Compound D in Balb/c mouse splenocytes as determined by STAT3 phosphorylation. Table 8 shows the potency of wild-type [His]-IL-10, Compound A, and Compound D in B57BL/6 mouse splenocytes as determined by STAT3 phosphorylation. Fig. 10A-C illustrate the profiling of pSTAT3 in splenocytes from Balb/c mice for wild-type IL-10 (filled circles), Compound A (open triangles), and Compound D (open squares) in CD8+ T cells, NK cells, and B cells, respectively. Fig. 11A-C illustrate the profiling of pSTAT3 in splenocytes from B57BL/6 mice for wild-type IL-10 (filled circles), Compound A (open triangles), and Compound D (open squares) in CD8+ T cells, NK cells, and B cells, respectively.

Table 6. Antibodies used to profile IL-10 conjugates in mouse spleen. Target Clone Fluor. 1X Dilution FcX Block 93 None 1:50 CD49b DX5 eF506 1:50 NKp46 (Balb/c) or NK1.1 (B6) 29A1.4 PK136 BV605SB600 1:50 CD62L MEL-14 BV421 1:200 CD25 PC61 FITC 1:100 CD3 17A2 APC/Cy7 1:400

Table 7. Activity of wild-type His-IL-10, compound A, and compound D in splenocytes of Balb/c mice was determined by STAT3 phosphorylation. Compound CD8 T cells EC ₅₀ (ng/ml) NK cells EC ₅₀ (ng/ml) B cells EC ₅₀ (ng/ml) His-IL-10 wild type 4.07 1.08 1.30 Connection A 37.5 12.3 12.5 Compound D 165 47.8 46.1

Table 8. Activity of wild-type His-IL-10, compound A, and compound D in B57BL/6 mouse splenocytes was determined by STAT3 phosphorylation. Compound CD8 T cells EC ₅₀ (ng/ml) NK cells EC ₅₀ (ng/ml) B cells EC ₅₀ (ng/ml) His-IL-10 wild type 3.65 1.06 0.83 Connection A 38.2 17.3 9.18 Compound D 244 60.9 36.9

EXAMPLE 5

[998] Profiling of IL-10 conjugates in the human leukoreduction system (LRS)

[999] This study assessed STAT3 phosphorylation of B cell, NK cell, and CD8+ T cell subsets in response to IL-10 stimulation against wild-type His-IL-10, Compound A, and Compound D. Treatments were performed on 1 LRS donor. The maximum concentration was 0.5 μg/mL for wild-type His-IL-10 and 30 μg/mL for Compound A and Compound D. LRS blood was diluted in PBS and 90 μL of cells were added to each well of a sterile 96-well U-bottom plate. Cells were incubated with compound dilutions for 45 min at 37°C, followed by fixation with Lyse/fix buffer. After 10 min at 37°C, cells were washed with staining buffer, followed by incubation with the appropriate antibody solutions as listed in Table 9 . Cells were incubated for 20 min protected from light, followed by two washes with staining buffer. Cells were then permeabilized with ice-cold Perm Buffer III (BD Biosciences) for 30 min protected from light. Cells were incubated with the appropriate intracellular antibody mixture as listed in Table 10 for 1 h in the dark. Finally, cells were washed with staining buffer and prepared for flow cytometric analysis. Figures 12A–C illustrate the concentration of wild-type His-IL-10, Compound A, and Compound D compared to pSTAT3 MFI in CD8+ T cells, NK cells, and B cells, respectively.

Table 9. Antibody mixture for staining membrane markers. Target Clone Fluor. 1X Dilution CD127 eBioRDR5 eFluor506 1:50 CD19 SJ25C1 BV785 1:100 CD3 UCHT1 APC/Cy7 1:500

Table 10. Antibody mixture for staining intracellular markers. Target Clone Fluor. 1X Dilution CD4 RPA-T4 PE/Cy7 1:200 CD8 RPA-T8 PerCP/Cy5.5 1:100 CD25 M-A251 P.E. 1:500 CD45RA HI100 A488 1:500 CD14 M5E2 BV605 1:50 CD56 HCD56 BV421 1:100 pSTAT3 4/P-Stat3 AF647 1:5

EXAMPLE 6

[1000] Measurement of memory-induced response in CMV IL-10 ⁺ cells

[1001] This study measured how the IL-10 conjugate, Compound A, altered the functional response of memory CD8+ T cells to the CMV peptide compared to wild-type His-IL-10. Donor CD8+ T cells were co-cultured with peptide-loaded non-CD8 cells and several concentrations of wild-type His-IL-10 or Compound A for 5 days. After incubation, cells were stained for IFN-gamma and PD1. CD8 T cells were purified from cryopreserved CMV ⁺ PBMCs by positive selection using a CD8 ⁺ T cell isolation kit according to the manufacturer’s recommendations (Miltenyi Biotech, Auburn, CA, USA). CD8 cells were adjusted to 4× ¹⁰⁶ cells/mL. The concentration of non-CD8 cells was adjusted based on the measured % CD14+ monocytes by FACS.

