IL-21 Polypeptide and Method of Use

Abstract

A variety of polypeptides, including IL-21 variants, are provided herein. Conjugates comprising a polypeptide and at least one additional moiety are further provided herein. Related nucleic acid molecules, vectors, transformed or host cells, pharmaceutical compositions and kits are further provided herein. Methods for preparing the polypeptides or conjugates described herein, and methods of treatment using them are also provided herein.

Description

Background Art

[0001] Background Interleukin-21 (IL-21) has a four-helix bundle structure and exists as a monomer. In humans, two isoforms of IL-21 are known, each of which is derived from a precursor molecule. The first IL-21 isoform contains 162 amino acids (aa) including a 29aa-long signal peptide. The second IL-21 isoform contains 153aa including a 29aa-long signal peptide. The amino acid sequences of isoform 1 and isoform 2 are provided herein as SEQ ID NO: 3 and SEQ ID NO: 4, respectively. IL-21 may also refer to a fragment of full-length IL-21 isoform 1 (SEQ ID NO: 1) containing a signal peptide and a 133-amino acid mature polypeptide, or a fragment of full-length IL-21 isoform 2 (SEQ ID NO: 2) containing a signal peptide and a 124-amino acid mature polypeptide. IL-21 is mainly expressed in follicular helper T (Tfh) cells, Th2 and Th17 cell subsets, and NK cells. IL-21 has an antitumor effect by enhancing the cytotoxicity of CD8 + T cells and NK cells and stimulating their maturation (see, for example, Vidard L et al., J Immunol. 2019;203(3):676-685). Low abundance of IL-21 contributes to the decreased activity of cytotoxic T lymphocytes in the tumor microenvironment. Since IL-21R is widely expressed on the surface of normal lymphoid tissues including spleen, thymus, lymph nodes, peripheral blood lymphocytes, T cells, B cells and NK cells, wild-type IL-21 can cause a systemic inflammatory effect in related cancer treatment.

Prior Art Documents

Non-Patent Documents

[0002]

Non-Patent Document 1

[0003] Abstract In response to the need for IL-21 related therapeutic agents having better safety and stability profiles, improved pharmacokinetics, and maximized pharmacodynamics profiles, as well as stronger tumor inhibitory efficacy, polypeptides are provided herein that include an IL-21 variant having an amino acid residue substitution at position P79 corresponding to wild-type human IL-21. In some embodiments, the wild-type human IL-21 includes the sequence shown in SEQ ID NO: 1 or 2.

[0004] In some embodiments, the amino acid residue substitution at position P79 is P79E or P79C. In some specific embodiments, the amino acid residue substitution at position P79 is P79E. In other specific embodiments, the amino acid residue substitution at position P79 is P79C, and the polypeptide further includes one or more amino acid residue substitutions with cysteine in the region of positions 1 to 10 corresponding to wild-type human IL-21. In some cases, the one or more amino acid residue substitutions are selected from the group consisting of R5C, H6C, and R9C. In some cases, the cysteine residues of the one or more amino acid residue substitutions in the region of positions 1 to 10 form a disulfide bond together with the cysteine residue at position P79.

[0005] In some embodiments, the polypeptide includes one or more amino acid residue substitutions at positions selected from the group consisting of R5, H6, R9, Q12, L13, D15, I16, S70, K72, K73, R76, and P78. In some embodiments, the polypeptide includes one or more amino acid residue substitutions at positions selected from the group consisting of S70, K72, K73, and R76.

[0006] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position K73 corresponding to wild-type human IL-21. In some cases, the amino acid residue substitution at position K73 is an aromatic amino acid. In certain cases, the amino acid residue substitution at position K73 is K73Y or K73F.

[0007] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position S70 corresponding to wild-type human IL-21. In some cases, the amino acid residue substitution at S70 is S70L or S70A.

[0008] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position R76 corresponding to wild-type human IL-21. In some cases, the amino acid residue substitution at R76 is R76F.

[0009] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position K72 corresponding to wild-type human IL-21. In some cases, the amino acid residue substitution at K72 is a non-aromatic amino acid containing a side-chain hydroxyl. In some specific cases, the amino acid residue substitution at K72 is K72S.

[0010] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position Q12 corresponding to wild-type human IL-21. In some cases, the amino acid residue substitution at Q12 is Q12W.

[0011] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position L13 corresponding to wild-type human IL-21. In some cases, the amino acid residue substitution at L13 is L13A.

[0012] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position D15 corresponding to wild-type human IL-21. In some cases, the amino acid residue substitution at D15 is D15L, D15K or D15R.

[0013] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position I16 corresponding to wild-type human IL-21. In some cases, the amino acid residue substitution at I16 is I16R or I16W.

[0014] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position P78 corresponding to wild-type human IL-21. In some cases, the amino acid residue substitution at P78 is P78G or P78E.

[0015] In some embodiments, the polypeptide is STNAGRRQKHR (SEQ ID NO: 71) replaced with P79E;K73Y, P79E;K73F, P79E;S70L, K73F, P79E;S70L, K73Y, P79E;S70L, K72S, K73F, P79E;S70L, K72S, K73Y, P79E;D15K, S70L, K73F, P79E;D15R, S70L, K73F, P79E;I16W, S70L, K73F, P79E;D15L, S70L, K73F, P79E;L13A, S70L, K73F, P79E;D15R, R76F, P78E, P79E;D15L, I16R, S70L, K73Y, P79E;D15K, I16W, S70L, R76F, P79E;D15R, I16W, S70L, R76F, P79E;GGGSEGGGS (SEQ ID NO: 72) (STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72)); P79E, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72); K73Y, P79E, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72); K73F, P79E, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72); S70L, K73F, P79E, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72); S70L, K73Y, P79E, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72); D15K, S70L, K73F, P79E, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72); D15R, S70L, K73F, P79E, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72); I16W, S70L, K73F, P79E, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72); S70L, K72S, K73F, P79E, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72); S70L, K72S, K73Y, P79E, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72); D15R, I16W, S70L, K73Y, P79E, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72);and further comprises an amino acid residue mutation selected from the group consisting of D15L, I16R, S70L, K73Y, P79E, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72).;

[0016] In some embodiments, the polypeptide further comprises an amino acid residue mutation selected from the group consisting of S70L, K73F, P79E; S70L, K72S, K73F, P79E; S70L, K73Y, P79E; and S70L, K72S, K73Y, P79E.

[0017] In some embodiments, the polypeptide further comprises an amino acid residue mutation selected from the group consisting of P79C; R5C, P79C; H6C, P79C; R9C, P79C; R5C, R76F, P79C; H6C, R76F, P79C; H6C, K73Y, P79C; H6C, K73F, P79C; H6C, S70L, K73Y, P79C; H6C, S70L, K73F, P79C; R9C, D15R, P78G, P79C; H6C, D15L, I16R, S70L, K73Y, P79C; R9C, D15L, I16R, S70L, K73Y, P79C; H6C, P79C, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72); H6C, K73Y, P79C, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72); H6C, K73F, P79C, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72); and H6C, S70L, P79C, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72).

[0018] In another aspect, provided herein is a polypeptide comprising an IL-21 variant having an amino acid residue substitution at position S70 or K73 corresponding to wild-type human IL-21. In some embodiments, wild-type human IL-21 comprises the sequence set forth in SEQ ID NO: 1 or 2.

[0019] In some embodiments, the polypeptide comprises an amino acid residue substitution with an aromatic amino acid at position K73 corresponding to wild-type human IL-21. In some cases, the amino acid residue substitution at position K73 is K73Y and K73F. In some embodiments, the polypeptide comprises an amino acid residue substitution at position S70. In some cases, the amino acid residue substitution at position S70 is S70L or S70A.

[0020] In some embodiments, the polypeptide comprises an amino acid residue substitution (e.g., S70L or S70A) at position S70 corresponding to wild-type human IL-21 and an amino acid residue substitution at position K73. In certain embodiments, the polypeptide comprises an amino acid residue substitution with an aromatic amino acid at position K73 corresponding to wild-type human IL-21. In certain cases, the amino acid residue substitution at position K73 is K73Y or K73F. In other embodiments, the polypeptide further comprises one, two, three, or more amino acid residue substitutions at positions selected from the group consisting of R5, H6, R9, Q12, L13, D15, I16, K72, R76, P78, and P79. In certain embodiments, the polypeptide further comprises one, two, three, or four amino acid residue substitutions at positions selected from the group consisting of Q12, D15, I16, and K72. In some cases, the polypeptide comprises an amino acid residue substitution selected from the group consisting of I16R and I16W. In other cases, the polypeptide comprises an amino acid residue substitution selected from the group consisting of D15L, D15K, and D15R. In still other cases, the polypeptide comprises an amino acid residue substitution selected from the group consisting of K72S and Q12W.

[0021] In some embodiments, the polypeptide comprises an amino acid residue substitution (e.g., S70L or S70A) at position S70 corresponding to wild-type human IL-21 and an amino acid residue substitution at position R76. In some cases, the amino acid residue substitution at position R76 is R76F. In some cases, the polypeptide further comprises one, two, three, or more amino acid residue substitutions at positions selected from the group consisting of R5, H6, R9, Q12, L13, D15, I16, K72, K73, P78, and P79. In some cases, the polypeptide further comprises one, two, three, or four amino acid residue substitutions at positions selected from the group consisting of Q12, D15, I16, and K72. In some cases, the polypeptide comprises an amino acid residue substitution selected from the group consisting of I16R and I16W. In other cases, the polypeptide comprises an amino acid residue substitution selected from the group consisting of D15L, D15K, and D15R. In still other cases, the polypeptide comprises an amino acid residue substitution selected from the group consisting of K72S and Q12W.

[0022] In some embodiments, the polypeptide comprises an amino acid residue mutation selected from the group consisting of K73Y; K73F; S70L, K73Y; S70L, K73F; S70L, K72S, K73Y; S70A, K72S, K73F; Q12W, S70A, R76F; D15K, S70A, R76F; D15L, S70A, R76F; D15R, S70A, R76F; I16R, S70L, K73Y; D15L, I16R, S70L, K73Y; D15K, I16W, S70L, K73Y; D15K, I16W, S70L, R76F; D15R, I16W, S70L, R76F; I16W, S70L, K72S, K73F; D15R, I16W, S70L, K73Y; and D15K, I16W, S70L, K72S, K73Y.

[0023] In some embodiments, the amino acid segment STNAGRRQKHR (SEQ ID NO: 71) at positions 80-90 corresponding to wild-type human IL-21 is replaced with a short peptide linker. In some embodiments, the length of the short peptide linker is 4-10 amino acid residues, e.g., 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In some embodiments, the length of the short peptide linker is 5-9 amino acid residues. In some embodiments, the length of the short peptide linker is 7-9 amino acid residues. In further embodiments, the short peptide linker comprises one or more Gly-Ser units and optionally one glutamic acid residue. In certain embodiments, the short peptide linker comprises a glutamic acid residue, and the glutamic acid residue is in the middle of the short peptide linker. In certain embodiments, the short peptide linker is selected from the group consisting of GGSEGGS (SEQ ID NO: 73), GSEGS (SEQ ID NO: 74), GGSGGS (SEQ ID NO: 75), GGGSEGGS (SEQ ID NO: 76), GGSEGGGS (SEQ ID NO: 77), GSGGS (SEQ ID NO: 78), GGGSGGS (SEQ ID NO: 79), and GGGSEGGGS (SEQ ID NO: 72).

[0024] In some embodiments, the polypeptide exhibits a lower binding affinity for the IL-21 receptor (IL-21R) than wild-type human IL-21. In certain embodiments, the polypeptide exhibits a decrease in binding affinity for the IL-21R from 1 / 2 to 1 / 1000 compared to wild-type human IL-21. In certain embodiments, the polypeptide has a higher K D value for the IL-21R compared to wild-type human IL-21.

[0025] In another aspect, provided herein is a polypeptide comprising an interleukin-21 (IL-21) variant, wherein the amino acid segment of STNAGRRQKHR (SEQ ID NO: 71) at positions 80 to 90 corresponding to wild-type human IL-21 is replaced with a short peptide linker. In some embodiments, the length of the short peptide linker is 4 to 10 or 5 to 9 amino acid residues. In some cases, the short peptide linker comprises one or more Gly-Ser units and optionally one glutamate residue. In certain cases, the short peptide linker comprises a glutamate residue, and the glutamate residue is in the middle of the short peptide linker. In certain cases, the short peptide linker is selected from the group consisting of GGSEGGS (SEQ ID NO: 73), GSEGS (SEQ ID NO: 74), GGSGGS (SEQ ID NO: 75), GGGSEGGS (SEQ ID NO: 76), GGSEGGGS (SEQ ID NO: 77), GSGGS (SEQ ID NO: 78), GGGSGGS (SEQ ID NO: 79), and GGGSEGGGS (SEQ ID NO: 72).

[0026] In some embodiments, the polypeptide comprises one or more amino acid residue substitutions at positions selected from the group consisting of R5, H6, R9, Q12, L13, D15, I16, S70, K72, K73, R76, P78, and P79. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position R5, and optionally, the substitution is R5C. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position H6, and optionally, the substitution is H6C. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position R9, and optionally, the substitution is R9C. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position Q12, and optionally, the substitution is Q12W. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position L13, and optionally, the substitution is L13A. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position D15, and optionally, the substitution is D15L, D15K, or D15R. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position I16, and optionally, the substitution is I16R or I16W. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position S70, and optionally, the substitution is S70L or S70A. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position K72, and optionally, the substitution is K72S. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position K73, and optionally, the substitution is K73Y or K73F. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position R76, and optionally, the substitution is R76F. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position P78, and optionally, the substitution is P78G or P78E. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position P79, and optionally, the substitution is P79E or P79C.

[0027] In some embodiments, the polypeptide exhibits improved thermal stability compared to wild-type human IL-21. In some embodiments, the polypeptide exhibits a lower binding affinity for the IL-21 receptor (IL-21R) than wild-type IL-21. In some embodiments, the polypeptide exhibits a decrease in binding affinity for IL-21R from one-half to one-thousandth compared to wild-type IL-21. In some embodiments, the polypeptide has a higher K D value compared to wild-type IL-21 for the IL-21R. In some embodiments, the polypeptide exhibits improved efficacy compared to wild-type human IL-21 in inhibiting tumor growth and reducing tumor volume. In some embodiments, the polypeptide exhibits improved efficacy and / or achieves a synergistic effect when fused or co-administered with at least one additional moiety / drug disclosed herein, such as an anti-PD1 antibody, in the treatment of tumors.

[0028] In another aspect, provided herein is a polypeptide comprising an interleukin-21 (IL-21) variant comprising a wild-type human IL-21 amino acid sequence having at least one mutation selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, wherein X1 is L, K, or R, X2 is R or W, X3 is L or A, X4 is Y or F, X5 is G or E, and X6 is E or C. In some embodiments, the wild-type human IL-21 comprises the sequence set forth in SEQ ID NO: 1 or 2.

[0029] In some embodiments, the amino acid segment STNAGRRQKHR (SEQ ID NO: 71) at positions 80-90 corresponding to wild-type human IL-21 is replaced with a short peptide linker. In some embodiments, the length of the short peptide linker is 4-10 amino acid residues, for example, 4, 5, 6, 7, 8, 9 or 10 amino acid residues. In some embodiments, the length of the short peptide linker is 5-9 amino acid residues. In some embodiments, the length of the short peptide linker is 7-9 amino acid residues. In some embodiments, the short peptide linker comprises one or more Gly-Ser units and optionally one glutamic acid residue. In some embodiments, the short peptide linker comprises a glutamic acid residue, and the glutamic acid residue is in the middle of the short peptide linker. In some embodiments, the short peptide linker is selected from the group consisting of GGSEGGS (SEQ ID NO: 73), GSEGS (SEQ ID NO: 74), GGSGGS (SEQ ID NO: 75), GGGSEGGS (SEQ ID NO: 76), GGSEGGGS (SEQ ID NO: 77), GSGGS (SEQ ID NO: 78), GGGSGGS (SEQ ID NO: 79), and GGGSEGGGS (SEQ ID NO: 72).

[0030] In some specific embodiments, the polypeptide comprises a sequence represented by any one of SEQ ID NOs: 5-62. In some specific embodiments, the polypeptide consists of a sequence represented by any one of SEQ ID NOs: 5-62.

[0031] In some embodiments, the polypeptide exhibits a lower binding affinity for the IL-21 receptor (IL-21R) than wild-type human IL-21. In some embodiments, the polypeptide exhibits a decrease in binding affinity for IL-21R from 1 / 2 to 1 / 1000 compared to wild-type human IL-21. In some embodiments, the polypeptide has a higher K D value for IL-21R compared to wild-type human IL-21.

[0032] In another aspect, conjugates are provided herein that include a polypeptide disclosed herein and at least one additional moiety. In some embodiments, the at least one additional moiety includes a crystallizable fragment (Fc) domain of an antibody. In some particular embodiments, the antibody is IgG, IgA, IgD, IgM, or IgE. In some particular embodiments, the IgG is IgG1, IgG2, IgG3, or IgG4.

[0033] In some embodiments, the at least one additional moiety includes an antigen-binding molecule. In certain embodiments, the antigen-binding molecule is an antibody or an antigen-binding fragment thereof. In certain embodiments, the antigen-binding molecule is a multispecific antigen-binding molecule.