100 μl/well of non-CD8 cells were transferred to another CMV bulk loading tube. CMV peptide was added at 2-fold final concentration, incubated for 2 h at room temperature with frequent agitation, and then fractions were pooled after normalization of CD8 T cell frequency to CD14 ⁺ monocyte frequency. After centrifugation, cells were resuspended in complete media, transferred to wells of a 96-well plate, and incubated with different compounds in complete media. The dose curve consisted of 3 dose points, 10-fold lower than the maximum dose. After 5 days of incubation at 37°C, cells were permeabilized and stained with the appropriate antibody mixture for 30 min at room temperature protected from light. An additional aliquot of additional CD8 cells and non-CD8 cells was used to confirm the purity of each fraction. Fig. 13A-B illustrate the release of IFNγ upon activation of the antigen-specific TCR with wild-type His-IL-10 or Compound A. Fig. 14A-B illustrate the activation of PD-1 following treatment with His-IL-10 or Compound A and demonstrate that such activation is independent of TCR activation.

EXAMPLE 7

[1002] Phase 1 Clinical Trial of IL-10 in Participants with Cancer

[1003] A clinical trial is conducted to examine the efficacy and safety of administering any of the modified IL-10 polypeptides or IL-10 conjugates described herein to participants with cancer. In some cases, the study is multicenter, randomized, double-blind, and vehicle-controlled. The purpose of the study is to administer 1 mcg/kg, 2 mcg/kg, 2.5 mcg/kg, 5 mcg/kg, 10 mcg/kg, 15 mcg/kg, 20 mcg/kg, 25 mcg/kg, 30 mcg/kg, 50 mcg/kg, 100 mcg/kg, 200 mcg/kg or more of a modified IL-10 polypeptide or IL-10 conjugate to participants with cancer, including melanoma, prostate cancer, ovarian cancer, renal cell carcinoma, colorectal carcinoma, pancreatic carcinoma, non-small cell lung carcinoma, solid tumors, and breast cancer.

Objectives and outcome measures

[1004] The purpose and outcome measures of administering modified IL-10 polypeptides or IL-10 conjugates to participants with cancer were determined based on the primary outcome (i.e., safety) and secondary outcome (i.e., efficacy). The primary outcome was determined by measuring the incidence and severity of adverse events, such as grade 3 and 4 adverse events. The secondary primary outcome was determined by pharmacokinetic (PK) parameters, including trough and peak serum drug concentrations ( _Cmin and _Cmax ), area under the serum concentration-time curve (AUC), and half-life. The secondary outcome was based on change in tumor burden, as determined by volumetric computed tomography (CT) or magnetic resonance imaging (MRI).

Inclusion Criteria

[1005] An eligible participant has histologically or cytologically confirmed advanced malignant solid tumor limited to melanoma, castration-resistant prostate cancer (CRPC), ovarian cancer (OVCA), renal cell carcinoma, colorectal cancer (CRC), pancreatic cancer, or non-small cell lung carcinoma (NSCLC) that is refractory, intolerable, for which there is no standard of care, or where the participant refuses existing treatment options. Participants must be at least 18 years of age or older and have adequate organ function.

Exclusion criteria

[1006] A participant is ineligible if they are pregnant or breastfeeding, have a diagnosed neurological disorder, have had a myocardial infarction within the last 6 months, have unstable angina requiring medication to control cardiac arrhythmia, have had surgery within the last 28 days, have any type of infection, have a history of bleeding diathesis within the last 6 months, or test positive for HIV, hepatitis C, or hepatitis B.

Research plan

[1007] Participants are randomly assigned to 1 of 3 treatment groups in a 1:1:1 ratio. During Phase A of the study, the first cohort of participants will receive daily subcutaneous (SC) injections of 1 mcg/kg of one of the modified IL-10 polypeptides or IL-10 conjugates described herein for 12 months. The second cohort of participants will receive daily SC injections of 10 mcg/kg of one of the modified IL-10 polypeptides or IL-10 conjugates described herein for 12 months. The third cohort of participants will receive daily SC injections of 50 mcg/kg of one of the modified IL-10 polypeptides or IL-10 conjugates described herein for 12 months. Dose escalation is aimed at determining the incidence and severity of adverse events in participants. During Phase B of the study, three cohorts will receive a daily SC injection of one of the modified IL-10 polypeptides or IL-10 conjugates and at least one additional immune checkpoint inhibitor selected from a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, an OX40 agonist, and a 4-1BB agonist.

[1008] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now be apparent to those skilled in the art without departing from the present invention. It will be understood that various alternatives to the embodiments of the present invention described herein may be used in practicing the present invention. The following claims are intended to define the scope of the present invention and thereby cover methods and structures within the scope of the claims, as well as their equivalents. The disclosures of all patents and scientific literature cited herein are expressly incorporated herein by reference in their entireties. To the extent that any incorporated material is inconsistent with the express teachings of the present disclosure, the express teachings shall control.

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SEQUENCE LIST

<110> SYNTHORX, INC.

CAFFARO, Carolina E., et al.

<120> INTERLEUKIN 10 CONJUGATES AND THEIR APPLICATION VARIANTS

<130> 01183-0077-00PCT

<150> US 62/930,322

<151> 2019-11-04

<150> US 62/953,095

<151> 2019-12-23

<160> 79

<170> PatentIn version 3.5

<210> 1

<211> 161

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<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<400> 1

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

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Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

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Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

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Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

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Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

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Arg Ala Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His

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Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe

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Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu

50 55 60

Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys

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Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro

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Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu

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Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg

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Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn

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Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu

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Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile

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Arg Asn

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Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

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Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

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Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

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Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

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Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

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Ala Glu Xaa Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

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Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

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Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

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Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

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Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

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Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

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Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

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Ala Glu Asn Gln Asp Pro Asp Ile Xaa Ala His Val Asn Ser Leu Gly

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Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

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Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

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Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

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Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

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Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

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Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Xaa His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

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Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

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Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

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Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Xaa Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

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Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

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<400> 7

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

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Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

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Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Xaa Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 8

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<223> Synthetic: IL-10_N126X

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<220>

<221> other_feature

<222> (127)..(127)

<223> Xaa is an unnatural amino acid

<400> 8

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Xaa Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 9

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N129X

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (130)..(130)

<223> Xaa is an unnatural amino acid

<400> 9

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Xaa Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 10

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K130X

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (131)..(131)

<223> Xaa is an unnatural amino acid

<400> 10

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Xaa Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 11

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N82[AzK]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (83)..(83)

<223> Xaa represents [AzK]

<400> 11

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Xaa Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 12

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K88[AzK]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (89)..(89)

<223> Xaa represents [AzK]

<400> 12

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Xaa Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 13

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_A89[AzK]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (90)..(90)

<223> Xaa represents [AzK]

<400> 13

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Xaa His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 14

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K99[AzK]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (100)..(100)

<223> Xaa represents [AzK]

<400> 14

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Xaa Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 15

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K125[AzK]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (126)..(126)

<223> Xaa represents [AzK]

<400> 15

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Xaa Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 16

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N126[AzK]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (127)..(127)

<223> Xaa represents [AzK]

<400> 16

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Xaa Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 17

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N129[AzK]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (130)..(130)

<223> Xaa represents [AzK]

<400> 17

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Xaa Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 18

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K130[AzK]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (131)..(131)

<223> Xaa represents [AzK]

<400> 18

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Xaa Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 19

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N82[AzK_PEG]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (83)..(83)

<223> Xaa is [Azk_PEG]

<400> 19

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Xaa Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 20

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K88[AzK_PEG]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (89)..(89)

<223> Xaa is [Azk_PEG]

<400> 20

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Xaa Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 21

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_A89[AzK_PEG]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (90)..(90)

<223> Xaa is [Azk_PEG]

<400> 21

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Xaa His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 22

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K99[AzK_PEG]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (100)..(100)

<223> Xaa is [Azk_PEG]

<400> 22

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Xaa Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 23

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K125[AzK_PEG]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (126)..(126)

<223> Xaa is [Azk_PEG]

<400> 23

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Xaa Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 24

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N126[AzK_PEG]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (127)..(127)

<223> Xaa is [Azk_PEG]

<400> 24

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Xaa Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 25

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N129[AzK_PEG]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (130)..(130)

<223> Xaa is [Azk_PEG]

<400> 25

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Xaa Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 26

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K130[AzK_PEG]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (131)..(131)

<223> Xaa is [Azk_PEG]

<400> 26

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Xaa Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 27

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N82[AzK_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (83)..(83)

<223> Xaa is [AzK_PEG20kDa]

<400> 27

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Xaa Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 28

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K88[AzK_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (89)..(89)

<223> Xaa is [AzK_PEG20kDa]

<400> 28

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Xaa Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 29

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_A89[AzK_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (90)..(90)

<223> Xaa is [AzK_PEG20kDa]

<400> 29

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Xaa His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 30

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K99[AzK_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (100)..(100)

<223> Xaa is [AzK_PEG20kDa]

<400> 30

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Xaa Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 31

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K125[AzK_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (126)..(126)

<223> Xaa is [AzK_PEG20kDa]

<400> 31

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Xaa Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 32

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N126[AzK_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (127)..(127)

<223> Xaa is [AzK_PEG20kDa]

<400> 32

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Xaa Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 33

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N129[AzK_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (130)..(130)

<223> Xaa is [AzK_PEG20kDa]

<400> 33

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Xaa Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 34

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K130[AzK_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (131)..(131)

<223> Xaa is [AzK_PEG20kDa]

<400> 34

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Xaa Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 35

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N82[AzK_PEG30kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (83)..(83)

<223> Xaa is [AzK_PEG30kDa]

<400> 35

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Xaa Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 36

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K88[AzK_PEG30kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (89)..(89)

<223> Xaa is [AzK_PEG30kDa]

<400> 36

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Xaa Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 37