[0034] In some embodiments, the antigen-binding molecule targets tumor cells or immune cells. In some embodiments, the antigen is a tumor-associated antigen. In other embodiments, the antigen is an immune checkpoint antigen or an immune checkpoint-related antigen. In certain embodiments, the antigen is involved in an immune checkpoint pathway. In certain embodiments, the antigen is selected from the group consisting of PD-1, PD-L1, TIGIT, CTLA-4, PD-L2, B7-H3, B7-H4, BTLA, LAG3, CD112, CD112R, CD96, TIM-3, CD47, and CEACAM1. In certain embodiments, the antigen is a costimulatory immune checkpoint target. In certain embodiments, the antigen is selected from the group consisting of CD155, ICOS, OX40, CD137, CD137L, CD27, CD28, and GITR.

[0035] In some embodiments, the at least one additional moiety is attached to the C-terminus and / or N-terminus of an IL-21 variant. In some embodiments, the at least one additional moiety is attached to the IL-21 variant directly or via a linker.

[0036] In another aspect, provided herein are nucleic acid molecules encoding the polypeptides disclosed herein or the conjugates disclosed herein. In some embodiments, the nucleic acid molecule is a DNA molecule. In some embodiments, the nucleic acid molecule is an RNA molecule. In some embodiments, the nucleic acid molecule is an mRNA molecule. In another aspect, provided herein are vectors comprising the nucleic acid molecules provided herein. In some embodiments, the vector comprises a viral vector. In another aspect, provided herein are transformed or host cells that express the polypeptides disclosed herein or the conjugates disclosed herein. In another aspect, provided herein are pharmaceutical compositions comprising the polypeptides disclosed herein or the conjugates disclosed herein and a pharmaceutically acceptable carrier. In another aspect, provided herein are kits comprising a polypeptide, a conjugate, a nucleic acid molecule, a vector, a transformed or host cell, or a pharmaceutical composition, combinations thereof, and a container.

[0037] In another aspect, provided herein is a method of preparing a polypeptide or conjugate described herein, the method comprising: (a) constructing a nucleic acid molecule and a vector described herein; (b) culturing a transformed or host cell described herein; and (c) recovering the polypeptide from the transformed or host cell.

[0038] In another aspect, provided herein is a method of treating a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition described herein in an amount effective to treat the subject. In some embodiments, the subject has a solid tumor.

[0039] Incorporation by reference All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. If the publications and patents or patent applications incorporated by reference are contrary to the present disclosure set forth herein, this specification is intended to supersede and / or prevail over any such contrary materials.

[0040] This application includes at least one drawing created in color. Copies of this patent or patent application publication with color drawings will be provided by the United States Patent and Trademark Office upon request and payment of the necessary fee.

[0041] The novel features of this disclosure are specifically set forth in the appended claims. A better understanding of the features and advantages of this disclosure will be obtained by reference to the following detailed description, which illustrates embodiments that are examples of the principles of this disclosure, and to the appended drawings (also the "FIG" and "FIGURE" in this specification).

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0055] DETAILED DESCRIPTION As used herein and in the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a plurality of such agents, reference to "the cell" includes reference to one or more cells (or a plurality of cells) and their equivalents known to those skilled in the art, and the like. When ranges are used herein with respect to physical properties, such as molecular weight, or chemical properties, such as chemical formula, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. The term "about" when referring to a number or numerical range means that the recited number or numerical range is an approximation within experimental variability (or within statistical experimental error), and thus, in some instances, the number or numerical range may vary between 1% and 15% of the specified number or numerical range. The term "comprising" (and related terms such as "comprise," "comprises," "having," or "including") is not intended to exclude other specific embodiments, such as embodiments of any material composition, composition, method, or process described herein, from being "consisting of" or "consisting essentially of" the recited features.

[0056] DEFINITIONS When used to describe specific combinatorial mutations with respect to the present invention, the phrase "mutation A, mutation B, mutation C" means that these mutations A, B, and C are present simultaneously in a single IL-21 variant. For example, the combinatorial mutation "S70L, K73Y, P79E" means that mutations S70L, K73Y, and P79E are all present in a single IL-21 variant. Also, when used with respect to the present invention, the term "STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72)" refers to a mutation in which the amino acid segment STNAGRRQKHR (SEQ ID NO: 71) at positions 80-90 corresponding to wild-type human IL-21 is replaced with GGGSEGGGS (SEQ ID NO: 72).

[0057] As used in this specification and the appended claims, unless otherwise specified, the following terms have the meanings set forth below.

[0058] "Wild-type" or "WT" or "wt" or "native" refers to an amino acid sequence found in nature, including allelic variation or natural isoforms. A wild-type protein or polypeptide has an amino acid sequence that has not been intentionally modified.

[0059] "Variant" or "mutant", which can be used interchangeably for an amino acid sequence (e.g., of a peptide, protein, or polypeptide), refers to an amino acid insertion variant, amino acid addition variant, amino acid deletion variant, and / or amino acid substitution variant. An amino acid insertion variant is characterized by the insertion of one or more amino acids into a particular amino acid sequence, e.g., a wild-type IL-21 sequence or a functional variant thereof. An amino acid addition variant includes N-terminal and / or C-terminal fusions of one or more amino acids. An amino acid deletion variant is characterized by the removal of one or more amino acids from the sequence. The deletion can be a deletion at any position in the protein sequence. Amino acid deletion variants include deletions at the N-terminal and / or C-terminal of the protein that result in N-terminal and / or C-terminal truncation variants. An amino acid substitution variant is characterized by the removal of one or more amino acids from the sequence and the insertion of one or more other amino acids in place of the original amino acids.

[0060] As used herein, "IL-21 variant" or "IL-21 mutein" or "IL-21 variant" or "IL-21 variant moiety" refers to a polypeptide in which one or more amino acids of wild-type human IL-21 or a functional variant thereof have been mutated to result in a variant or mutant of wild-type human IL-21. In some embodiments, the IL-21 variant has a sequence that is at least 80% identical to the sequence of wild-type human IL-21 (e.g., SEQ ID NO: 1 or 2). In some embodiments, the IL-21 variant polypeptides provided herein include a signal peptide. In some particular embodiments, the signal peptide is the naturally occurring signal peptide of wild-type human IL-21. In some particular embodiments, the signal peptide is any signal peptide known in the art. In some embodiments, the IL-21 variant polypeptides provided herein do not include a signal peptide.

[0061] "Natural amino acid" refers to an amino acid among the 20 naturally occurring amino acids in proteins. Naturally occurring amino acids are generally classified into the following four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are classified as aromatic amino acids.

[0062] "Unnatural amino acid" refers to an amino acid other than the 20 naturally occurring amino acids in proteins.

[0063] "Percentage identity" refers to the percentage of identical amino acid residues between two sequences compared after optimal alignment of the sequences. Optimal alignment of the sequences can be brought about manually or by a computer program using a sequence alignment algorithm (e.g., ClustalW, T-coffee, COBALT, BestFit, FASTA, BLASTP, BLASTN, and TFastA). The percentage identity can be calculated by determining the number of positions that are identical between the two sequences being compared, dividing this number by the number of positions compared, and multiplying the resulting value by 100 to obtain the percentage identity between the two sequences.

[0064] The term "subject" or "patient" includes mammals. Examples of mammals include any member of the class Mammalia: humans, non-human primates such as chimpanzees, as well as other apes and monkey species; farm animals such as cows, horses, sheep, goats, pigs; household animals such as rabbits, dogs, and cats; and laboratory animals including rodents such as rats, mice, and guinea pigs, but are not limited thereto. In one aspect, the mammal is a human.

[0065] As used herein, "treatment" or "treating" are used interchangeably. These terms refer to an approach for obtaining a beneficial or desired result, including but not limited to a therapeutic benefit and / or a prophylactic benefit. "Therapeutic benefit" means eradication or alleviation of the underlying disorder being treated. Also, a therapeutic benefit is realized by eradication or alleviation of one or more of the physiological symptoms associated with the underlying disorder such that improvement is observed in the patient even though the patient still suffers from the underlying disorder. For prophylactic benefit, the compositions are administered in some embodiments to patients at risk of developing a particular disease or to patients reporting one or more of the physiological symptoms of a disease, even if a diagnosis of such disease has not been made.

[0066] IL-21 IL-21 is a member of a large family of cytokines (IL-2, IL-4, IL-7, IL-9 and IL-15) whose receptors share the common receptor γ-chain (γc). IL-21 is a pleiotropic type I cytokine mainly produced by T cells and natural killer T (NKT) cells. This cytokine has diverse effects on a wide range of cell types including, but not limited to, CD4+ and CD8+ T cells, B cells, macrophages, monocytes, and dendritic cells (DC). Specifically, IL-21 can drive the differentiation of B cells into plasma cells, regulate immunoglobulin production, control the proliferation and / or effector functions of both CD4+ and CD8+ T cells, limit the differentiation of Tregs, and stimulate epithelial cells and fibroblasts to produce inflammatory mediators (see, for example, Leonarda and Wan, F1000Res. 2016; 5: F1000 Faculty Rev-224; Stolfi et al, Oncoimmunology. 2012 May 1; 1(3): 351-354).

[0067] The IL-21 receptor (IL-21R) forms a heterodimeric receptor complex together with the common γc, which is also a subunit of the receptors for IL-2, IL-4, IL-7, IL-9 and IL-15. The IL-21R portion is the ligand recognition binding site, and γc is the signal transduction unit. The IL-21R subunit is mainly expressed on the surface of normal lymphoid tissues including the spleen, thymus, lymph nodes, peripheral blood lymphocytes, T cells, B cells and NK cells. IL-21R acts through the JAK / STAT pathway and activates its target genes by JAK1, JAK3 and STAT3 dimers.

[0068] IL-21R transmits the growth-promoting signal of IL-21, which plays an important role in regulating B cell proliferation, promoting the proliferation and differentiation of multiple T cell subsets, regulating the survival of NK cells, and improving the cytotoxic activity of NK cells. Studies in IL-21R knockout mice have demonstrated an important role in regulating immunoglobulin production. Furthermore, IL-21 has been reported to have a potent antitumor effect due to its ability to expand the pool of cytotoxic CD8+ T cells, NK cells and NKT cells.

[0069] Compared with the affinity of wild-type IL-21 protein for IL-21R, the affinity of the IL-21 variants of the present disclosure for IL-21R is decreased. Therefore, in some embodiments, due to the decreased affinity of the IL-21 variants described herein for IL-21R in normal lymphoid tissues, the polypeptides described herein have lower systemic toxicity. Thus, in some embodiments, the polypeptides with lower systemic toxicity described herein are advantageous in the therapeutic window when administered as a dosing strategy, as a single drug or in combination therapy, and are also more suitable candidates for conjugating with another functional moiety (e.g., an antibody, a small molecule inhibitor, or a nucleic acid) to form a therapeutic bifunctional or multifunctional molecule.

[0070] In some embodiments, the thermal stability of the IL-21 variants of the present disclosure is improved as compared to wild-type IL-21. In some embodiments, the pharmacodynamics of the IL-21 variants of the present disclosure are improved. Thus, the drug discovery potential of the IL-21 variants is optimized as compared to wild-type IL-21.

[0071] IL-21 variant IL-21 variant having a substitution at position P79 Provided herein are polypeptides comprising an IL-21 variant having an amino acid residue substitution at position P79 corresponding to wild-type human IL-21. In some embodiments, wild-type human IL-21 comprises the sequence set forth in SEQ ID NO: 1 or 2.

[0072] In some embodiments, the amino acid residue substitution at position P79 is P79E or P79C. In some particular embodiments, the amino acid residue substitution at position P79 is P79E. In other particular embodiments, the amino acid residue substitution at position P79 is P79C.

[0073] In some embodiments where the amino acid residue substitution at position P79 is P79C, the polypeptide further comprises one or more amino acid residue substitutions with cysteine in the region of positions 1 to 10 corresponding to wild-type human IL-21. In some cases, the polypeptide further comprises Q1C. In some cases, the polypeptide further comprises G2C. In some cases, the polypeptide further comprises Q3C. In some cases, the polypeptide further comprises D4C. In some cases, the polypeptide further comprises R5C. In some cases, the polypeptide further comprises H6C. In some cases, the polypeptide further comprises M7C. In some cases, the polypeptide further comprises I8C. In some cases, the polypeptide further comprises R9C. In some cases, the polypeptide further comprises M10C. In some cases, the polypeptide further comprises a combination of two or more of the above cysteine substitutions. In some cases, the one or more amino acid residue substitutions are selected from the group consisting of R5C, H6C and R9C.

[0074] In some cases, one or more cysteine residues with amino acid residue substitutions in the 1st to 10th positions form disulfide bonds together with the cysteine residue at position P79. In some specific cases, the disulfide bond is formed by the cysteine residue at position P79 and the cysteine residue at position 1. In some specific cases, the disulfide bond is formed by the cysteine residue at position P79 and the cysteine residue at position 2. In some specific cases, the disulfide bond is formed by the cysteine residue at position P79 and the cysteine residue at position 3. In some specific cases, the disulfide bond is formed by the cysteine residue at position P79 and the cysteine residue at position 4. In some specific cases, the disulfide bond is formed by the cysteine residue at position P79 and the cysteine residue at position 5. In some specific cases, the disulfide bond is formed by the cysteine residue at position P79 and the cysteine residue at position 6. In some specific cases, the disulfide bond is formed by the cysteine residue at position P79 and the cysteine residue at position 7. In some specific cases, the disulfide bond is formed by the cysteine residue at position P79 and the cysteine residue at position 8. In some specific cases, the disulfide bond is formed by the cysteine residue at position P79 and the cysteine residue at position 9. In some specific cases, the disulfide bond is formed by the cysteine residue at position P79 and the cysteine residue at position 10.

[0075] In some embodiments where the polypeptide has an amino acid residue substitution at position P79 (e.g., P79E or P79C), the polypeptide further comprises one or more amino acid residue substitutions at positions selected from the group consisting of R5, H6, R9, Q12, L13, D15, I16, S70, K72, K73, R76, and P78. In some specific embodiments where the polypeptide has an amino acid residue substitution at position P79 (e.g., P79E or P79C), the polypeptide further comprises one or more amino acid residue substitutions at positions selected from the group consisting of S70, K72, K73, and R76.

[0076] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position K73 corresponding to wild-type human IL-21. In some cases, the amino acid residue substitution at position K73 is an aromatic amino acid. In certain cases, the amino acid residue substitution at position K73 is K73Y or K73F.

[0077] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position S70 corresponding to wild-type human IL-21. In some cases, the amino acid residue substitution at S70 is S70L or S70A.

[0078] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position R76 corresponding to wild-type human IL-21. In some cases, the amino acid residue substitution at R76 is R76F.

[0079] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position K72 corresponding to wild-type human IL-21. In some cases, the amino acid residue substitution at K72 is a non-aromatic amino acid containing a side-chain hydroxyl. In some specific cases, the amino acid residue substitution at K72 is K72S.

[0080] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position Q12 corresponding to wild-type human IL-21. In some cases, the amino acid residue substitution at Q12 is Q12W.

[0081] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position L13 corresponding to wild-type human IL-21. In some cases, the amino acid residue substitution at L13 is L13A.

[0082] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position D15 corresponding to wild-type human IL-21. In some cases, the amino acid residue substitution at D15 is D15L, D15K or D15R.

[0083] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position I16 corresponding to wild-type human IL-21. In some cases, the amino acid residue substitution at I16 is I16R or I16W.

[0084] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position P78 corresponding to wild-type human IL-21. In some cases, the amino acid residue substitution at P78 is P78G or P78E.

[0085] In some embodiments, the amino acid segment STNAGRRQKHR (SEQ ID NO: 71) at positions 80-90 corresponding to wild-type human IL-21 is replaced with a short peptide linker. In some embodiments, the length of the short peptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In some embodiments, the length of the short peptide linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues or less. In some embodiments, the length of the short peptide linker is 4-10 amino acid residues. In some embodiments, the length of the short peptide linker is 5-9 amino acid residues. In further embodiments, the short peptide linker comprises one or more Gly-Ser units. In further embodiments, the short peptide linker comprises one or more glutamic acid residues. In further embodiments, the short peptide linker comprises one glutamic acid residue. In certain embodiments, the short peptide linker comprises a glutamic acid residue, and the glutamic acid residue is in the middle of the short peptide linker. In some embodiments where the short peptide linker has an odd (i.e., 2n + 1) length, the glutamic acid residue is at the n + 1 position. In an exemplary embodiment where the short peptide linker is 5 amino acids in length, the glutamic acid residue is at the 3 position. In an exemplary embodiment where the short peptide linker is 7 amino acids in length, the glutamic acid residue is at the 4 position. In some embodiments where the short peptide linker has an even (i.e., 2n) length, the glutamic acid residue is at either the n or n + 1 position. In an exemplary embodiment where the short peptide linker is 4 amino acids in length, the glutamic acid residue is at the 2 or 3 position. In an exemplary embodiment where the short peptide linker is 6 amino acids in length, the glutamic acid residue is at the 3 or 4 position. In certain embodiments, the short peptide linker is selected from the group consisting of GGSEGGS (SEQ ID NO: 73), GSEGS (SEQ ID NO: 74), GGSGGS (SEQ ID NO: 75), GGGSEGGS (SEQ ID NO: 76), GGSEGGGS (SEQ ID NO: 77), GSGGS (SEQ ID NO: 78), GGGSGGS (SEQ ID NO: 79), and GGGSEGGGS (SEQ ID NO: 72).

[0086] In some embodiments, the polypeptide exhibits a lower binding affinity for the IL-21 receptor (IL-21R) than wild-type human IL-21. In certain embodiments, the polypeptide exhibits a decrease in binding affinity for the IL-21R from one-half to one-thousandth compared to wild-type human IL-21. In certain embodiments, the polypeptide has a higher K D value for the IL-21R compared to wild-type human IL-21.