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_A89[AzK_PEG30kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (90)..(90)

<223> Xaa is [AzK_PEG30kDa]

<400> 37

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Xaa His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 38

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K99[AzK_PEG30kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (100)..(100)

<223> Xaa is [AzK_PEG30kDa]

<400> 38

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Xaa Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 39

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K125[AzK_PEG30kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (126)..(126)

<223> Xaa is [AzK_PEG30kDa]

<400> 39

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Xaa Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 40

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N126[AzK_PEG30kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (127)..(127)

<223> Xaa is [AzK_PEG30kDa]

<400> 40

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Xaa Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 41

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N129[AzK_PEG30kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (130)..(130)

<223> Xaa is [AzK_PEG30kDa]

<400> 41

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Xaa Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 42

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K130[AzK_PEG30kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (131)..(131)

<223> Xaa is [AzK_PEG30kDa]

<400> 42

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Xaa Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 43

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N82[AzK_L1_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (83)..(83)

<223> Xaa is [AzK_L1_PEG20kDa]

<400> 43

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Xaa Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 44

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K88[AzK__L1_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (89)..(89)

<223> Xaa is [AzK_L1_PEG20kDa]

<400> 44

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Xaa Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 45

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_A89[AzK_L1_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (90)..(90)

<223> Xaa is [AzK_L1_PEG20kDa]

<400> 45

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Xaa His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 46

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K99[AzK_L1_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (100)..(100)

<223> Xaa is [AzK_L1_PEG20kDa]

<400> 46

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Xaa Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 47

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K125[AzK_L1_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (126)..(126)

<223> Xaa is [AzK_L1_PEG20kDa]

<400> 47

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Xaa Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 48

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N126[AzK_L1_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (127)..(127)

<223> Xaa is [AzK_L1_PEG20kDa]

<400> 48

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Xaa Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 49

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N129[AzK_L1_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (130)..(130)

<223> Xaa is [AzK_L1_PEG20kDa]

<400> 49

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Xaa Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 50

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K130[AzK_L1_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (131)..(131)

<223> Xaa is [AzK_L1_PEG20kDa]

<400> 50

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Xaa Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 51

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N82[AzK_L1_PEG30kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (83)..(83)

<223> Xaa is [AzK_L1_PEG30kDa]

<400> 51

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Xaa Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 52

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K88[AzK__L1_PEG30kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (89)..(89)

<223> Xaa is [AzK_L1_PEG30kDa]

<400> 52

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Xaa Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 53

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_A89[AzK_L1_PEG30kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (90)..(90)

<223> Xaa is [AzK_L1_PEG30kDa]

<400> 53

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Xaa His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 54

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K99[AzK_L1_PEG30kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (100)..(100)

<223> Xaa is [AzK_L1_PEG30kDa]

<400> 54

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Xaa Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 55

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K125[AzK_L1_PEG30kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (126)..(126)

<223> Xaa is [AzK_L1_PEG30kDa]

<400> 55

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Xaa Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 56

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N126[AzK_L1_PEG30kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (127)..(127)

<223> Xaa is [AzK_L1_PEG30kDa]

<400> 56

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Xaa Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 57

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N129[AzK_L1_PEG30kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (130)..(130)

<223> Xaa is [AzK_L1_PEG30kDa]

<400> 57

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Xaa Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 58

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K130[AzK_L1_PEG30kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (131)..(131)

<223> Xaa is [AzK_L1_PEG30kDa]

<400> 58

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Xaa Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 59

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N82[AzK_L1_PEG]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (83)..(83)

<223> Xaa is [AzK_L1_PEG]

<400> 59

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Xaa Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 60

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K88[AzK_L1_PEG]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (89)..(89)

<223> Xaa is [AzK_L1_PEG]

<400> 60

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Xaa Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 61

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_A89[AzK_L1_PEG]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (90)..(90)

<223> Xaa is [AzK_L1_PEG]

<400> 61

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Xaa His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 62

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K99[AzK_L1_PEG]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (100)..(100)

<223> Xaa is [AzK_L1_PEG]

<400> 62

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Xaa Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 63

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K125[AzK_L1_PEG]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (126)..(126)

<223> Xaa is [AzK_L1_PEG]

<400> 63

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Xaa Asn Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 64

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N126[AzK_L1_PEG]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (127)..(127)

<223> Xaa is [AzK_L1_PEG]

<400> 64

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Xaa Ala

115 120 125

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 65

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_N129[AzK_L1_PEG]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (130)..(130)

<223> Xaa is [AzK_L1_PEG]

<400> 65

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Xaa Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 66

<211> 161

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: IL-10_K130[AzK_L1_PEG]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (131)..(131)

<223> Xaa is [AzK_L1_PEG]

<400> 66

Met Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe

1 5 10 15

Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser

20 25 30

Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu

35 40 45

Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln

50 55 60

Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln

65 70 75 80

Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly

85 90 95

Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe

100 105 110

Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala

115 120 125

Phe Asn Xaa Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

130 135 140

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg

145 150 155 160

Asn

<210> 67

<211> 176

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: His-IL-10

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<400> 67

Met His His His His His Gly Ser Ser Glu Asn Leu Tyr Phe Gln

1 5 10 15

Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro

20 25 30

Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

35 40 45

Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

50 55 60

Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

65 70 75 80

Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala

85 90 95

Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu

100 105 110

Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

115 120 125

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe

130 135 140

Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

145 150 155 160

Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn

165 170 175

<210> 68

<211> 176

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: His-IL-10_N82[AzK_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (98)..(98)

<223> Xaa is [AzK_PEG20kDa]

<400> 68

Met His His His His His Gly Ser Ser Glu Asn Leu Tyr Phe Gln

1 5 10 15

Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro

20 25 30

Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

35 40 45

Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

50 55 60

Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

65 70 75 80

Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala

85 90 95

Glu Xaa Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu

100 105 110

Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

115 120 125

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe

130 135 140

Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

145 150 155 160

Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn

165 170 175

<210> 69

<211> 176

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: His-IL-10_K99[AzK_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (115)..(115)

<223> Xaa is [AzK_PEG20kDa]

<400> 69

Met His His His His His Gly Ser Ser Glu Asn Leu Tyr Phe Gln

1 5 10 15

Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro

20 25 30

Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

35 40 45

Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

50 55 60

Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

65 70 75 80

Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala

85 90 95

Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu

100 105 110

Asn Leu Xaa Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

115 120 125

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe

130 135 140

Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

145 150 155 160

Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn

165 170 175

<210> 70

<211> 176

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: His-IL-10_K125[AzK_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (141)..(141)

<223> Xaa is [AzK_PEG20kDa]

<400> 70

Met His His His His His Gly Ser Ser Glu Asn Leu Tyr Phe Gln

1 5 10 15

Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro

20 25 30

Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

35 40 45

Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

50 55 60

Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

65 70 75 80

Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala

85 90 95

Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu

100 105 110

Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

115 120 125

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Xaa Asn Ala Phe

130 135 140

Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

145 150 155 160

Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn

165 170 175

<210> 71

<211> 176

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: His-IL-10_N129[AzK_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (145)..(145)

<223> Xaa is [AzK_PEG20kDa]

<400> 71

Met His His His His His Gly Ser Ser Glu Asn Leu Tyr Phe Gln

1 5 10 15

Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro

20 25 30

Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

35 40 45

Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

50 55 60

Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

65 70 75 80

Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala

85 90 95

Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu

100 105 110

Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

115 120 125

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe

130 135 140

Xaa Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

145 150 155 160

Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn

165 170 175

<210> 72

<211> 176

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: His-IL-10_K130[AzK_PEG20kDa]

<220>

<221> other_feature

<222> (1)..(1)

<223> Met may or may not be present; as

alternatively, the Met residue can substitute for and replace Ser at the N-terminus

<220>

<221> other_feature

<222> (146)..(146)

<223> Xaa is [AzK_PEG20kDa]

<400> 72

Met His His His His His Gly Ser Ser Glu Asn Leu Tyr Phe Gln

1 5 10 15

Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro

20 25 30

Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

35 40 45

Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

50 55 60

Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

65 70 75 80

Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala

85 90 95

Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu

100 105 110

Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

115 120 125

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe

130 135 140

Asn Xaa Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

145 150 155 160

Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn

165 170 175

<210> 73

<211> 176

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Synthetic: His-IL-10_N82[AzK_PEG10kDa]

<220>

<221> other_feature

<222> (98)..(98)

<223> Xaa is [AzK_PEG10kDa]