[0087] An IL-21 variant having a substitution at position 70 In another aspect, provided herein is a polypeptide comprising an IL-21 variant having an amino acid residue substitution at position S70 corresponding to wild-type human IL-21. In some embodiments, wild-type human IL-21 comprises the sequence set forth in SEQ ID NO: 1 or 2. In some cases, the amino acid residue substitution at position S70 is S70L or S70A.

[0088] In some embodiments, the polypeptide comprises an amino acid residue substitution (e.g., S70L or S70A) at position S70 corresponding to wild-type human IL-21 and an amino acid residue substitution at position K73. In certain embodiments, the polypeptide comprises an amino acid residue substitution with an aromatic amino acid at position K73 corresponding to wild-type human IL-21. In certain cases, the amino acid residue substitution at position K73 is K73Y or K73F. In other embodiments, the polypeptide further comprises one, two, three, or more amino acid residue substitutions at positions selected from the group consisting of R5, H6, R9, Q12, L13, D15, I16, K72, R76, P78, and P79. In certain embodiments, the polypeptide further comprises one, two, three, or four amino acid residue substitutions at positions selected from the group consisting of Q12, D15, I16, and K72. In some cases, the polypeptide comprises an amino acid residue substitution selected from the group consisting of I16R and I16W. In other cases, the polypeptide comprises an amino acid residue substitution selected from the group consisting of D15L, D15K, and D15R. In still other cases, the polypeptide comprises an amino acid residue substitution selected from the group consisting of K72S and Q12W.

[0089] In some embodiments, the polypeptide comprises an amino acid residue substitution (e.g., S70L or S70A) at position S70 corresponding to wild-type human IL-21 and an amino acid residue substitution at position R76. In some cases, the amino acid residue substitution at position R76 is R76F. In some cases, the polypeptide further comprises one, two, three, or more amino acid residue substitutions at positions selected from the group consisting of R5, H6, R9, Q12, L13, D15, I16, K72, K73, P78, and P79. In some cases, the polypeptide further comprises one, two, three, or four amino acid residue substitutions at positions selected from the group consisting of Q12, D15, I16, and K72. In some cases, the polypeptide comprises an amino acid residue substitution selected from the group consisting of I16R and I16W. In other cases, the polypeptide comprises an amino acid residue substitution selected from the group consisting of D15L, D15K, and D15R. In still other cases, the polypeptide comprises an amino acid residue substitution selected from the group consisting of K72S and Q12W.

[0090] In some embodiments, the amino acid segment STNAGRRQKHR (SEQ ID NO: 71) at positions 80-90 corresponding to wild-type human IL-21 is replaced with a short peptide linker. In some embodiments, the length of the short peptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues. In some embodiments, the length of the short peptide linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues or less. In some embodiments, the length of the short peptide linker is 4-10 amino acid residues. In some embodiments, the length of the short peptide linker is 5-9 amino acid residues. In further embodiments, the short peptide linker comprises one or more Gly-Ser units. In further embodiments, the short peptide linker comprises one or more glutamic acid residues. In further embodiments, the short peptide linker comprises one glutamic acid residue. In certain embodiments, the short peptide linker comprises a glutamic acid residue, and the glutamic acid residue is in the middle of the short peptide linker. In some embodiments where the short peptide linker has an odd (i.e., 2n+1) length, the glutamic acid residue is at the n+1 position. In an exemplary embodiment where the short peptide linker is 5 amino acids in length, the glutamic acid residue is at the 3 position. In an exemplary embodiment where the short peptide linker is 7 amino acids in length, the glutamic acid residue is at the 4 position. In some embodiments where the short peptide linker has an even (i.e., 2n) length, the glutamic acid residue is at either the n or n+1 position. In an exemplary embodiment where the short peptide linker is 4 amino acids in length, the glutamic acid residue is at the 2 or 3 position. In an exemplary embodiment where the short peptide linker is 6 amino acids in length, the glutamic acid residue is at the 3 or 4 position. In certain embodiments, the short peptide linker is selected from the group consisting of GGSEGGS (SEQ ID NO: 73), GSEGS (SEQ ID NO: 74), GGSGGS (SEQ ID NO: 75), GGGSEGGS (SEQ ID NO: 76), GGSEGGGS (SEQ ID NO: 77), GSGGS (SEQ ID NO: 78), GGGSGGS (SEQ ID NO: 79), and GGGSEGGGS (SEQ ID NO: 72).

[0091] In some embodiments, the polypeptide exhibits a lower binding affinity for the IL-21 receptor (IL-21R) than wild-type human IL-21. In certain embodiments, the polypeptide exhibits a decrease in binding affinity for the IL-21R from one-half to one-thousandth compared to wild-type human IL-21. In certain embodiments, the polypeptide has a higher K D value for the IL-21R compared to wild-type human IL-21.

[0092] IL-21 variant having a peptide linker In another aspect, provided herein is a polypeptide comprising an interleukin-21 (IL-21) variant, wherein the amino acid segment STNAGRRQKHR (SEQ ID NO: 71) at positions 80-90 corresponding to wild-type human IL-21 is replaced with a short peptide linker. In some embodiments, the length of the short peptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In some embodiments, the length of the short peptide linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues or less. In some embodiments, the length of the short peptide linker is 4-10 amino acid residues. In some embodiments, the length of the short peptide linker is 5-9 amino acid residues. In some embodiments, the length of the short peptide linker is 6-9 amino acid residues. In some embodiments, the length of the short peptide linker is 7-9 amino acid residues. In further embodiments, the short peptide linker comprises one or more Gly-Ser units. In further embodiments, the short peptide linker comprises one or more glutamic acid residues. In further embodiments, the short peptide linker comprises one glutamic acid residue. In certain embodiments, the short peptide linker comprises a glutamic acid residue, and the glutamic acid residue is in the middle of the short peptide linker. In some embodiments where the short peptide linker has an odd (i.e., 2n+1) length, the glutamic acid residue is at the n+1 position. In an exemplary embodiment where the short peptide linker is 5 amino acids in length, the glutamic acid residue is at the 3 position. In an exemplary embodiment where the short peptide linker is 7 amino acids in length, the glutamic acid residue is at the 4 position. In some embodiments where the short peptide linker has an even (i.e., 2n) length, the glutamic acid residue is at either the n or n+1 position. In an exemplary embodiment where the short peptide linker is 4 amino acids in length, the glutamic acid residue is at the 2 or 3 position. In an exemplary embodiment where the short peptide linker is 6 amino acids in length, the glutamic acid residue is at the 3 or 4 position.In certain embodiments, the short peptide linker is selected from the group consisting of GGSEGGS (SEQ ID NO: 73), GSEGS (SEQ ID NO: 74), GGSGGS (SEQ ID NO: 75), GGGSEGGS (SEQ ID NO: 76), GGSEGGGS (SEQ ID NO: 77), GSGGS (SEQ ID NO: 78), GGGSGGS (SEQ ID NO: 79), and GGGSEGGGS (SEQ ID NO: 72).

[0093] In some embodiments, the polypeptide comprises one or more amino acid residue substitutions at positions selected from the group consisting of R5, H6, R9, Q12, L13, D15, I16, S70, K72, K73, R76, P78, and P79. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position R5, and optionally, the substitution is R5C. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position H6, and optionally, the substitution is H6C. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position R9, and optionally, the substitution is R9C. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position Q12, and optionally, the substitution is Q12W. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position L13, and optionally, the substitution is L13A. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position D15, and optionally, the substitution is D15L, D15K, or D15R. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position I16, and optionally, the substitution is I16R or I16W. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position S70, and optionally, the substitution is S70L or S70A. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position K72, and optionally, the substitution is K72S. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position K73, and optionally, the substitution is K73Y or K73F. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position R76, and optionally, the substitution is R76F. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position P78, and optionally, the substitution is P78G or P78E. In certain embodiments, the polypeptide comprises an amino acid residue substitution at position P79, and optionally, the substitution is P79E or P79C.

[0094] In some embodiments, the polypeptide exhibits improved thermal stability compared to wild-type human IL-21. In some embodiments, the polypeptide exhibits lower binding affinity for the IL-21 receptor (IL-21R) than wild-type IL-21. In some embodiments, the polypeptide exhibits a decrease in binding affinity for IL-21R from one-half to one-thousandth compared to wild-type IL-21. In some embodiments, the polypeptide has a higher K D value compared to wild-type IL-21.

[0095] IL-21 variant having one or more specific substitutions In another aspect, provided herein is a polypeptide comprising an interleukin-21 (IL-21) variant comprising a wild-type human IL-21 amino acid sequence having at least one mutation selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, wherein X1 is L, K, or R, X2 is R or W, X3 is L or A, X4 is Y or F, X5 is G or E, and X6 is E or C. In some embodiments, the wild-type human IL-21 comprises the sequence set forth in SEQ ID NO: 1 or 2.

[0096] In some embodiments, the polypeptide described herein comprises only one mutation selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, wherein X1 is L, K, or R, X2 is R or W, X3 is L or A, X4 is Y or F, X5 is G or E, and X6 is E or C.

[0097] In some embodiments, the polypeptide described herein contains two mutations, each of the two mutations being selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, where X1 is L, K, or R, X2 is R or W, X3 is L or A, X4 is Y or F, X5 is G or E, and X6 is E or C.

[0098] In some embodiments, the polypeptide described herein contains three mutations, each of the three mutations being selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, where X1 is L, K, or R, X2 is R or W, X3 is L or A, X4 is Y or F, X5 is G or E, and X6 is E or C.

[0099] In some embodiments, the polypeptide described herein contains four mutations, each of the four mutations being selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, where X1 is L, K, or R, X2 is R or W, X3 is L or A, X4 is Y or F, X5 is G or E, and X6 is E or C.

[0100] In some embodiments, the polypeptide described herein contains five mutations, each of the five mutations being selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, where X1 is L, K, or R, X2 is R or W, X3 is L or A, X4 is Y or F, X5 is G or E, and X6 is E or C.

[0101] In some embodiments, the polypeptide described herein comprises six mutations, each of the six mutations being selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, where X1 is L, K, or R, X2 is R or W, X3 is L or A, X4 is Y or F, X5 is G or E, and X6 is E or C.

[0102] In some embodiments, the polypeptide described herein comprises seven mutations, each of the two mutations being selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, where X1 is L, K, or R, X2 is R or W, X3 is L or A, X4 is Y or F, X5 is G or E, and X6 is E or C.

[0103] In some embodiments, the polypeptide described herein comprises eight mutations, each of the eight mutations being selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, where X1 is L, K, or R, X2 is R or W, X3 is L or A, X4 is Y or F, X5 is G or E, and X6 is E or C.

[0104] In some embodiments, the polypeptide described herein comprises nine mutations, each of the nine mutations being selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, where X1 is L, K, or R, X2 is R or W, X3 is L or A, X4 is Y or F, X5 is G or E, and X6 is E or C.

[0105] In some embodiments, the polypeptide described herein comprises 10 mutations, each of the 10 mutations being selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, where X1 is L, K, or R, X2 is R or W, X3 is L or A, X4 is Y or F, X5 is G or E, and X6 is E or C.

[0106] In some embodiments, the polypeptide described herein comprises 11 mutations, each of the 11 mutations being selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, where X1 is L, K, or R, X2 is R or W, X3 is L or A, X4 is Y or F, X5 is G or E, and X6 is E or C.

[0107] In some embodiments, the polypeptide described herein comprises 12 mutations, each of the 12 mutations being selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, where X1 is L, K, or R, X2 is R or W, X3 is L or A, X4 is Y or F, X5 is G or E, and X6 is E or C.

[0108] In some embodiments, the polypeptide described herein comprises 13 mutations, each of the 13 mutations being selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, where X1 is L, K, or R, X2 is R or W, X3 is L or A, X4 is Y or F, X5 is G or E, and X6 is E or C.

[0109] In some embodiments, the amino acid segment STNAGRRQKHR (SEQ ID NO: 71) at positions 80-90 corresponding to wild-type human IL-21 is replaced with a short peptide linker. In some embodiments, the length of the short peptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In some embodiments, the length of the short peptide linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid residues or less. In some embodiments, the length of the short peptide linker is 4-10 amino acid residues. In some embodiments, the length of the short peptide linker is 5-9 amino acid residues. In some embodiments, the length of the short peptide linker is 7-9 amino acid residues. In further embodiments, the short peptide linker comprises one or more Gly-Ser units. In further embodiments, the short peptide linker comprises one or more glutamic acid residues. In further embodiments, the short peptide linker comprises one glutamic acid residue. In certain embodiments, the short peptide linker comprises a glutamic acid residue, and the glutamic acid residue is in the middle of the short peptide linker. In some embodiments where the short peptide linker has an odd (i.e., 2n + 1) length, the glutamic acid residue is at the n + 1 position. In an exemplary embodiment where the short peptide linker is 5 amino acids in length, the glutamic acid residue is at the 3 position. In an exemplary embodiment where the short peptide linker is 7 amino acids in length, the glutamic acid residue is at the 4 position. In some embodiments where the short peptide linker has an even (i.e., 2n) length, the glutamic acid residue is at either the n or n + 1 position. In an exemplary embodiment where the short peptide linker is 4 amino acids in length, the glutamic acid residue is at the 2 or 3 position. In an exemplary embodiment where the short peptide linker is 6 amino acids in length, the glutamic acid residue is at the 3 or 4 position.In certain embodiments, the short peptide linker is selected from the group consisting of GGSEGGS (SEQ ID NO: 73), GSEGS (SEQ ID NO: 74), GGSGGS (SEQ ID NO: 75), GGGSEGGS (SEQ ID NO: 76), GGSEGGGS (SEQ ID NO: 77), GSGGS (SEQ ID NO: 78), GGGSGGS (SEQ ID NO: 79), and GGGSEGGGS (SEQ ID NO: 72).

[0110] In another aspect, polypeptides comprising an IL-21 variant and comprising the amino acid residue mutation P79E are provided herein. In another aspect, polypeptides comprising an IL-21 variant and comprising the amino acid residue mutations P79E K73Y and P79E are provided herein. In another aspect, polypeptides comprising an IL-21 variant and comprising the amino acid residue mutations K73F and P79E are provided herein. In another aspect, polypeptides comprising an IL-21 variant and comprising the amino acid residue mutations S70L, K73F, and P79E are provided herein. In another aspect, polypeptides comprising an IL-21 variant and comprising the amino acid residue mutations S70L, K73Y, and P79E are provided herein. In another aspect, polypeptides comprising an IL-21 variant and comprising the amino acid residue mutations S70L, K72S, K73F, and P79E are provided herein. In another aspect, polypeptides comprising an IL-21 variant and comprising the amino acid residue mutations S70L, K72S, K73Y, and P79E are provided herein. In another aspect, polypeptides comprising an IL-21 variant and comprising the amino acid residue mutations D15K, S70L, K73F, and P79E are provided herein. In another aspect, polypeptides comprising an IL-21 variant and comprising the amino acid residue mutations D15R, S70L, K73F, and P79E are provided herein. In another aspect, polypeptides comprising an IL-21 variant and comprising the amino acid residue mutations I16W, S70L, K73F, and P79E are provided herein. In another aspect, polypeptides comprising an IL-21 variant and comprising the amino acid residue mutations D15L, S70L, K73F, and P79E are provided herein. In another aspect, polypeptides comprising an IL-21 variant and comprising the amino acid residue mutations L13A, S70L, K73F, and P79E are provided herein.In another aspect, provided herein are polypeptides comprising an IL-21 variant, wherein the polypeptide comprises amino acid residue mutations D15R, R76F, P78E, and P79E. In another aspect, provided herein are polypeptides comprising an IL-21 variant, wherein the polypeptide comprises amino acid residue mutations D15L, I16R, S70L, K73Y, and P79E. In another aspect, provided herein are polypeptides comprising an IL-21 variant, wherein the polypeptide comprises amino acid residue mutations D15K, I16W, S70L, R76F, and P79E. In another aspect, provided herein are polypeptides comprising an IL-21 variant, wherein the polypeptide comprises amino acid residue mutations D15R, I16W, S70L, R76F, and P79E. In another aspect, provided herein are polypeptides comprising an IL-21 variant, wherein the polypeptide comprises the amino acid residue mutation STNAGRRQKHR (SEQ ID NO: 71) substituted with GGGSEGGGS (SEQ ID NO: 72) (STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72)). In another aspect, provided herein are polypeptides comprising an IL-21 variant, wherein the polypeptide comprises the amino acid residue mutations P79E and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). In another aspect, provided herein are polypeptides comprising an IL-21 variant, wherein the polypeptide comprises the amino acid residue mutations K73Y, P79E, and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). In another aspect, provided herein are polypeptides comprising an IL-21 variant, wherein the polypeptide comprises the amino acid residue mutations K73F, P79E, and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). In another aspect, provided herein are polypeptides comprising an IL-21 variant, wherein the polypeptide comprises the amino acid residue mutations S70L, K73F, P79E, and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72).In another aspect, provided herein are polypeptides comprising an IL-21 variant, the polypeptide comprising amino acid residue mutations S70L, K73Y, P79E, and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). In another aspect, provided herein are polypeptides comprising an IL-21 variant, the polypeptide comprising amino acid residue mutations D15K, S70L, K73F, P79E, and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). In another aspect, provided herein are polypeptides comprising an IL-21 variant, the polypeptide comprising amino acid residue mutations D15R, S70L, K73F, P79E, and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). In another aspect, provided herein are polypeptides comprising an IL-21 variant, the polypeptide comprising amino acid residue mutations I16W, S70L, K73F, P79E, and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). In another aspect, provided herein are polypeptides comprising an IL-21 variant, the polypeptide comprising amino acid residue mutations S70L, K72S, K73F, P79E, and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). In another aspect, provided herein are polypeptides comprising an IL-21 variant, the polypeptide comprising amino acid residue mutations S70L, K72S, K73Y, P79E, and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). In another aspect, provided herein are polypeptides comprising an IL-21 variant, the polypeptide comprising amino acid residue mutations D15R, I16W, S70L, K73Y, P79E, and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). In another aspect, provided herein are polypeptides comprising an IL-21 variant, the polypeptide comprising amino acid residue mutations D15L, I16R, S70L, K73Y, P79E, and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72).

[0111] In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutation P79C. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations R5C and P79C. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations H6C and P79C. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations R9C and P79C. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations R5C, R76F, and P79C. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations H6C, R76F, and P79C. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations H6C, K73Y, and P79C. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations H6C, K73F, and P79C. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations H6C, S70L, K73Y, and P79C. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations H6C, S70L, K73F, P79C; R9C, D15R, P78G, and P79C. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations H6C, D15L, I16R, S70L, K73Y, and P79C. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations R9C, D15L, I16R, S70L, K73Y, and P79C.In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising amino acid residue mutations H6C, P79C, and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising amino acid residue mutations H6C, K73Y, P79C, and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising amino acid residue mutations H6C, K73F, P79C, and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising amino acid residue mutations H6C, S70L, P79C, and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72).

[0112] In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutation K73Y. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutation K73F. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutation S70L. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutation S70A. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations S70L and K73Y. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations S70L and K73F. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations S70L, K72S, and K73Y. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations S70A, K72S, and K73F. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations Q12W, S70A, and R76F. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations D15K, S70A, and R76F. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations D15L, S70A, and R76F. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations D15R, S70A, and R76F. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising the amino acid residue mutations I16R, S70L, and K73Y.In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising amino acid residue mutations D15L, I16R, S70L, and K73Y. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising amino acid residue mutations D15K, I16W, S70L, and K73Y. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising amino acid residue mutations D15K, I16W, S70L, and R76F. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising amino acid residue mutations D15R, I16W, S70L, and R76F. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising amino acid residue mutations I16W, S70L, K72S, and K73F. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising amino acid residue mutations D15R, I16W, S70L, and K73Y. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, the polypeptide comprising amino acid residue mutations D15K, I16W, S70L, K72S, and K73Y.

[0113] In another aspect, provided herein are polypeptides comprising an IL-21 variant, wherein the polypeptide comprises the sequence shown by any one of SEQ ID NOs: 5 to 62. In another aspect, provided herein are polypeptides comprising an IL-21 variant, wherein the polypeptide consists of the sequence shown by any one of SEQ ID NOs: 5 to 62. In some embodiments, the polypeptide comprises or consists of the sequence shown by SEQ ID NO: 5. In some embodiments, the polypeptide comprises or consists of the sequence shown by SEQ ID NO: 6. In some embodiments, the polypeptide comprises or consists of the sequence shown by SEQ ID NO: 7. In some embodiments, the polypeptide comprises or consists of the sequence shown by SEQ ID NO: 8. In some embodiments, the polypeptide comprises or consists of the sequence shown by SEQ ID NO: 9. In some embodiments, the polypeptide comprises or consists of the sequence shown by SEQ ID NO: 10. In some embodiments, the polypeptide comprises or consists of the sequence shown by SEQ ID NO: 11. In some embodiments, the polypeptide comprises or consists of the sequence shown by SEQ ID NO: 12. In some embodiments, the polypeptide comprises or consists of the sequence shown by SEQ ID NO: 13. In some embodiments, the polypeptide comprises or consists of the sequence shown by SEQ ID NO: 14. In some embodiments, the polypeptide comprises or consists of the sequence shown by SEQ ID NO: 15. In some embodiments, the polypeptide comprises or consists of the sequence shown by SEQ ID NO: 16. In some embodiments, the polypeptide comprises or consists of the sequence shown by SEQ ID NO: 17. In some embodiments, the polypeptide comprises or consists of the sequence shown by SEQ ID NO: 18. In some embodiments, the polypeptide comprises or consists of the sequence shown by SEQ ID NO: 19. In some embodiments, the polypeptide comprises or consists of the sequence shown by SEQ ID NO: 20. In some embodiments, the polypeptide comprises or consists of the sequence shown by SEQ ID NO: 21. In some embodiments, the polypeptide comprises or consists of the sequence shown by SEQ ID NO: 22.In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 23. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 24. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 25. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 26. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 27. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 28. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 29. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 30. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 31. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 32. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 33. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 34. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 35. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 36. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 37. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 38. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 39. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 40. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 41. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 42. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 43.In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 44. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 45. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 46. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 47. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 48. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 49. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 50. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 51. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 52. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 53. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 54. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 55. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 56. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 57. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 58. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 59. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 60. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 61. In some embodiments, the polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 62.

[0114] In some embodiments, the polypeptide exhibits a lower binding affinity for the IL-21 receptor (IL-21R) than wild-type human IL-21. In some embodiments, the polypeptide exhibits a decrease in binding affinity for the IL-21R from one-half to one-thousandth compared to wild-type human IL-21. In some embodiments, the polypeptide has a higher K D value for the IL-21R compared to wild-type human IL-21.

[0115] In some embodiments, wild-type human IL-21 comprises the amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, wild-type human IL-21 consists of the amino acid sequence of SEQ ID NO: 1 or 2. In one embodiment, the IL-21 variant comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1 or 2. In some embodiments, the IL-21 polypeptide comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 5-62.

[0116] In some embodiments, the amino acid residues of the IL-21 variants described herein are mutated to non-natural amino acids. In some embodiments, the polypeptides described herein contain non-natural amino acids, the cytokine is conjugated to a protein, and the point of attachment is not a non-natural amino acid. In some embodiments, the polypeptides described herein contain non-natural amino acids, the cytokine is conjugated to a protein, and the point of attachment is a non-natural amino acid. In some embodiments, the amino acid residues of the IL-21 variants described herein are mutated prior to binding to (reacting with) an additional moiety. In some embodiments, the mutation to a non-natural amino acid reduces the likelihood of a self-antigen response to the immune system or minimizes the self-antigen response.

[0117] Non-limiting examples of non-natural amino acids include p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, p-methoxyphenylalanine, O-methyl-L-tyrosine, p-propynyloxyphenylalanine, p-propynyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, 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-boronophenylalanine, O-propynyltyrosine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, selenocysteine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, azido-lysine (AzK), non-natural analogs of tyrosine amino acids; non-natural analogs of glutamine amino acids; non-natural analogs of phenylalanine amino acids; non-natural analogs of serine amino acids; non-natural analogs of threonine amino acids; alkyl, aryl, acyl, azide, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl (alkynl), ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino-substituted amino acids, or combinations thereof; amino acids having photoactivatable crosslinkers; spin-labeled amino acids; fluorescent amino acids; metal-binding amino acids; metal-containing amino acids; radioactive amino acids; photo-caged and / or photo-isomerizable amino acids; biotin or biotin analog-containing amino acids; keto-containing amino acids; amino acids containing polyethylene glycol or polyether; heavy atom-substituted amino acids; chemically cleavable or photocleavable amino acids; amino acids having extended side chains; amino acids containing toxic groups; sugar-substituted amino acids; carbon-linked sugar-containing amino acids; redox-active amino acids; α-hydroxyamino acids; aminothio acids; α,α-disubstituted amino acids; β-amino acids; cyclic amino acids other than proline or histidine;Aromatic amino acids other than phenylalanine, tyrosine or tryptophan; N6-azidoethoxy-L-lysine (AzK), N6-propylgylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propylgyloxyphenylalanine, p-propylgyl-phenylalanine, 3-methyl-phenylalanine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic acid, and selenocysteine.;

[0118] In some embodiments, the non-natural amino acids include p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, O-methyl-L-tyrosine, p-methoxyphenylalanine, p-propynyloxyphenylalanine, p-propynyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, 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, and / or isopropyl-L-phenylalanine. In some embodiments, the non-natural amino acid is 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxy-phenylalanine, or 3-iodotyrosine. In some embodiments, the non-natural amino acid is phenylselenocysteine. In some embodiments, the non-natural amino acid is a benzophenone, ketone, iodide, methoxy, acetyl, benzoyl or azide-containing phenylalanine derivative. In some embodiments, the non-natural amino acid is a benzophenone, ketone, iodide, methoxy, acetyl, benzoyl or azide-containing lysine derivative.

[0119] In some embodiments, the unnatural amino acid comprises a selectively reactive group or a reactive group for site-selective labeling of a target polypeptide. In some embodiments, the chemistry is a biorthogonal reaction (e.g., a biocompatible and selective reaction). In some cases, the chemistry is a Cu(I)-catalyzed or "copper-free" alkyne-azide triazole formation reaction, a Staudinger ligation, an inverse electron demand Diels-Alder (IEDDA) reaction, "photoclick" chemistry, or a metal-mediated process, such as olefin metathesis and Suzuki-Miyaura or Sonogashira cross-coupling. In some embodiments, the unnatural amino acid comprises a photoreactive group that crosslinks upon irradiation, for example, with UV. In some embodiments, the unnatural amino acid comprises a photo-caged amino acid.

[0120] In some embodiments, the unnatural amino acid is a para-substituted, meta-substituted, or ortho-substituted amino acid derivative.

[0121] IL-21 Binding and Activity In some embodiments, the polypeptides disclosed herein exhibit a decreased affinity for IL-21R as compared to wild-type human IL-21. In some embodiments, the decreased affinity is a decrease of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or more than that, in the binding affinity for IL-21R as compared to wild-type human IL-21.

[0122] The binding affinity of the polypeptides provided herein for a target receptor or a subunit thereof can be evaluated by measuring the dissociation constant (K D ). In some embodiments, the polypeptides provided herein have a K DK is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, or at least 1000-fold that of wild-type IL-21 and binds to IL-21R. D In some embodiments, the polypeptides provided herein have a K that is D about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 200-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, or about 1000-fold that of wild-type IL-21 and binds to IL-21R. D In some embodiments, the polypeptides provided herein bind to IL-21R with a K of at least 10

[0123] μM, at least 10 -10 μM, at least 10 -9 μM, at least 10 -8 μM, at least 10 -7 μM, at least 10 -6 μM, at least 10 -5 μM, at least 10 -4 μM, at least 10 -3 μM, at least 10 -2 μM, at least 10 -1 μM, or greater and binds to IL-21R. In some embodiments, the polypeptides provided herein bind to IL-21R with a K of about 10 D μM, about 10 -10 μM, about 10 -9 μM, about 10 -8 μM, about 10 -7 μM, about 10 -6 μM, about 10 -5 μM, about 10 -4 μM, about 10 -3M, about 10 -2 M, or about 10 -1 M of K D and binds to IL-21R. In some embodiments, the polypeptides provided herein are 10 -10 M to 10 -1 M, 10 -10 M to 10 -2 M, 10 -10 M to 10 -3 M, 10 -10 M to 10 -4 M, 10 -10 M to 10 -5 M, 10 -10 M to 10 -6 M, 10 -10 M to 10 -7 M, 10 -10 M to 10 -8 M, 10 -10 M to 10 -9 M, 10 -9 M to 10 -1 M, 10 -9 M to 10 -2 M, 10 -9 M to 10 -3 M, 10 -9 M to 10 -4 M, 10 -9 M to 10 -5 M, 10 -9 M to 10 -6 M, 10 -9 M to 10 -7 M, 10 -9 M to 10 -8 M, 10 -8 M to 10 -1 M, 10 -8 M to 10 -2 M, 10 -8 M to 10 -3 M, 10 -8 M to 10 -4 M, 10 -8 M to 10 -5 M, 10 -8 M to 10 -6 M, 10 -8 M to 10 -7 M, 10 -7 M to 10 -1 M, 10 -7 M to 10 -2 M, 10 -7 M to 10-3 M, 10 -7 M to 10 -4 M, 10 -7 M to 10 -5 M, 10 -7 M to 10 -6 M, 10 -6 M to 10 -1 M, 10 -6 M to 10 -2 M, 10 -6 M to 10 -3 M, 10 -6 M to 10 -4 M or 10 -6 M to 10 -5 K of M D binds to IL-21R.

[0124] The IL-21 signaling activity by the IL-21 variants provided herein can be evaluated by IL21-mediated phosphorylation of STAT3. In some embodiments, HuT78 cells can be treated with the IL-21 polypeptides provided herein, and then the phosphorylation of STAT3 can be analyzed. STAT3 phosphorylation can be used to calculate the half-maximal effective concentration or EC 50 can be calculated. The relative activity of the IL-21 variants provided herein can be determined by comparing the EC 50 values.

[0125] In some embodiments, the IL-21 variants provided herein have a relative activity that is at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold lower than that of wild-type IL-2. In some embodiments, the IL-21 variants provided herein have an EC of STAT3 phosphorylation activity that is at least 1-fold, at least 1.5-fold, 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least 5-fold, at least 5.5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold that of wild-type IL-2 50 having.

[0126] conjugate In another aspect, conjugates are provided herein that comprise a polypeptide disclosed herein and at least one additional moiety. In other embodiments, the at least one additional moiety comprises a protein or a binding fragment thereof. In additional embodiments, the at least one additional moiety comprises a peptide. In additional embodiments, the at least one additional moiety comprises a nucleic acid. In additional embodiments, the at least one additional moiety comprises a small molecule.

[0127] In some embodiments, at least one additional moiety is attached to a non-natural or natural amino acid in the polypeptide. In some embodiments, the polypeptide comprises at least one additional moiety attached to a natural amino acid in the polypeptide. In some embodiments, the polypeptide comprises at least one additional moiety attached to a non-natural amino acid in the polypeptide. In some embodiments, at least one additional moiety is attached to the N-terminal or C-terminal amino acid of the polypeptide. In some embodiments, various combinations of sites are disclosed herein, for example, a first additional moiety is attached to an amino acid in the polypeptide and a second additional moiety is attached to the N-terminal or C-terminal of the polypeptide. In some embodiments, a single additional moiety is attached to multiple residues of the polypeptide (e.g., a staple). In some embodiments, at least one additional moiety is attached to both the N-terminal amino acid and the C-terminal amino acid of the polypeptide.

[0128] In some embodiments, at least one additional moiety can be linked to the N-terminus of the IL-21 variant moiety. In some embodiments, at least one additional moiety can be linked to the C-terminus of the IL-21 variant moiety.

[0129] Water-soluble polymer

[0130] In some embodiments, at least one additional moiety is a water-soluble polymer. In some embodiments, the water-soluble polymer is non-peptidic, non-toxic, and biocompatible. A substance is considered biocompatible when, when evaluated by a clinician, e.g., a physician, toxicologist, or clinical development specialist, the beneficial effects associated with the use (e.g., administration to a subject) of the substance alone or in combination with another substance (e.g., an active agent such as an IL-21 or polypeptide disclosed herein) on living tissue outweigh any harmful effects.

[0131] In some embodiments, the water-soluble polymer is non-immunogenic. A substance is considered non-immunogenic when its intended in vivo use, as evaluated by a clinician, such as a physician, toxicologist, or clinical development specialist, does not result in an unwanted immune response (e.g., antibody formation), or when an immune response is produced and the response is considered clinically insignificant or not important.

[0132] In some examples, the water-soluble polymer is characterized by having from about 2 to about 300 termini. Non-limiting examples of water-soluble polymers include poly(alkylene glycols), such as polyethylene glycol (PEG), poly(propylene glycol) (PPG), and copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinic alcohols), poly(vinylpyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharides), poly(α-hydroxy acids), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA), polydimethylacrylamide (PDAAm), polyphosphazenes, polyoxazolines (POZ), poly(N-acryloylmorpholine), and any combination thereof.

[0133] In some embodiments, 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 bifunctional PEG), branched or multi-armed (e.g., fork-shaped PEG, or PEG attached to a polyol core), dendritic (or star-shaped) in structure, each having one or more degradable bonds or not. Further, the internal structure of the water-soluble polymer can be configured in any number of different repeating patterns, such as a homopolymer, alternating copolymer, random copolymer, block copolymer, alternating terpolymer, random terpolymer, or block terpolymer.

[0134] In some embodiments, the weight average molecular weight of the water-soluble polymer is from about 100 Daltons (Da) to about 150,000 Da. Non-limiting examples of the weight average molecular weight range include from about 5,000 Da to about 100,000 Da, from about 6,000 Da to about 90,000 Da, from about 10,000 Da to about 85,000 Da, from about 10,000 Da to about 85,000 Da, from about 20,000 Da to about 85,000 Da, from about 53,000 Da to about 85,000 Da, from about 25,000 Da to about 120,000 Da, from about 29,000 Da to about 120,000 Da, from about 35,000 Da to about 120,000 Da, and from about 40,000 Da to about 120,000 Da.