<400> 73

Met His His His His His Gly Ser Ser Glu Asn Leu Tyr Phe Gln

1 5 10 15

Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro

20 25 30

Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

35 40 45

Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

50 55 60

Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

65 70 75 80

Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala

85 90 95

Glu Xaa Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu

100 105 110

Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

115 120 125

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe

130 135 140

Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

145 150 155 160

Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn

165 170 175

<210> 74

<211> 575

<212> PROTEIN

<213> Phaeodactylum tricornutum

<220>

<221> other_feature

<223> non-truncated PtNTT2; CCAP 1055/1 Phaeodactylum tricornutum

<400> 74

Met Arg Pro Tyr Pro Thr Ile Ala Leu Ile Ser Val Phe Leu Ser Ala

1 5 10 15

Ala Thr Arg Ile Ser Ala Thr Ser Ser His Gln Ala Ser Ala Leu Pro

20 25 30

Val Lys Lys Gly Thr His Val Pro Asp Ser Pro Lys Leu Ser Lys Leu

35 40 45

Tyr Ile Met Ala Lys Thr Lys Ser Val Ser Ser Ser Phe Asp Pro Pro

50 55 60

Arg Gly Gly Ser Thr Val Ala Pro Thr Thr Pro Leu Ala Thr Gly Gly

65 70 75 80

Ala Leu Arg Lys Val Arg Gln Ala Val Phe Pro Ile Tyr Gly Asn Gln

85 90 95

Glu Val Thr Lys Phe Leu Leu Ile Gly Ser Ile Lys Phe Phe Ile Ile

100 105 110

Leu Ala Leu Thr Leu Thr Arg Asp Thr Lys Asp Thr Leu Ile Val Thr

115 120 125

Gln Cys Gly Ala Glu Ala Ile Ala Phe Leu Lys Ile Tyr Gly Val Leu

130 135 140

Pro Ala Ala Thr Ala Phe Ile Ala Leu Tyr Ser Lys Met Ser Asn Ala

145 150 155 160

Met Gly Lys Lys Met Leu Phe Tyr Ser Thr Cys Ile Pro Phe Phe Thr

165 170 175

Phe Phe Gly Leu Phe Asp Val Phe Ile Tyr Pro Asn Ala Glu Arg Leu

180 185 190

His Pro Ser Leu Glu Ala Val Gln Ala Ile Leu Pro Gly Gly Ala Ala

195 200 205

Ser Gly Gly Met Ala Val Leu Ala Lys Ile Ala Thr His Trp Thr Ser

210 215 220

Ala Leu Phe Tyr Val Met Ala Glu Ile Tyr Ser Ser Val Ser Val Gly

225 230 235 240

Leu Leu Phe Trp Gln Phe Ala Asn Asp Val Val Asn Val Asp Gln Ala

245 250 255

Lys Arg Phe Tyr Pro Leu Phe Ala Gln Met Ser Gly Leu Ala Pro Val

260 265 270

Leu Ala Gly Gln Tyr Val Val Arg Phe Ala Ser Lys Ala Val Asn Phe

275 280 285

Glu Ala Ser Met His Arg Leu Thr Ala Ala Val Thr Phe Ala Gly Ile

290 295 300

Met Ile Cys Ile Phe Tyr Gln Leu Ser Ser Ser Tyr Val Glu Arg Thr

305 310 315 320

Glu Ser Ala Lys Pro Ala Ala Asp Asn Glu Gln Ser Ile Lys Pro Lys

325 330 335

Lys Lys Lys Pro Lys Met Ser Met Val Glu Ser Gly Lys Phe Leu Ala

340 345 350

Ser Ser Gln Tyr Leu Arg Leu Ile Ala Met Leu Val Leu Gly Tyr Gly

355 360 365

Leu Ser Ile Asn Phe Thr Glu Ile Met Trp Lys Ser Leu Val Lys Lys

370 375 380

Gln Tyr Pro Asp Pro Leu Asp Tyr Gln Arg Phe Met Gly Asn Phe Ser

385 390 395 400

Ser Ala Val Gly Leu Ser Thr Cys Ile Val Ile Phe Phe Gly Val His

405 410 415

Val Ile Arg Leu Leu Gly Trp Lys Val Gly Ala Leu Ala Thr Pro Gly

420 425 430

Ile Met Ala Ile Leu Ala Leu Pro Phe Phe Ala Cys Ile Leu Leu Gly

435 440 445

Leu Asp Ser Pro Ala Arg Leu Glu Ile Ala Val Ile Phe Gly Thr Ile

450 455 460

Gln Ser Leu Leu Ser Lys Thr Ser Lys Tyr Ala Leu Phe Asp Pro Thr

465 470 475 480