[0135] Non-limiting examples of the weight average molecular weight for the water-soluble polymer include about 100 Da, about 200 Da, about 300 Da, about 400 Da, about 500 Da, about 600 Da, about 700 Da, about 750 Da, about 800 Da, about 900 Da, about 1,000 Da, about 1,500 Da, about 2,000 Da, about 2,200 Da, about 2,500 Da, about 3,000 Da, about 4,000 Da, about 4,400 Da, about 4,500 Da, about 5,000 Da, about 5,500 Da, about 6,000 Da, about 7,000 Da, about 7,500 Da, about 8,000 Da, about 9,000 Da, about 10,000 Da, about 11,000 Da, about 12,000 Da, about 13,000 Da, about 14,000 Da, about 15,000 Da, about 20,000 Da, about 22,500 Da, about 25,000 Da, about 30,000 Da, about 35,000 Da, about 40,000 Da, about 45,000 Da, about 50,000 Da, about 55,000 Da, about 60,000 Da, about 65,000 Da, about 70,000 Da, and about 75,000 Da. Branched versions of the water-soluble polymer having the total molecular weight of any of the foregoing (e.g., a branched 40,000 Da water-soluble polymer composed of two 20,000 Da polymers) can also be used. In some embodiments, the conjugate has no PEG moiety that is directly or indirectly linked to a PEG having a weight average molecular weight of less than about 6,000 Da.

[0136] PEG can contain several (OCH2CH2) monomers or (CH2CH2O) monomers. The number of repeating units can be specified by the subscript "n" in "(OCH2CH2)n". Thus, the value of "n" can typically fall within one or more of the following ranges: 2 to about 3400, about 100 to about 2300, about 100 to about 2270, about 136 to about 2050, about 225 to about 1930, about 450 to about 1930, about 1200 to about 1930, about 568 to about 2727, about 660 to about 2730, about 795 to about 2730, about 795 to about 2730, about 909 to about 2730, and about 1,200 to about 1,900. For any given polymer of known molecular weight, the number of repeating units (i.e., "n") can be determined by dividing the total weight average molecular weight of the polymer by the molecular weight of the repeating monomer.

[0137] In some examples, 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 C1-C6 alkoxy group or a hydroxyl group. For example, the water-soluble polymer can be methoxy-PEG (mPEG), which is in a linear form where one end of the PEG is a methoxy (-OCH3) group while the other end is a hydroxyl or other functional group that can be chemically modified as needed. Non-limiting examples of water-soluble polymers include linear or branched discrete PEG (dPEG); linear, branched or fork-shaped PEG; and Y-shaped PEG derivatives.

[0138] In some embodiments, the polypeptides described herein are conjugated to a water-soluble polymer selected from poly(alkylene glycol), such as polyethylene glycol (PEG), poly(propylene glycol) (PPG), and copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyol), poly(olefinic alcohol), poly(vinyl pyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(saccharide), poly(α-hydroxy acid), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA), polydimethylacrylamide (PDAAm), polyphosphazene, polyoxazoline (POZ), poly(N-acryloylmorpholine), and any combination thereof. For example, the IL-21 polypeptide is conjugated to PEG (e.g., PEGylated).

[0139] In some embodiments, the water-soluble polymer comprises polyglycerol (PG), such as HPG, LPG, moderately functional PG, linear-block-hyperbranched PG, or side-chain functional PG. In some cases, the polyglycerol is hyperbranched PG (HPG).

[0140] In some embodiments, the water-soluble polymer is a degradable synthetic PEG alternative. Non-limiting examples of degradable synthetic PEG alternatives include poly[oligo(ethylene glycol) methyl methacrylate] (POEGMA); backbone-modified PEG derivatives produced by polymerization of telechelic or di-end-functionalized PEG-based macromonomers; PEG derivatives containing comonomers with degradable linkages, such as poly[(ethylene oxide)-co-(methylene ethylene oxide)] [P(EO-co-MEO)], 5,6-benzo-2-methylene-1,3-dioxepane (BMDO), 2-methylene-1,3-dioxepane (MDO), and 2-methylene-4-phenyl-1,3-dioxolane (MPDL), which are cyclic ketene acetals copolymerized with OEGMA, or poly-(s-caprolactone)-graft-poly(ethylene oxide) (PCL-g-PEO).

[0141] In some embodiments, the water-soluble polymer comprises a poly(zwitterion). Non-limiting examples of poly(zwitterions) include poly(sulfobetaine methacrylate) (PSBMA), poly(carboxybetaine methacrylate) (PCBMA), and poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC).

[0142] In some embodiments, the water-soluble polymer comprises a polycarbonate. A non-limiting example of a polycarbonate is pentafluorophenyl 5-methyl-2-oxo-1,3-dioxane-5-carboxylate (MTC-OC6F5).

[0143] In some embodiments, the water-soluble polymer includes a polymer hybrid, such as a polycarbonate / PEG polymer hybrid, a peptide / protein-polymer conjugate, or a hydroxyl-containing and / or zwitterion-derivatized polymer (e.g., a hydroxyl-containing and / or zwitterion-derivatized PEG polymer).

[0144] In some embodiments, the water-soluble polymer comprises a polysaccharide. Non-limiting examples of polysaccharides include dextran, poly(sialic acid) (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES).

[0145] In some embodiments, the water-soluble polymer comprises a glycan. Non-limiting examples of glycans include N-linked glycans, O-linked glycans, glycolipids, O-GlcNAc, and glycosaminoglycans.

[0146] In some embodiments, the water-soluble polymer comprises a polyoxazoline polymer. The polyoxazoline polymer is a linear synthetic polymer, similar to PEG, and includes a low polydispersity. In some examples, the polyoxazoline polymer is a polydisperse polyoxazoline polymer characterized by an average molecular weight. In some cases, the average molecular weight of the polyoxazoline polymer is, for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, 100,000, 200,000, 300,000, 400,000, or 500,000 Da. In some embodiments, the polyoxazoline polymer comprises poly(2-methyl-2-oxazoline) (PMOZ), poly(2-ethyl-2-oxazoline) (PEOZ), or poly(2-propyl-2-oxazoline) (PPOZ). In some cases, a cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to the polyoxazoline polymer.

[0147] In some embodiments, the water-soluble polymer is a polyacrylic acid polymer.

[0148] In some embodiments, the water-soluble polymer comprises a polyamine. The polyamine is an organic polymer containing two or more primary amino 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.

[0149] Lipid In some embodiments, at least one additional moiety is a lipid. The lipid can be a fatty acid, e.g., a saturated or unsaturated fatty acid. Such fatty acids can have from 6 to 26 carbon atoms, from 6 to 24 carbon atoms, from 6 to 22 carbon atoms, from 6 to 20 carbon atoms, from 6 to 18 carbon atoms, from 20 to 26 carbon atoms, from 12 to 26 carbon atoms, from 12 to 24 carbon atoms, from 12 to 22 carbon atoms, from 12 to 20 carbon atoms, or from 12 to 18 carbon atoms. In some cases, the fatty acid has 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 carbon atoms. Non-limiting examples of fatty acids include 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), arachidonic acid (eicosanoic acid), heneicosylic acid (heneicosanoic acid), behenic acid (docosanoic acid), tricosylic acid (tricosanoic acid), lignoceric acid (tetracosanoic acid), pentacosylic acid (pentacosanoic acid), and cerotic acid (hexacosanoic acid). In some embodiments, the lipid binds to one or more serum proteins, thereby increasing serum stability and / or serum half-life.

[0150] Protein In some embodiments, at least one additional moiety described herein is a protein or a binding fragment thereof. Non-limiting examples of such proteins include albumin, transferrin, or transthyretin. In some embodiments, the protein or binding fragment thereof includes an antibody or a binding fragment thereof.

[0151] In some embodiments, at least one additional moiety described herein is a tag useful for functions such as purification or detection that is extended at the N-terminus, C-terminus, or both the N-terminus and C-terminus of the polypeptides of the present disclosure. In some embodiments, the tag can be a polyglutamate tag, FLAG tag, HA tag, polyHis tag (having about 5-10 histidines) (SEQ ID NO: 80), hexahistidine tag (HHHHHH) (SEQ ID NO: 81), 8X-His tag (HHHHHHHH) (SEQ ID NO: 82), Myc tag, glutathione-S-transferase tag, green fluorescent protein tag, maltose binding protein tag, thioredoxin tag, or Fc tag. In some embodiments, at least one additional moiety is an Fc tag. In some particular embodiments, the Fc fragment is IgG, IgA, IgD, IgM, or IgE. In some particular embodiments, IgG is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the extension or additional moiety can be an N-terminal signal peptide fused to the protein to enhance expression. Such signal peptides are often cleaved during expression in cells, although some polypeptides can contain cytokines with an intact signal peptide.

[0152] In some embodiments, at least one additional moiety comprises an antigen-binding molecule. In certain embodiments, the antigen-binding molecule is an antibody or an antigen-binding fragment thereof. In certain embodiments, the antigen-binding molecule is a multispecific antigen-binding molecule. In certain cases, the antigen-binding molecule is a bispecific antibody or a bispecific antigen-binding fragment thereof. In other certain cases, the antigen-binding molecule is an antibody-drug conjugate (ADC). In other embodiments, at least one additional moiety is selected from the group consisting of scFv, (scFv)2, Fab, Fab’, F(ab’)2, Fv, dAb, Fd fragment, diabody, F(ab’)3, disulfide-linked Fv, sdAb (VHH or nanobody), CDR, di-scFv, bi-scFv, tascFv (tandem scFv), tribody, tetrabody, V-NAR domain, Fcab, IgGACH2, DVD-Ig, probody, DARPin, centyrin, afibody, affilin, affitin, anticalin, avimer, finomer, knotted domain peptide, monobody (or adnectin), tribody, and nanofitin.

[0153] In other embodiments, at least one additional portion is a fragment that retains the ability to bind to the target antigen. Exemplary functional fragments include Fab fragments (e.g., antibody fragments containing an antigen-binding domain and comprising a light chain and a portion of a heavy chain cross-linked by a disulfide bond); Fab' (e.g., antibody fragments containing a single antigen-binding domain and comprising Fab and an additional portion of the heavy chain through the hinge region); F(ab')2 (e.g., two Fab' molecules joined by an interchain disulfide bond within the hinge region of the heavy chain; these Fab' molecules can be directed to the same or different epitopes); bispecific Fab (e.g., Fab molecules having two antigen-binding domains, each of which can be directed to a different epitope); single-chain containing variable regions, also known as scFv (e.g., the variable, antigen-binding determining regions of a single light chain and heavy chain of an antibody linked to each other by a chain of 10 to 25 amino acids); disulfide-linked Fv, or dsFv (e.g., the variable, antigen-binding determining regions of a single light chain and heavy chain of an antibody linked to each other by a disulfide bond); camelized VH (e.g., the variable, antigen-binding determining region of a single heavy chain of an antibody, where some amino acids at the VH interface are those found in the heavy chain of a naturally occurring camel antibody); bispecific scFv (e.g., scFv or dsFv molecules having two antigen-binding domains, each of which can be directed to a different epitope); diabody (e.g., a dimerized scFv formed when the VH domain of a first scFv associates with the VL domain of a second scFv and the VL domain of the first scFv associates with the VH domain of the second scFv; the two antigen-binding regions of the diabody can be directed to the same or different epitopes); triabody (e.g., a trimerized scFv formed in the same manner as a diabody, but with three antigen-binding domains created within a single complex; the three antigen-binding domains can be directed to the same or different epitopes); and tetrabody (e.g., a tetramerized scFv formed in the same manner as a diabody, but with four antigen-binding domains created within a single complex; the four antigen-binding domains can be directed to the same or different epitopes).

[0154] In some embodiments, the antigen-binding molecule conjugated to the polypeptide described herein targets tumor cells. In some embodiments, the antigen is a tumor-associated antigen. In certain embodiments, the tumor-associated antigen is selected from the group consisting of human epidermal growth factor receptor 2 (HER2), CD20, CD33, B cell maturation antigen (BCMA), prostate-specific membrane (PSMA), DLL3, ganglioside GD2 (GD2), CD123, anoctamin-1 (Anol), mesothelin, carbonic anhydrase IX (CAIX), tumor-associated calcium signal transducer 2 (TROP2), carcinoembryonic antigen (CEA), claudin 18.2, receptor tyrosine kinase-like orphan receptor 1 (ROR1), trophoblast glycoprotein (5T4), glycoprotein non-metastatic melanoma protein B (GPNMB), folate receptor-alpha (FR-alpha), pregnancy-associated plasma protein A (PAPP-A), CD37, epithelial cell adhesion molecule (EpCAM), CD2, CD19, CD30, CD38, CD40, CD52, CD70, CD79b, fms-like tyrosine kinase 3 (FLT3), glypican 3 (GPC3), B7 homolog 6 (B7H6), C-C chemokine receptor type 4 (CCR4), C-X-C motif chemokine receptor 4 (CXCR4), receptor tyrosine kinase-like orphan receptor 2 (ROR2), CD133, HLA class I histocompatibility antigen, alpha chain E (HLA-E), epidermal growth factor receptor (EGFR / ERBB-1), insulin-like growth factor 1 receptor (IGF1R), and human epidermal growth factor 3.

[0155] In some embodiments, the antigen-binding molecule conjugated to the polypeptide described herein targets tumor cells. In some embodiments, the antigen is an immune checkpoint antigen or an immune checkpoint-related antigen. In certain embodiments, the antigen is involved in an immune checkpoint pathway. In certain embodiments, the antigen is selected from the group consisting of PD-1, PD-L1, TIGIT, CTLA-4, PD-L2, B7-H3, B7-H4, BTLA, LAG3, CD112, CD112R, CD96, TIM-3, CD47, and CEACAM1. In certain embodiments, the antigen is a costimulatory immune checkpoint target. In certain embodiments, the antigen is selected from the group consisting of CD155, ICOS, OX40, CD137, CD137L, CD27, CD28, and GITR.

[0156] In some embodiments, at least one additional moiety is attached to the C-terminus and / or N-terminus of the IL-21 variant. In some embodiments, at least one additional moiety is attached to the IL-21 variant directly or via a linker.

[0157] In certain examples, multiple additional moieties disclosed herein can be attached to a single polypeptide disclosed herein. In certain examples, multiple polypeptides disclosed herein can be attached to a single additional moiety disclosed herein. In certain examples, a single additional moiety disclosed herein is attached to a single polypeptide disclosed herein. In some cases, the ratio of the additional moiety to the polypeptide is 1:1. In some cases, the ratio of the additional moiety to the polypeptide is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some cases, the ratio of the additional moiety to the polypeptide is about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0158] In some embodiments, at least one additional moiety is albumin or a functional fragment thereof. Albumin is a family of water-soluble globular proteins. Albumin is commonly found in plasma and constitutes about 55-60% of total plasma proteins. Human serum albumin (HSA) is a 585 amino acid polypeptide containing 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 contains a binding site that can interact either reversibly or irreversibly with an endogenous ligand, such as a fatty acid, bilirubin or hemin, or an exogenous compound, such as a heterocyclic or aromatic compound.

[0159] In some embodiments, at least one additional moiety is transferrin. Transferrin is a 679 amino acid polypeptide with a size of approximately 80 kDa and contains two Fe 3+ binding sites, one in the N-terminal domain and the other in the C-terminal domain. Human transferrin has a half-life of about 7-12 days.

[0160] In some embodiments, at least one additional moiety is transthyretin (TTR). Transthyretin is a transport protein found in serum and cerebrospinal fluid that transports the thyroid hormone thyroxine (T4) and retinol bound to retinol-binding protein.

[0161] In some embodiments, a conjugate comprising a polypeptide described herein and an antibody or an antigen-binding fragment thereof described herein is enriched in a relevant tumor tissue by the action of the antibody or an antigen-binding fragment thereof described herein. Thus, the antitumor activity of the polypeptide described herein can be enhanced.

[0162] In some embodiments, at least one additional moiety comprises a bioconjugate (e.g., a toll-like receptor (TLR) agonist, e.g., a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 agonist; or a synthetic ligand, e.g., Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, poly I:C, poly A:U, AGP, MPLA, RC-529, MDF2p, CFA, or flagellin).

[0163] Moieties categorized by function In some cases, at least one additional moiety reduces the interaction of IL-21 with one or more IL-21 receptor domains or subunits. In some embodiments, at least one additional moiety blocks the interaction of IL-21 with one or more IL-21 domains or subunits in the presence of its cognate receptor. In some cases, at least one additional moiety extends serum half-life and / or improves stability in vivo.

[0164] Masking moiety In some embodiments, at least one additional moiety is a masking moiety. A masking moiety can block, occlude, inhibit, reduce, or otherwise decrease (i.e., mask) the activity or binding of IL-21 to its cognate receptor or protein. For example, the polypeptides disclosed herein can be activatable by proteases at a target site, e.g., in the tumor microenvironment, by including a protease-cleavable linker. In some embodiments, the protease-cleavable linker couples the polypeptide to the masking moiety. When the linker cleavable at the target site is cleaved by proteolysis, the polypeptides disclosed herein can be activated, whereby the polypeptides can bind to their cognate receptor or protein.

[0165] The masking portion may include a cleavable peptide and / or a linker. In some embodiments, the cleavable linker includes a cleavable peptide. A cleavable peptide is a polypeptide that includes a cleavage site such as a protease cleavage site. The cleavage site is a site recognizable for cleavage of a portion of the cleavable peptide of the cleavable linker described herein. In some embodiments, the cleavable peptide includes more than one cleavage site. In some embodiments, the cleavage site is an amino acid sequence that is recognized and cleaved by a cleavage agent. Exemplary cleavage agents include proteins, enzymes, DNAzymes, RNAzymes, metals, acids, and bases.

[0166] In some embodiments, the protease cleavage site is a matrix metalloprotease (MMP) cleavage site, a disintegrin and metalloprotease domain-containing (ADAM) metalloprotease cleavage site, a prostate-specific antigen (PSA) protease cleavage site, a urokinase-type plasminogen activator (uPA) protease cleavage site, a membrane-type serine protease 1 (MT-SP1) protease cleavage site, a matriptase protease cleavage site (ST14), or a legumain protease cleavage site. In embodiments, the matrix metalloprotease (MMP) cleavage site is an MMP9 cleavage site, an MMP13 cleavage site, or an MMP2 cleavage site. In embodiments, the disintegrin and metalloprotease domain-containing (ADAM) metalloprotease cleavage site is an ADAM9 metalloprotease cleavage site, an ADAM10 metalloprotease cleavage site, or an ADAM17 metalloprotease cleavage site. The protease cleavage site can be indicated by a specific amino acid sequence.

[0167] Half-life extension domain In some embodiments, at least one additional moiety is a half-life extension domain that extends the serum half-life and / or improves the stability of the polypeptides disclosed herein. The half-life extension domain can be fused to the N-terminus or C-terminus of the polypeptides disclosed herein. In some embodiments, a proteolytically cleavable linker couples the polypeptides disclosed herein to the half-life extension domain.

[0168] In some embodiments, the half-life extension domain is an albumin polypeptide or a functional fragment thereof. Albumin is a natural carrier protein that has an extended serum half-life of approximately three weeks due to its size and its susceptibility to FcRn-mediated recycling, and has a low likelihood of intracellular degradation. Thus, the serum half-life of an IL-21 polypeptide can be significantly extended by conjugating the IL-21 polypeptide to albumin.

[0169] In some embodiments, the IL-21 polypeptide herein comprises an IL-21 mutein fused to a heterologous polypeptide (i.e., a polypeptide that is not IL-21, preferably a variant of IL-21, e.g., a polypeptide that is not the portion to be conjugated as described herein). The heterologous polypeptide can extend the circulating half-life of the IL-21 polypeptide. For example, the polypeptide that extends the circulating half-life can be serum albumin, e.g., HSA.

[0170] In some embodiments, the IL-21 polypeptides provided herein comprise an amino acid sequence that is heterologous to wild-type human IL-21 or a functional variant thereof. The amino acid sequence can be, for example, a half-life extension moiety that increases the stability of the polypeptide by extending the in vivo half-life of the polypeptide when administered to a subject. The half-life extension moiety can be a protein, an antibody, albumin, an immunoglobulin or a fragment thereof, transferrin, or PEG. The antibody can be an anti-albumin antibody. The albumin can be serum albumin, for example, mouse serum albumin or human serum albumin. In some embodiments, the immunoglobulin fragment is the Fc domain of a human immunoglobulin, for example, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM.

[0171] In some embodiments, the polypeptides disclosed herein comprise a half-life extension domain that comprises an albumin polypeptide or a fragment or variant thereof. In some embodiments, the albumin polypeptide is mouse serum albumin. In some embodiments, the albumin polypeptide is human serum albumin.

[0172] In some embodiments, the albumin polypeptide is linked to a masking moiety. In some embodiments, the masking moiety is linked to the N-terminus or C-terminus of the albumin polypeptide. In some embodiments, the albumin polypeptide is linked to the masking moiety via a linker. In some embodiments, the linker is linked to the amino terminus or carboxy terminus of the albumin polypeptide. In some embodiments, the N-terminal or C-terminal spacer domain of the linker is linked to the N-terminus or C-terminus of the albumin polypeptide. In some embodiments, the cleavable peptide of the linker is linked to the N-terminus or C-terminus of the albumin polypeptide. In some embodiments, the albumin polypeptide is linked to a cytokine or a functional fragment thereof (e.g., IL-21 or a mutein thereof). In some embodiments, the cytokine or a functional fragment thereof is linked to the N-terminus or C-terminus of the albumin polypeptide. In some embodiments, the albumin polypeptide is linked to the cytokine or a functional fragment thereof via a linker. In some embodiments, the linker is linked to the amino terminus or carboxy terminus of the albumin polypeptide.

[0173] In some embodiments, the conjugate comprises a polypeptide disclosed herein and a combination of two or more of the above moieties.

[0174] Synergistic effect In some embodiments, the conjugates described herein exhibit better pharmacodynamic properties than the polypeptides described herein. In some embodiments, the conjugates described herein exhibit better pharmacodynamic properties than at least one additional moiety. In certain embodiments, the conjugates described herein exhibit stronger antitumor activity than the polypeptides described herein. In some embodiments, the conjugates described herein exhibit stronger antitumor activity than at least one additional moiety. In certain embodiments, the conjugates described herein result in fewer side effects than the polypeptides described herein. In some embodiments, the conjugates described herein result in fewer side effects than at least one additional moiety.

[0175] Thermal stability In some embodiments, the polypeptides or conjugates described herein exhibit improved thermal stability compared to wild-type human IL-21. In other embodiments, the polypeptides or conjugates described herein are more stable in vitro compared to wild-type human IL-21. In other embodiments, the polypeptides or conjugates described herein are more stable in vivo compared to wild-type human IL-21.

[0176] In some embodiments, the thermal stability of the polypeptides or conjugates described herein is evaluated by NanoTemper assay (Prometheus). In some embodiments, the thermal stability of the polypeptides or conjugates described herein is evaluated by qPCR with Protein Thermal Shift (PTS) research solution (Thermo Fisher). In some embodiments, the thermal stability of the polypeptides or conjugates described herein is evaluated by differential scanning calorimetry (DSC). In some embodiments, the thermal stability of the polypeptides or conjugates described herein is evaluated by differential scanning fluorimetry (DSF). In some specific embodiments, the melting temperature of the mutant IL21 polypeptide described herein is higher than that of the wild-type IL21 equivalent. In some specific embodiments, the melting temperature of the mutant IL21 conjugate described herein is higher than that of the wild-type IL21 equivalent.

[0177] Properties and efficacy In some embodiments, the polypeptides or conjugates described herein exhibit any one or more of the following, for example, 1, 2, 3, 4, 5, 6, or 7: (1) better pharmacokinetic properties than wild-type IL-21 or wild-type IL-21 conjugate; (2) better pharmacokinetic properties than at least one additional moiety; (3) stronger antitumor activity than wild-type IL-21 or wild-type IL-21 conjugate; (4) stronger antitumor activity than at least one additional moiety; (5) fewer side effects than wild-type IL-21 or wild-type IL-21 conjugate; (6) a synergistic effect in combination with at least one additional agent compared to any of them administered alone; and (7) improved thermal stability compared to wild-type IL-21 or wild-type IL-21 conjugate.

[0178] Nucleic acids, vectors, and transformed or host cells In another aspect, nucleic acid molecules encoding the polypeptides disclosed herein or the conjugates disclosed herein are provided herein. In another aspect, vectors comprising the nucleic acid molecules provided herein are provided herein. In some embodiments, the vector comprises a viral vector. In another aspect, transformed or host cells expressing the polypeptides disclosed herein or the conjugates disclosed herein are provided herein.

[0179] The nucleic acid molecule can encode the polypeptides described herein. The nucleic acid molecule can be codon-optimized to encode the polypeptides described herein. Such nucleic acid molecules can be inserted into an expression vector, which can then be transformed into or incorporated into a host cell. The expression vector can be a plasmid. Host cells comprising the expression vector can be used to express the IL-21 variants described herein. The host cell can be E. coli or other suitable bacterial cells.

[0180] Unless otherwise specified, the present invention is described, for example, in Michael R. Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); Current Protocols in Molecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley and Sons, Inc.), Current Protocols in Immunology (CPI) (John E.Coligan, et. al., ed. John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998), Methods in Molecular biology, Vol.180, Transgenesis Techniques by Alan R. Clark editor, second edition, 2002, Humana Press, and Methods in Meolcular Biology, Vo.In 203, 2003, Transgenic Mouse, edited by Marten H. Hofker and Jan van Deursen, it can be carried out using standard procedures known to those skilled in the art, and all of these references are hereby incorporated by reference in their entirety.

[0181] Kit In another aspect, provided herein are kits comprising a polypeptide, conjugate, nucleic acid molecule, vector, transformed or host cell, or pharmaceutical composition, combinations thereof, and a container.

[0182] In some embodiments, provided herein are kits or articles of manufacture comprising the polypeptides described herein, including their pharmaceutical compositions. The kit may include instructions for using the polypeptide, for example, in the methods provided herein. Thus, in some embodiments, the kit includes instructions for using the polypeptide in a method of treating a disorder (e.g., cancer) described herein in a subject in need of treatment by administering a therapeutically effective amount of the polypeptide to the subject. In some embodiments, the subject is human. In some embodiments, the disorder is cancer.

[0183] The kit may further include a container. Non-limiting examples of suitable containers include vials, vials (e.g., dual-chamber vials), syringes (e.g., single-chamber or dual-chamber syringes), test tubes, and intravenous (IV) bags. The container can be formed from various materials such as glass or plastic. The container can hold a formulation of the IL-21 polypeptide. In some embodiments, the formulation is a lyophilized formulation. In some embodiments, the formulation is a frozen formulation. In some embodiments, the formulation is a liquid formulation.

[0184] The kit may further include a label or package insert, which may be present on or associated with the container and may indicate instructions for reconstitution and / or use of the formulation. The label or package insert may further indicate that the formulation is useful for or intended for intravenous, subcutaneous or other modes of administration for treating a disorder (e.g., cancer) in a subject. The container holding the formulation may be a single-use vial or a multi-use vial that may allow for repeated administration of the reconstituted formulation. The kit may further include a second container containing a suitable diluent. The kit may further include other materials desirable from a commercial, therapeutic and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0185] In some embodiments, kits for single-dose administration units are provided herein. Such kits include a container of an aqueous formulation of a therapeutic IL-21 polypeptide, including, but not limited to, a single or multi-chamber pre-filled syringe.

[0186] In some embodiments, articles of manufacture or kits are provided herein that include the formulations described herein for administration with an autoinjector device. An autoinjector can be described as an injection device that delivers its contents without further need for an action by the patient or administrator upon activation. They are particularly suitable for self-administration of therapeutic formulations where the delivery rate must be constant and the delivery time exceeds a few minutes.

[0187] Pharmaceutical composition In another aspect, pharmaceutical compositions are provided herein that include a polypeptide disclosed herein or a conjugate disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions of the present disclosure can be a combination of a compound, e.g., a polypeptide described herein, and other chemical components, e.g., carriers, stabilizers, diluents, dispersants, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates administration of the compound to a living being.

[0188] The pharmaceutical preparation for administration may contain an aqueous solution of the active compound in water-soluble form. A suspension of the active compound can be prepared as an oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. The aqueous injection suspension may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. The suspension may also contain suitable stabilizers or suitable agents that increase the solubility of the compound to enable the preparation of a highly concentrated solution. The active ingredient may be in powder form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use.

[0189] In practicing the treatment or use methods provided herein, a therapeutically effective amount of the compounds described herein is administered in a pharmaceutical composition to a subject having a disease or condition to be treated. In some embodiments, the subject is a mammal, such as a human. The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used, as well as other factors.

[0190] The pharmaceutical composition can be formulated using one or more physiologically acceptable carriers, excipients, and auxiliaries that facilitate the processing of the active compound into a preparation that can be used pharmaceutically. The formulation can be modified depending on the chosen route of administration. The pharmaceutical composition containing the compounds described herein can be manufactured, for example, by processes such as mixing, dissolving, emulsifying, encapsulating, entrapping, or compressing.

[0191] The pharmaceutical composition can include at least one pharmaceutically acceptable carrier, diluent, or excipient, and the compound described herein in free base or pharmaceutically acceptable salt form. The pharmaceutical composition may contain solubilizing agents, stabilizers, tonicity enhancing agents, buffers, and preservatives.

[0192] The method for the preparation of a composition comprising a compound described herein involves formulating the compound using one or more inert pharmaceutically acceptable excipients or carriers to form a solid, semi-solid or liquid composition. Examples of solid compositions include, for example, powders, tablets, dispersible granules, capsules, and cachets. Examples of liquid compositions include, for example, solutions in which the compound is dissolved, emulsions containing the compound, or solutions containing liposomes, micelles or nanoparticles containing the compound, as disclosed herein. Examples of semi-solid compositions include, for example, gels, suspensions, and creams. The composition may exist in the form of a liquid solution or suspension, in a solid form suitable for dissolution or suspension in a liquid prior to use, or as an emulsion. These compositions may also contain small amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and other pharmaceutically acceptable additives.

[0193] Non-limiting examples of dosage forms suitable for use in the present disclosure include liquids, powders, gels, nanosuspensions, nanoparticle formulations, microgels, aqueous or oily suspensions, emulsions, and any combination thereof.

[0194] Non-limiting examples of pharmaceutically acceptable excipients suitable for use in the present disclosure include binders, disintegrants, anti-adhesion agents, anti-static agents, surfactants, antioxidants, coating agents, colorants, plasticizers, preservatives, suspending agents, emulsifying agents, antimicrobial agents, spheronizing agents, and any combination thereof.

[0195] The compositions of the present disclosure can be, for example, immediate-release forms or controlled-release formulations. Immediate-release formulations can be formulated to act rapidly after administration of the compound. Non-limiting examples of immediate-release formulations include readily soluble formulations. Controlled-release formulations are pharmaceutical formulations that are adapted or programmed to provide release of the active agent at a rate and release profile that can conform to physiological and chronotherapeutic requirements. Non-limiting examples of controlled-release formulations include granules, delayed-release granules, hydrogel agents (e.g., of synthetic or natural origin), other gelling agents (e.g., gelling dietary fibers), matrix-based formulations (e.g., formulations containing a polymeric material in which at least one active ingredient is dispersed), granules within a matrix, polymer mixtures, and agglomerates.

[0196] In some embodiments, the controlled-release formulation is in a delayed-release form. Delayed-release forms can be formulated to delay the action of the compound over an extended period of time. Delayed-release forms can be formulated to delay the release of an effective dose of one or more compounds for, for example, about 4 hours, about 8 hours, about 12 hours, about 16 hours, or about 24 hours.

[0197] The controlled-release formulation can be in a sustained-release form. Sustained-release forms can be formulated to maintain, for example, the action of the compound over an extended period of time. Sustained-release forms can be formulated to provide an effective dose of any of the compounds described herein for about 4, about 8, about 12, about 16, or about 24 hours (e.g., provide a physiologically effective blood profile).

[0198] The compounds described herein can be conventionally formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers. For example, reference is made to Remington's Pharmaceutical Sciences, latest edition, by E.W. Martin Mack Pub. Co., Easton, PA, which is hereby incorporated by reference in its entirety, as it discloses pharmaceutically acceptable excipients and carriers, as well as methods for preparing pharmaceutical compositions. Such carriers can be carriers for the administration of the compositions to humans and non-humans, including solutions, such as sterile water, saline, and buffered solutions at physiological pH. The pharmaceutical compositions can also include one or more additional active ingredients, such as antimicrobial agents, anti-inflammatory agents, and anesthetics.

[0199] Pharmaceutical formulations can also include additional carriers, as well as thickeners, diluents, buffers, preservatives, and surfactants, in addition to the agents disclosed herein.

[0200] The pharmaceutical compositions disclosed herein can be administered in therapeutically effective amounts by various forms and routes, including, for example, oral, topical, parenteral, intravenous injection, intravenous infusion, subcutaneous injection, subcutaneous infusion, intramuscular injection, intramuscular infusion, intradermal injection, intradermal infusion, intraperitoneal injection, intraperitoneal infusion, intracerebral injection, intracerebral infusion, subarachnoid injection, subarachnoid infusion, intraocular injection, intraspinal injection, intrasternal injection, ophthalmic administration, endothelial administration, topical administration, intranasal administration, pulmonary administration, rectal administration, intraarterial administration, intrathecal administration, inhalation, intralesional administration, intradermal administration, epidural administration, absorption from epithelial or skin mucosal layers (e.g., oral mucosa, rectal and intestinal mucosa), intracapsular administration, subcapsular administration, intracardiac administration, transtracheal administration, subepidermal administration, subarachnoid administration, subcapsular administration, intraspinal administration, or intrasternal administration.

[0201] The pharmaceutical composition can be administered in a local manner, for example, by injection of the compound directly into an organ, if necessary, as a depot or sustained-release formulation or implant. The pharmaceutical composition can be provided in the form of an immediate-release formulation, in the form of a sustained-release formulation, or in the form of an intermediate-release formulation. The immediate-release form can provide immediate release. The sustained-release formulation can provide controlled release or sustained-release delayed release.

[0202] The compounds herein can be administered in combination with one or more therapeutic agents, such as cytokines, antiviral agents, or antifungal agents. The term "therapeutic agent" includes any agent administered to treat a symptom or disease in an animal that requires such treatment. The compounds can also be administered as a component of a vaccine, i.e., in combination with essentially any preparation for active immunoprophylaxis.

[0203] The toxicity and therapeutic efficacy of the compounds herein can be determined by standard pharmacological procedures in cell culture or experimental animals. Cell culture assays and animal studies can be used to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic effect and the therapeutic effect is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Compounds herein showing a high therapeutic index are preferred. The data obtained from these cell culture assays and animal studies can be used to formulate a dosage range suitable for use in humans. The dosage of such compounds preferably lies within a range of circulating concentrations that includes the ED50 with little or no toxicity. The dosage can vary within this range depending on various factors such as the dosage form utilized, the route of administration utilized, and the condition of the subject, etc.