Thr Gln Met Ala Tyr Ile Pro Leu Asp Asp Glu Ser Lys Val Lys Gly

485 490 495

Lys Ala Ala Ile Asp Val Leu Gly Ser Arg Ile Gly Lys Ser Gly Gly

500 505 510

Ser Leu Ile Gln Gln Gly Leu Val Phe Val Phe Gly Asn Ile Ile Asn

515 520 525

Ala Ala Pro Val Val Gly Val Val Tyr Tyr Ser Val Leu Val Ala Trp

530 535 540

Met Ser Ala Ala Gly Arg Leu Ser Gly Leu Phe Gln Ala Gln Thr Glu

545 550 555 560

Met Asp Lys Ala Asp Lys Met Glu Ala Lys Thr Asn Lys Glu Lys

565 570 575

<210> 75

<211> 531

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic: Natural His-TEV-IL-10

<400> 75

atgcatcatc accatcatca tggtagcagc gaaaatctgt attttcagag ccctggtcag 60

ggcacccaga gcgaaaattc atgtacccat tttccgggta atctgccgaa tatgctgcgc 120

gatctgcgtg atgcatttag ccgtgttaaa acctttttcc agatgaaaga tcagctggat 180

aatctgctgc tgaaagaaag cctgctggaa gatttcaaag gttatctggg ttgtcaggca 240

ctgagcgaaa tgattcagtt ttatctggaa gaagttatgc cgcaggcaga aaatcaggat 300

ccggatatta aagcacatgt taatagcctg ggcgaaaatc tgaaaaccct gcgtctgcgc 360

ctgcgtcgtt gtcatcgttt tctgccgtgt gaaaacaaaa gcaaagcagt tgaacaggtg 420

aaaaacgcct ttaacaaact gcaagagaaa ggcatctata aagccatgag cgaattcgac 480

atcttcatca actatatcga agcctacatg accatgaaaa tccgcaatta a 531

<210> 76

<211> 486

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic: Natural IL-10

<400> 76

atgagccctg gtcagggaac ccaatccgaa aattcatgta cccatttcc gggtaatctg 60

ccgaatatgc tgcgcgatct gcgtgatgca tttagccgtg ttaaaacctt tttccagatg 120

aaagatcagc tggataatct gctgctgaaa gaaagcctgc tggaagattt caaaggttat 180

ctgggttgtc aggcactgag cgaaatgatt cagttttatc tggaagaagt tatgccgcag 240

gcagaaaatc aggatccgga tattaaagca catgttaata gcctgggcga aaatctgaaa 300

accctgcgtc tgcgcctgcg tcgttgtcat cgttttctgc cgtgtgaaaa caaaagcaaa 360

gcagttgaac aggtgaaaaa cgcctttaac aaactgcaag agaaaggcat ctataaagcc 420

atgagcgaat tcgacatctt catcaactat atcgaagcct acatgaccat gaaaatccgc 480

aattaa 486

<210> 77

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Repeat motif for the conjugated fragment of elastin-like

polypeptide

<220>

<221>MISC_FEATURE

<222> (4)..(4)

<223> X represents any amino acid except proline

<400> 77

Val Pro Gly Xaa Gly

1 5

<210> 78

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Illustrative cytoplasmic transduction peptide

<220>

<221>MISC_FEATURE

<222> (6)..(6)

<223> O-glycosylation site

<220>

<221>MISC_FEATURE

<222> (12)..(12)

<223> O-glycosylation site

<220>

<221>MISC_FEATURE

<222> (17)..(17)

<223> O-glycosylation site

<220>

<221>MISC_FEATURE

<222> (23)..(23)

<223> O-glycosylation site

<400> 78

Phe Gln Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser Pro

1 5 10 15

Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

20 25 30

<210> 79

<211> 31

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Illustrative cytoplasmic transduction peptide

<220>

<221>MISC_FEATURE

<222> (7)..(7)

<223> O-glycosylation site

<220>

<221>MISC_FEATURE

<222> (13)..(3)

<223> O-glycosylation site

<220>

<221>MISC_FEATURE

<222> (18)..(18)

<223> O-glycosylation site

<220>

<221>MISC_FEATURE

<222> (24)..(24)

<223> O-glycosylation site

<400> 79

Phe Gln Asp Ser Ser Ser Ser Lys Ala Pro Pro Pro Ser Leu Pro Ser

1 5 10 15

Pro Ser Arg Leu Pro Gly Pro Ser Asp Thr Pro Ile Leu Pro Gln

20 25 30

<---

Claims (75)