[0204] A therapeutically effective amount can first be estimated from cell culture assays by determining the IC50. Next, in an animal model, a dosage can be formulated to achieve a range of circulating plasma concentrations that includes the IC50 determined in cell culture. Such information can be used to more accurately determine a dosage useful in humans. Plasma levels can be measured, for example, by HPLC. The exact formulation, route of administration, and dosage will be selected by the individual physician in view of the patient's condition.

[0205] The attending physician of a patient being treated with a compound herein will know the methods and times for terminating, interrupting, or adjusting administration due to, for example, toxicity and organ failure. Conversely, the attending physician will also know to adjust treatment to a higher level if the clinical response is inadequate (while preventing toxicity). The magnitude of the dosage administered in the management of the disorder of interest will vary depending on the severity of the condition to be treated and the route of administration, among other things. The severity of the condition can be assessed, for example, in part, by standard prognostic evaluation methods. Further, the dosage and perhaps the frequency of administration will also vary depending on the age, weight, and response of the individual patient.

[0206] The compounds herein can be administered to an individual alone as a pharmaceutical preparation appropriately formulated for the route of delivery and for the condition to be treated. Suitable routes include oral, rectal, transdermal, vaginal, transmucosal, or enteral administration; and parenteral delivery, including intramuscular, subcutaneous, intramedullary injection, and intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection. For transmucosal administration, suitable penetration enhancers for crossing the barrier can be used in the formulation.

[0207] The compounds herein can be formulated as a liquid using a carrier which may contain a buffer and / or a salt, such as phosphate buffered saline. Alternatively, the compounds herein can be formulated as a solid using a carrier or filler, such as lactose, a binder, such as starch, and / or a lubricant, such as talc or magnesium stearate, and optionally a stabilizer.

[0208] For oral delivery, the final products to be formulated can be tablets, pills, capsules, dragees, liquids, gels, syrups, slurries, and suspensions, etc. Also, push-fit capsules made of gelatin, as well as sealed soft capsules made of gelatin and a plasticizer, such as glycerol or sorbitol, can be used. The push-fit capsules can contain the active ingredient as a mixture with the above fillers, while in the soft capsules, the active compound can be dissolved or suspended in a suitable liquid, such as a fatty oil, liquid paraffin, or liquid polyethylene glycol.

[0209] Formulations for oral delivery can include conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, and lyophilization processes, etc. The compounds herein can also be mixed with a solid excipient, and optionally, the resulting mixture can be ground and, if desired, suitable auxiliaries can be added, and then the mixture of granules can be processed to obtain tablets or dragee cores. Suitable excipients include, in particular, sugars, such as lactose, sucrose, mannitol, and sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc., and polyvinylpyrrolidone (PVP), etc., and mixtures of any two or more of these. If desired, disintegrants, such as cross-linked polyvinylpyrrolidone, agar, and alginic acid or its salts, such as sodium alginate, etc., can be added.

[0210] When injection is desired, the compounds herein can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or saline buffer. In addition, a suspension of the active compound can be prepared as a suitable oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain compounds that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to enable the preparation of highly concentrated solutions.

[0211] Method In another aspect, provided herein is a method of preparing a polypeptide or conjugate described herein, the method comprising: (a) constructing a nucleic acid molecule and vector described herein; (b) culturing a transformed or host cell described herein; and (c) recovering the polypeptide from the transformed or host cell.

[0212] In another aspect, provided herein is a method of treating a subject in need of treatment, the method comprising administering to the subject a pharmaceutical composition described herein in an amount effective to treat the subject. In some embodiments, the subject has a solid tumor.

[0213] In some embodiments, the subject described herein has or is suspected of having a proliferative disease or condition. In some embodiments, the proliferative disease or condition is a neoplastic disease, such as cancer. In some cases, the cancer is metastatic cancer. In some cases, the cancer is recurrent or refractory cancer. In some embodiments, the cancer is untreated cancer. Untreated cancer can be cancer that has not been treated by therapy.

[0214] In some embodiments, the cancer is leukemia, lymphoma, sarcoma, myeloma, glioma, glioblastoma, glioblastoma multiforme, gliosarcoma, head and neck cancer, colorectal cancer, colon cancer, prostate cancer, castration-resistant prostate cancer, pancreatic cancer, melanoma, breast cancer (e.g., triple-negative, ER-positive, ER-negative, chemotherapy-resistant, trastuzumab-resistant, HER2-positive, doxorubicin-resistant, tamoxifen-resistant, ductal carcinoma, lobular carcinoma, primary, metastatic), neuroblastoma, lung cancer (e.g., non-small cell lung cancer, squamous cell carcinoma of the lung (e.g., head, neck, or esophagus), adenocarcinoma, large cell lung cancer, small cell lung cancer, carcinoid, sarcoma), ovarian cancer, bone cancer (e.g., osteosarcoma, chondrosarcoma, Ewing sarcoma), bladder cancer, cervical cancer, liver cancer (e.g., hepatocellular carcinoma), kidney cancer, skin cancer, testicular cancer, adrenal cancer, adenoid cystic carcinoma, anal cancer, brain cancer, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, oral cancer, thyroid cancer, retinoblastoma, parathyroid cancer, pituitary cancer, bile duct cancer, uterine cancer, acute myeloid leukemia, lymphoma, B-cell lymphoma, multiple myeloma, mesothelioma, medulloblastoma, Hodgkin's disease, non-Hodgkin lymphoma, neuroblastoma, rhabdomyosarcoma, essential thrombocythemia, Waldenström macroglobulinemia, primary brain tumor, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, premalignant skin lesions, neuroblastoma, genitourinary cancer, hypercalcemia of malignancy, adrenocortical cancer, neoplasm of the endocrine or exocrine pancreas, medullary thyroid cancer, medullary carcinoma of the thyroid, papillary thyroid cancer, hepatocellular carcinoma, Paget's disease of the nipple, phyllodes tumor, lobular carcinoma, ductal carcinoma, pancreatic stellate cell carcinoma, or hepatic stellate cell carcinoma. Additional non-limiting examples of cancers include cancers of the thyroid, endocrine system, brain, breast, cervix, colon, head and neck, esophagus, liver, kidney, ovary, stomach, or uterus.

[0215] In some embodiments, the cancer is a solid tumor. Non-limiting examples of solid tumors include 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, and prostate cancer.

[0216] In some embodiments, the cancer is a hematological malignancy, such as 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. Non-limiting examples of hematological malignancies include chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenström macroglobulinemia, multiple myeloma, extranodal marginal zone B cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt lymphoma, non-Burkitt high-grade B cell lymphoma, primary mediastinal B cell lymphoma (PMBL), immunoblastic large cell lymphoma, precursor B lymphoblastic lymphoma, B cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, plasmacytic myeloma, plasma cell neoplasms, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

[0217] In some embodiments, the subject described herein has or is suspected of having an inflammatory or autoimmune disease. In some embodiments, the inflammatory or autoimmune disease is atherosclerosis, obesity, inflammatory bowel disease (IBD), rheumatoid arthritis, allergic encephalitis, psoriasis, atopic skin disease, osteoporosis, peritonitis, hepatitis, lupus, celiac disease, Sjogren's syndrome, polymyalgia rheumatica, multiple sclerosis (MS), ankylosing spondylitis, type 1 diabetes, alopecia areata, vasculitis, and temporal arteritis, graft-versus-host disease (GVHD), asthma, COPD, paraneoplastic autoimmune disease, cartilage inflammation, juvenile arthritis, juvenile rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile reactive arthritis, juvenile Reiter's syndrome, seronegative enthesopathy and arthropathy (SEA) syndrome, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, pauciarticular rheumatoid arthritis, systemic rheumatoid arthritis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myolitis, polymyolitis, dermatomyolitis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, psoriasis, plaque psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, Crohn's disease, ulcerative colitis, celiac disease, rhinitis, rhinitis with polyps, eosinophilic esophagitis, eosinophilic bronchitis, Guillain-Barré disease, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, transplant rejection, kidney injury, or hepatitis C-induced vasculitis.

Example

[0218] (Example 1) Recombinant expression and preparation of IL-21 variants

[0219] The full-length amino acid sequences of the IL-21 variants are shown in Table 1. The alignment between the variants and the wild type is shown in Table 2.

Table 1-1

Table 1-2

Table 1-3

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 2-1

Table 2-2

[0220] Exemplary WT IL-21 and IL-21 variants are shown in Tables 1 and 2. They were generated by directly adding human IgG1 Fc (SEQ ID NO: 63) to the C-terminus of each of WT IL-21 and IL-21 variants using an Fc tag, and directly adding an 18-aa signal peptide (SEQ ID NO: 64) to the N-terminus of each of mature WT IL-21 and IL-21 variants (the signal peptide was added to facilitate expression in a mammalian cell line and then cleaved in the expression product).

[0221] The sequence of IgG1 Fc is, from N-terminus to C-terminus:

Chemical formula

[0222] The sequence of the 18-aa signal peptide is, from N-terminus to C-terminus: MHSSALLCCLVLLTGVRA (SEQ ID NO: 64) is as follows.

[0223] An exemplary amino acid sequence of the encoded IL-21-Fc construct is, from N-terminus to C-terminus:

Chemical formula

[0224] All variants shared the above construct structure with the same signal peptide and Fc tag sequences, but each variant had its own mature IL-21 sequence as shown in Tables 1 and 2.

[0225] 1) Construction of the expression plasmid.

[0226] Based on the amino acid sequences of the encoded IL-21-Fc constructs (wild-type IL-21 and IL-21 variants), DNA sequences were synthesized and confirmed by gene sequencing.

[0227] Exemplary expression sequences of the IL-21-Fc constructs are, from the N-terminus to the C-terminus:

Chemical Formula

[0228] The confirmed DNA sequences were constructed into the expression vector pcDNA3.1 (Thermo Fisher, catalog number V79020). Plasmids containing the IL-21-Fc gene (the exemplary WT IL-21 recombinant expression vector shown in Figure 1) were transformed into E. coli DH5α cells. Large amounts of both wild-type -Fc plasmids and variant -Fc plasmids were obtained by culturing E. coli DH5α cells for amplification and plasmid purification. The preparation of plasmid constructs was carried out according to the protocol from Michael R. Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (4 th edition, 2012). This reference is also incorporated herein by reference in its entirety.

[0229] 2) Expression of wild-type IL-21-Fc and IL-21 variant-Fc proteins in HEK293F cells.

[0230] The cells were seeded into 300 mL of medium (Gibco™ FreeStyle™ 293 Expression Medium, catalog number 12338018) in a 1 L shake flask at an inoculum volume of 0.5×10 6 cells / ml. The cells were incubated in a shaker incubator at 37 °C, 120 rpm and 5% carbon dioxide concentration for 24 hours until the cell density reached 1×10 6 cells / ml. Then, 300 μg of wild-type-Fc or variant-Fc expression plasmid was added to 30 ml of medium. The mixture was vortexed for 3 seconds to mix thoroughly. Approximately 1.2 ml (0.5 mg / ml) of the transfection reagent PEI MAX 40K (Polysciences, catalog number 24765) was added to the transfection reagent / plasmid mixture. The mixture was allowed to stand for 20 minutes and then added to the HEK293F cells. After transfection, the cells were incubated in a shaker incubator at 37 °C, 120 rpm and 5% carbon dioxide concentration for 48 - 54 hours. After incubation, the supernatant of the culture medium was isolated by centrifugation and prepared for protein purification.

[0231] 3) Purification and detection of wild-type IL-21-Fc and IL-21 variant-Fc proteins.

[0232] Wild-type IL-21-Fc and IL-21 variant-Fc proteins were purified using Protein A agarose microspheres. An appropriate amount of Protein A agarose microspheres was added to the supernatant from the previous step and incubated at 4 °C for 10 minutes. The wild-type IL-21-Fc and IL-21 variant-Fc proteins attached to the Protein A agarose microspheres were collected by centrifuging the mixture at low speed (1000 rpm) for 3 minutes. Subsequently, an appropriate volume of washing buffer (50 mM PBS, pH 7.4) was added to remove non-specific proteins. Finally, an appropriate volume of elution buffer (glycine HCl, pH 3) was added to elute wild-type-Fc or variant-Fc from the microspheres, the pH was adjusted to 7.4, and purified wild-type-Fc or variant-Fc was obtained.

[0233] The designed wild-type IL-21 or IL-21 variant in Table 1 was prepared in the IL-21-Fc form. The purified wild-type IL-21-Fc and IL-21 variant-Fc proteins were detected by SDS-PAGE protein gels. As representative results, the expression data of wild-type IL-21-Fc, m92-Fc, m98-Fc, m99-Fc, m100-Fc, and m133-Fc are shown in Table 3. The data indicate that these IL-21 variant-Fc proteins had higher expression than WT IL21-Fc. The results of SDS-PAGE protein gel detection indicate that the sizes of WT IL-21-Fc, m92-Fc, m98-Fc, m99-Fc, m100-Fc, and m133-Fc were consistent with the theoretical molecular weights for both their reduced and non-reduced samples (see Figures 2A - 2F).

[0234] The purity and protein aggregation state of wild-type IL-21-Fc and IL-21 variant-Fc proteins were further detected by size exclusion chromatography (SEC). The SEC results at 280 nm showed that wild-type IL-21-Fc, m92-Fc, m98-Fc, m99-Fc, m100-Fc, and m133-Fc all reached a peak at approximately 8.5 - 9 minutes. The data indicate that these proteins are monomeric proteins and did not generate aggregated proteins (see Figures 3A - 3F).

Table 3

[0235] Alternative Preparation 1

[0236] The WT IL-21 or IL-21 variant of the present disclosure was also prepared and purified using human IgG4 Fc.

[0237] The full-length amino acid sequences of the IL-21 variants are shown in Table 1. The alignment of the variants with the wild-type is shown in Table 2. Exemplary WT IL-21 and IL-21 variants are shown in Tables 1 and 2. They were generated by fusing human IgG4 Fc (SEQ ID NO: 68) to the C-terminus of each of WT IL-21 and the IL-21 variants using an Fc tag, and further directly adding an 18-aa signal peptide (SEQ ID NO: 64) to the N-terminus of each of the mature WT IL-21 and IL-21 variants (the signal peptide was added to facilitate expression in a mammalian cell line and then cleaved in the expression product).

[0238] Sequence of IgG4 Fc (from N-terminus to C-terminus):

Chem.

[0239] The sequence of the 18-aa signal peptide is (from N-terminus to C-terminus): MHSSALLCCLVLLTGVRA (SEQ ID NO: 64) It is as follows.

[0240] An exemplary amino acid sequence of the encoded IL-21-IgG4 Fc construct is (from N-terminus to C-terminus):

Chemical formula

Chemical formula

[0241] 1) Construction of the expression plasmid. Based on the amino acid sequences of wild-type IL-21-IgG4 Fc and IL-21 variant-IgG4 Fc, a DNA sequence was synthesized and confirmed by gene sequencing. The confirmed DNA sequence was constructed into the expression vector pcDNA3.1 (Thermo Fisher, catalog number V79020). Plasmids containing the IL-21-IgG4 Fc gene were transformed into E. coli DH5α cells. Large amounts of both wild-type plasmids and variant plasmids were obtained by culturing E. coli DH5α cells for amplification and plasmid purification.

[0242] 2) Expression of wild-type IL-21-IgG4 Fc and IL-21 variant-IgG4 Fc proteins in CHO cells. CHO cells (CHO-K1, Taizhou Biointron Biological, Inc.) were seeded at 0.5×10 in 30 mL of medium (Taizhou Biointron Biological, Inc.) in a 100 mL shaking flask.6 Inoculated with an inoculum volume of cells / ml. The cells were incubated at 37 °C, 120 rpm and 5% carbon dioxide concentration in a shaker incubator for 24 hours until the cell density reached 1.5×10 6 cells / ml. Then, 60 μg of wild-type-IgG4 Fc or variant-IgG4 Fc expression plasmid was added to 1 ml of medium. The mixture was vortexed for 3 seconds to mix thoroughly. Approximately 15 μl of transfection reagent (Taizhou Biointron Biological, Inc.) was added to 1 ml of medium (was added to was added to 1 ml of medium). The plasmid mixture was added to the transfection reagent mixture. The transfection reagent / plasmid mixture was allowed to stand for 20 minutes and then added to CHO cells. After transfection, the cells were incubated at 37 °C, 120 rpm and 5% carbon dioxide concentration in a shaker incubator for 48 - 54 hours. After incubation, the supernatant of the culture medium was isolated by centrifugation and prepared for protein purification.

[0243] 3) Purification and detection of wild-type IL-21-IgG4 Fc and IL-21 variant-IgG4 Fc proteins.

[0244] Wild-type IL-21-IgG4 Fc and IL-21 variant-IgG4 Fc proteins were purified using Protein A agarose microspheres. An appropriate amount of Protein A agarose microspheres was added to the supernatant from the previous step and incubated at 4 °C for 10 minutes. The wild-type IL-21-IgG4 Fc and IL-21 variant-IgG4 Fc proteins attached to the Protein A agarose microspheres were collected by centrifuging the mixture at low speed (1000 rpm) for 3 minutes. Subsequently, an appropriate volume of wash buffer (50 mM PBS, pH 7.4) was added to remove non-specific proteins. Finally, an appropriate volume of elution buffer (glycine HCl, pH 3) was added to elute wild-type-IgG4 Fc or variant-IgG4 Fc from the microspheres, the pH was adjusted to 7.4, and purified wild-type-IgG4 Fc or variant-IgG4 Fc was obtained.