1. An IL-10 conjugate for the treatment of cancer comprising an IL-10 polypeptide comprising the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 polypeptide is replaced by a structure of formula (I) (I), wherein at least one amino acid residue replaced by the structure of formula (I) is located in the IL-10 polypeptide at a position selected from N82, K99, K125, N129 and K130; Z represents CH ₂ and Y represents ; Y represents CH ₂ and Z represents ; Z represents CH ₂ and Y represents ; or Y represents CH ₂ and Z represents ; W is a PEG group having a molecular weight selected from about 5 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, and about 60 kDa; q is equal to 1, 2, or 3; X has a structure ; X-1 indicates the point of attachment to the preceding amino acid residue; and X+1 indicates the point of attachment to the next amino acid residue. 2. The IL-10 conjugate of claim 1, wherein Z is CH ₂ and Y is . 3. The IL-10 conjugate of claim 1, wherein Y is CH ₂ and Z is . 4. The IL-10 conjugate of claim 1, wherein Z is CH ₂ and Y is . 5. The IL-10 conjugate of claim 1, wherein Y is CH ₂ and Z is . 6. The IL-10 conjugate of any one of claims 1-5, wherein the PEG group has a molecular weight selected from about 5 kDa, about 10 kDa, about 20 kDa, and about 30 kDa. 7. The IL-10 conjugate of claim 6, wherein the PEG group has a molecular weight selected from about 10 kDa and about 20 kDa. 8. An IL-10 conjugate according to any one of claims 1-7, wherein the position is selected from N82, K99, N129 and K130. 9. The IL-10 conjugate of claim 8, wherein the position is selected from N82 and N129. 10. The IL-10 conjugate of claim 1, wherein the structure of formula (I) has a structure of formula (X) or formula (XI): (X); (XI); where q is 1, 2, or 3; n is an integer such that the PEG group having the structure -( _OCH2CH2 ) _{n -OCH3 has} a molecular weight of about 5 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, and about 60 kDa; and Wavy lines indicate covalent bonds with amino acid residues within SEQ ID NO: 1 that are not replaced. 11. The IL-10 conjugate of claim 10, wherein the position is selected from N82, K99, N129 and K130. 12. The IL-10 conjugate of claim 11, wherein the position is selected from N82 and N129. 13. The IL-10 conjugate of any one of claims 10-12, wherein n is an integer such that the PEG group having the structure -(OCH ₂ CH ₂ ) _n -OCH ₃ has a molecular weight of about 10 kDa or about 20 kDa. 14. A pharmaceutical composition for treating cancer, comprising an effective amount of a mixture of IL-10 conjugates according to any one of claims 10-13, wherein the mixture comprises IL-10 conjugates in which the structure of formula (I) has a structure of formula (X), and IL-10 conjugates in which the structure of formula (I) has a structure of formula (XI). 15. The IL-10 conjugate of claim 1, wherein the structure of formula (I) has a structure of formula (XII) or formula (XIII): (XII); (XIII); where q is 1, 2, or 3; n is an integer such that the PEG group having the structure -( _OCH2CH2 ) _{n -OCH3 has} a molecular weight of about 5 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, and about 60 kDa; and Wavy lines indicate covalent bonds with amino acid residues within SEQ ID NO: 1 that are not replaced. 16. The IL-10 conjugate of claim 15, wherein the position is selected from N82, K99, N129 and K130. 17. The IL-10 conjugate of claim 15 or 16, wherein the position is selected from N82 and N129. 18. The IL-10 conjugate of any one of claims 15-17, wherein n is an integer such that the PEG group having the structure -(OCH ₂ CH ₂ ) _n -OCH ₃ has a molecular weight of about 10 kDa or about 20 kDa. 19. A pharmaceutical composition for treating cancer, comprising an effective amount of a mixture of IL-10 conjugates according to any one of claims 15-18, wherein the mixture comprises IL-10 conjugates in which the structure of formula (I) has a structure of formula (XII), and IL-10 conjugates in which the structure of formula (I) has a structure of formula (XIII). 20. The IL-10 conjugate of any one of claims 1-13 and 15-18, wherein q is 1. 21. The IL-10 conjugate of any one of claims 1-13 and 15-18, wherein q is 2. 22. The IL-10 conjugate of any one of claims 1-13 and 15-18, wherein q is 3. 23. The pharmaceutical composition according to claim 14 or 19, where q is equal to 1. 24. The pharmaceutical composition according to claim 14 or 19, where q is equal to 2. 25. The pharmaceutical composition according to claim 14 or 19, where q is equal to 3. 26. A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of an IL-10 conjugate according to any one of claims 1-13, 15-18, and 20-22. 27. A method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition according to any one of claims 14, 19 and 23-25. 28. The method of claim 26 or 27, wherein the cancer is selected from renal cell carcinoma (RCC), non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin's lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability cancer, microsatellite stable cancer, microsatellite stable colorectal cancer, gastric cancer, cervical cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), melanoma, small cell lung cancer (SCLC), esophageal cancer, glioblastoma, mesothelioma, breast cancer, triple-negative breast cancer, prostate cancer, bladder cancer, ovarian cancer, tumors with a moderate to low mutational burden, cutaneous squamous cell carcinoma (CSCC), cutaneous squamous cell carcinoma (SCSC), tumors with low to absent PD-L1 expression, tumors systemically disseminating to the liver and CNS beyond their primary site of anatomical origin, and diffuse large B-cell lymphoma. 29. The method of any one of claims 26-28, wherein the IL-10 conjugate is administered to the subject once daily, twice daily, three times daily, once weekly, once every two weeks, once every three weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, or once every 8 weeks. 30. The method according to any one of claims 26-29, wherein the IL-10 conjugate is administered to the subject by intravenous administration. 31. The method according to any one of claims 26-29, wherein the IL-10 conjugate is administered to the subject by subcutaneous administration. 32. A method for producing an IL-10 conjugate, comprising carrying out a reaction of an IL-10 polypeptide containing an unnatural amino acid of the formula at position X of the IL-10 polypeptide, wherein the IL-10 polypeptide comprises the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid residue in the IL-10 polypeptide is replaced with a non-natural amino acid, position X-1 indicates the point of attachment to the previous amino acid residue; position X+1 indicates the point of attachment to the next amino acid residue, and position X indicates the position of the amino acid that is replaced by the non-natural amino acid, with mPEG-DBCO formulas or , where q is 1, 2, or 3 and n is such that mPEG-DBCO comprises PEG having a molecular weight of about 5 kDa, about 10 kDa, about 15 kDa, about 20 kDa, about 25 kDa, about 30 kDa, about 35 kDa, about 40 kDa, about 45 kDa, about 50 kDa, or about 60 kDa, thus obtaining an IL-10 conjugate, where the position of X is selected from N82, K99, K125, N129 and K130. 33. The method according to item 32, where q is equal to 1. 34. The method according to item 32, where q is equal to 2. 35. The method according to item 32, where q is equal to 3. 36. The method of claim 32, wherein the IL-10 conjugate is an IL-10 conjugate according to any one of claims 1-13, 15-18, and 20-22.