[0245] The designed wild-type IL-21 and IL-21 variants in Table 1 were prepared in the IL-21-IgG4 Fc form. The purified wild-type IL-21-IgG4 Fc and IL-21 variant-IgG4 Fc proteins were detected by SDS-PAGE protein gels. Quantification is shown in Table 7. The data show that the m98-IgG4 Fc protein had a higher expression than WT IL-21-IgG4 Fc. The results of SDS-PAGE protein gel detection show that the sizes of WT IL-21-IgG4 Fc and m98-IgG4 Fc were consistent with the theoretical molecular weights for both their reduced and non-reduced samples (see Figures 7A - 7B).

[0246] The purity of WT IL-21 IgG4 Fc and m98-IgG4 Fc proteins was further detected by size exclusion chromatography (SEC). The SEC results at 280 nm showed that WT IL-21 IgG4 Fc reached a peak at 8.6 minutes and m98-IgG4 Fc reached a peak at 8 minutes. The data show that these proteins were monomeric proteins that were not aggregated (see Figures 8A - 8B).

[0247]

Table 7

[0248] Alternative Modulation 2

[0249] The WT IL-21 or IL-21 variant of the present disclosure can also be prepared and purified using other well-known tags such as His-tag.

[0250] The IL-21 variant was designed such that a GGGGS linker (SEQ ID NO: 91) and a His6 tag (SEQ ID NO: 81) were linked to the C-terminus.

[0251] An exemplary sequence of IL-21-His tag is

Figure

[0252] 1) Construction of expression plasmid.

[0253] Based on the amino acid sequences of wild-type IL-21-His and IL-21 variant-His (including GGGGS linker (SEQ ID NO: 91) and His tag), DNA sequences were synthesized and confirmed by gene sequencing. The confirmed DNA sequences were constructed into the expression vector pcDNA3.1 (Thermo Fisher, catalog number V79020). Plasmids containing the IL-21-His gene were transformed into E. coli DH5α cells. Large amounts of both wild-type plasmids and variant plasmids were obtained by culturing E. coli DH5α cells for amplification and plasmid purification.

[0254] 2) Expression of wild-type IL-21-His and IL-21 variant-His in HEK293F cells.

[0255] The cells were inoculated into 300 mL of medium (Gibco™ FreeStyle™ 293 Expression Medium, catalog number 12338018) in a 1 L shake flask at an inoculum volume of 0.5×10 6 cells / mL. The cells were incubated in a shaker incubator at 37 °C, 120 rpm and 5% carbon dioxide concentration for 24 hours until the cell density reached 1×10 6 cells / mL. Then, 300 μg of wild-type or mutant expression plasmid was added to 30 mL of PBS. The mixture was vortexed for 3 seconds to mix thoroughly. Approximately 1.2 mL (0.5 mg / mL) of transfection reagent PEI MAX 40K (Polysciences, catalog number 24765) was added to the PBS / plasmid mixture. The mixture was allowed to stand for 20 minutes and then added to the HEK293F cells. After transfection, the cells were incubated in a shaker incubator at 37 °C, 120 rpm and 5% carbon dioxide concentration for 48 - 54 hours. After incubation, the supernatant of the culture medium was isolated by centrifugation and prepared for protein purification.

[0256] 3) Purification and detection of wild-type IL-21-His and IL-21 variant-His.

[0257] Wild-type IL-21 and IL-21 variants containing His-tag were purified using Ni-NTA agarose microspheres (Thermo Fisher, catalog number R901). An appropriate amount of Ni-NTA agarose microspheres was added to the supernatant in the previous step and incubated at 4 °C for 30 minutes. Wild-type IL-21 and IL-21 variants attached to the Ni-NTA agarose microspheres were collected by centrifuging the mixture at low speed (1000 rpm) for 3 minutes. Then, an appropriate volume of washing buffer (50 mM PBS, pH 7.4, 10 mM imidazole) was added to remove non-specific proteins. Finally, an appropriate volume of elution buffer (50 mM PBS, pH 7.4, 250 mM imidazole) was added to elute the wild-type or variant from the microspheres, and the purified wild-type or variant was obtained.

[0258] The designed wild-type IL-21 and some IL-21 variants in Table 1 were prepared in the IL-21-His form. The purified wild-type IL-21-His and IL-21 variant-His proteins were detected by SDS-PAGE protein gels. The results of SDS-PAGE protein gel detection showed that the sizes of WT IL-21-His, m98-his, m103-his and m153-his were consistent with the theoretical molecular weights for both their reduced and non-reduced samples (see Figures 9A - 9D).

[0259] (Example 2) Recombinant expression and preparation of mutant IL-21-antibody fusion proteins. IL-21 or IL-21 variants are shown in Table 1 and Table 2. IL-21 or IL-21 variants can be fused with an antibody (e.g., anti-PD-1 antibody). Specifically, IL-21 or IL-21 variants can be conjugated to the N-terminus and / or C-terminus of the heavy chain and / or light chain of the antibody.

[0260] 1) Construction of expression plasmids.

[0261] Based on the amino acid sequences of wild-type IL-21 and IL-21 variants, DNA sequences were synthesized and designed to contain the sequences of the heavy or light chains of the antibody. The sequences were confirmed by gene sequencing. The confirmed DNA sequences were constructed into the expression vector pcDNA3.1 (Thermo Fisher, catalog number V79020). Plasmids containing the IL-21 gene were transformed into E. coli DH5α cells. Large amounts of both wild-type-antibody plasmids and variant-antibody plasmids were obtained by culturing E. coli DH5α cells for amplification and plasmid purification.

[0262] 2) Expression of wild-type IL-21-antibody and IL-21 variant-antibody fusion proteins in HEK293F cells.

[0263] Cells were seeded into 300 mL of medium (Gibco™ FreeStyle™ 293 Expression Medium, catalog number 12338018) in a 1 L shake flask at an inoculum volume of 0.5×10 6 cells / ml. The cells were incubated in a shaker incubator at 37 °C, 120 rpm, and 5% carbon dioxide concentration for 24 hours until the cell density reached 1×10 6 cells / ml. Then, 300 μg of wild-type-antibody or variant-antibody expression plasmid was added to 30 ml of PBS. The mixture was vortexed for 3 seconds to mix thoroughly. Approximately 1.2 ml (0.5 mg / ml) of the transfection reagent PEI MAX 40K (Polysciences, catalog number 24765) was added to the PBS / plasmid mixture. The mixture was allowed to stand for 20 minutes and then added to the HEK293F cells. After transfection, the cells were incubated in a shaker incubator at 37 °C, 120 rpm, and 5% carbon dioxide concentration for 48 - 54 hours. After incubation, the supernatant of the culture medium was isolated by centrifugation and prepared for protein purification.

[0264] 3) Purification of wild-type IL-21-antibody and IL-21 variant-antibody.

[0265] The wild-type IL-21-antibody and IL-21 variant-antibody fusion proteins were purified using Protein A agarose microspheres. An appropriate amount of Protein A agarose microspheres was added to the supernatant from the previous step and incubated at 4 °C for 10 minutes. The wild-type IL-21-antibody and IL-21 variant-antibody fusion proteins attached to the Protein A agarose microspheres were collected by centrifuging the mixture at low speed (1000 rpm) for 3 minutes. Then, an appropriate volume of wash buffer (50 mM PBS, pH 7.4) was added to remove non-specific proteins. Finally, an appropriate volume of elution buffer (glycine HCl, pH 3) was added to elute the wild-type-antibody or variant-antibody fusion protein from the microspheres, and the purified wild-type-antibody or variant-antibody fusion protein was obtained.

[0266] The PD-1 antibody-IL-21 fusion protein was constructed such that IL-21 was linked to the C-terminus of the heavy chain of the mouse PD-1 antibody via a linker (GGGGS (SEQ ID NO: 91)). The fusion proteins (anti-mPD1 antibody_WT IL21 fusion protein, anti-mPD1 antibody-IL21m98 fusion protein, anti-mPD1 antibody-IL21m100 fusion protein, and anti-mPD1 antibody-IL21m153 fusion protein) were prepared using wild-type (WT IL21) and IL21 variants (m98, m100, and m153) and the following anti-mPD1 antibody sequences: Anti-mPD1 antibody heavy chain sequence:

Chemical formula

Chemical formula

[0267] Among the IL-21_PD-1 antibody fusion proteins prepared in this way, the anti-mPD1 antibody-IL21m98 fusion protein is given as an example to illustrate the full-length sequence of the anti-mPD-1 heavy chain fused with IL-21: [Chemical formula] [Chemical formula] (The underlined segment represents the mature IL-21 sequence, the italicized part represents the flexible linker, and the rest is the anti-mPD1 Ab)

[0268] The purified fusion proteins (anti-mPD1 antibody IL21 fusion protein, anti-mPD1 antibody-IL21m98 fusion protein, anti-mPD1 antibody-IL21m100 fusion protein and anti-mPD1 antibody-IL21m153 fusion protein) were detected by SDS-PAGE protein gel. The results of SDS-PAGE protein gel detection showed that the size of the fusions was consistent with the theoretical molecular weight for both their reduced and non-reduced samples (see Figures 10A - 10D).

[0269] (Example 3) Determination of the thermal stability of IL-21 mutant proteins. To evaluate the stability of the three-dimensional structure, the melting temperature Tm (°C) value indicating the structural stability of the sample was obtained by monitoring the intrinsic tryptophan and tyrosine fluorescence at emission wavelengths of 330 nm and 350 nm.

[0270] The stability of the IL-21 variant protein was evaluated by detecting small changes in tryptophan and tyrosine fluorescence in the variant IL-21 protein using label-free nanoDSF technology (Prometheus, PR NT.48). Briefly, wild-type IL-21 or variant IL-21 protein was diluted to 0.4 mg / ml with PBS. 10 μL of wild-type IL-21 or variant IL-21 protein was loaded into a capillary and then placed in a sample holder. A temperature gradient of 1 °C / min from 25 to 95 °C was applied, and the intrinsic protein fluorescence was recorded at 330 and 350 nm. The data were analyzed using the data analysis software provided by the NT.48 instrument.

[0271] As shown in Table 8, m98-His and m153-His had Tm values of 62.02 °C and 63.12 °C, respectively, which were significantly higher than that of WT IL-21 His (Tm 48.47 °C), demonstrating high thermal stability.

[0272]

Table 8

[0273] (Example 4) Determination of the affinity of the variant IL-21 protein for human IL-21R.

[0274] The affinity of variant IL-21 for human IL-21R (ACRO, catalog: ILR-H5226) was determined as follows by label-free biolayer interferometry (Octet® R8, BLI):

[0275] 1: Wild-type and variant IL-21-Fc proteins were prepared and diluted to a final concentration of 5 μg / ml with buffer (10 mM PBS + 0.02% TW-20 + 0.1% BSA, pH 7.4).

[0276] 2: The IL-21R protein was diluted in a buffer (10 mM PBS + 0.02% Tween®-20 + 0.1% BSA, pH 7.4) in a 4-fold gradient from an initial concentration of 1000 nM to 3.9 nM, yielding a total of five concentrations: 1000 nM, 250 nM, 62.5 nM, 15.6 nM, and 3.9 nM, respectively.

[0277] 3: The Protein A sensor was immersed in the buffer (10 mM PBS + 0.02% Tween®-20 + 0.1% BSA, pH 7.4) for 10 minutes to equilibrate.

[0278] 4: The IL-21 mutant-Fc protein was loaded onto the Protein A sensor. The loading time was 90 seconds. Then, the mutant-Fc protein was eluted and then equilibrated for 30 seconds. Then, the mutant-Fc protein was combined with four different concentrations of the IL-21R protein. The binding time was 120 seconds. Finally, the bound IL-21R protein was eluted with the buffer. The dissociation time was 180 seconds.

[0279] 5: Octet® R8's Octet Data Acquisition Software was used for data processing and data fitting.

[0280] The affinity data in Table 4 indicate that the affinity (K D ) of WT IL-21-Fc for IL-21R was 4.57E-10 and that the affinity of the IL-21 mutant-Fc for IL-21R was lower than that of wild-type IL-21-Fc for IL-21R.

[0281] The interaction between WT IL-21-Fc and IL-21R was "fast Kon, slow Koff". However, the interactions between the mutants (e.g., m92-Fc, m98-Fc, m99-Fc, m100-Fc, and m133-Fc) and IL-21R were fast Kon, fast Koff (see Figures 4A - 4F).

Table 4-1

Table 4-2

[0282] (Example 5) Determination of p-STAT3 phosphorylation in HuT78 cells by the IL-21 variant. IL-21R is expressed on the surface of HuT78 cells. IL-21 can stimulate the phosphorylation of Tyr705 of STAT3 protein, a downstream signaling protein of IL-21R in HuT78 cells, by binding to IL-21R. The phosphorylation effect of the phosphorylation of Tyr705 of STAT3 protein was measured using the phospho-STAT3(Tyr705) kit / 62AT3PET (Cisbio), which served as a proxy for evaluating the ability of the IL-21 variant to activate the downstream signaling pathway of IL-21R. The phosphor-STAT3(Tyr705) assay kit was used to detect STAT3 phosphorylation according to the manufacturer's recommendation (Cisbio, catalog: 64NT3PEH).

[0283] STAT3 Phosphorylation Assay

[0284] 1) The IL-21 Fc fusion protein was diluted in IMDM complete medium at a 10-fold gradient from an initial concentration of 1000 nM to 0.0001 nM, resulting in a total of 8 concentrations: 1000 nM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, 0.001 nM, and 0.0001 nM, respectively.

[0285] 2) Seed HuT78 cells at a density of 33,000 cells per well (8 μl) in a HTRF 96-well low-volume white plate (Cisbio, catalog number 66PL96100). Then, stimulate the HuT78 cells with 12 μL / well of the IL-21-Fc fusion at 37 °C for 30 minutes. Next, add 4 μl of supplemented lysis buffer (4X) (Cisbio, catalog number 62AT3PET) per well and incubate with shaking at room temperature for 30 minutes. Finally, add 4 μl of Premix antibody solution (Cisbio, catalog number 62AT3PET) and incubate overnight at room temperature.

[0286] 3) Detect the FRET signal from the assay using an EnVision Multilable Plate Reader (Perkin Elmer). First, determine the HTRF ratio as recommended by Cisbio, and then analyze the data by calculating the magnification relative to the background value using data from unstimulated cells. Calculate the EC50 value by fitting the dependent data to a four-parameter logistic model using dotmatics software.

[0287] The experimental results showed that the EC 50 of WT IL-21-Fc for activating STAT3 phosphorylation in HuT78 cells was 0.01412 nM. Since the affinity of the IL-21 mutant-Fc for the IL-21R protein decreased to varying degrees, the EC 50 values of the IL-21 mutant-Fc for activating STAT3 phosphorylation in HuT78 cells were all higher. (See Figure 5 and Table 5).

Table 5

[0288] (Example 6) Evaluation of the in vivo antitumor activity of the IL-21-antibody fusion protein The in vivo efficacy of the IL-21-antibody fusion proteins (anti-mPD1 antibody-IL21m98 fusion protein, anti-mPD1 antibody-IL21m100 fusion protein, and anti-mPD1 antibody-IL21m153 fusion protein) prepared in Example 2 was further evaluated and compared with the anti-mPD1 antibody_WT IL21 fusion protein.

[0289] An hIL21R KI mouse model was established and tumor-bearing mice were divided into a control group and an experimental group. The control group was administered a vehicle, and the experimental group was further divided into (1) WT IL21_antibody fusion protein, (2) antibody alone, and (3) IL21 variant_antibody fusion protein. In each of the three subgroups, various concentrations and / or dosing regimens were optimized. Subcutaneous tumors were preferred to monitor tumor burden over time without sacrificing the animals easily. The following parameters for efficacy and safety were measured at multiple time points before and after injection of the IL21 variant_antibody conjugate: percent test / control (%T / C) tumor weight calculated on each day the tumor was measured, tumor growth delay, net log cell kill, median number of days to defined tumor weight or to a specified number of tumor doublings, tumor regression, animal survival rate, and baseline health status (e.g., body weight and locomotion). For tumors that were not subcutaneous, the tumors were excised and physically weighed, and then the same criteria were used to determine the tumor boundaries. Alternatively, tumors can be monitored using imaging techniques (e.g., bioluminescence, ultrasound, magnetic resonance imaging).

[0290] Specifically, MC38 mouse colon cancer cells (1×10 6 cells) were C57BL / 6-Il21r tm1(IL21R)It was subcutaneously implanted into the flanks of Bcgen mice (Beijing Biocytogen Co., Ltd., stock number 110766). Mice bearing MC38 cells were treated twice a week for 2 weeks (total of 4 doses) with PBS, 3 mg / kg of anti-mPD1 antibody, 3.65 mg / kg of anti-mPD1 antibody_WT IL21 fusion protein, 3.65 mg / kg of anti-mPD1 antibody_IL21m98 fusion protein, 3.65 mg / kg of anti-mPD1 antibody_IL21m100 fusion protein, or 3.65 mg / kg of anti-mPD1 antibody_IL21m153 fusion protein. Body weight and tumor volume were measured every 3 or 4 days throughout the study. Tumor measurements (length (L) and width (W)) were collected three times a week using digital calipers, and tumor volume was calculated as: (L × W × W) / 2. The results are shown in Table 6 below.

Table 6

[0291] Figure 6 shows tumor growth inhibition (error bars, SEM) in MC38 tumor-bearing mice upon treatment with anti-mPD1 antibody_IL21m fusion proteins and anti-mPD1 antibody_WT IL21 fusion protein at the indicated concentrations. n = 8 mice / group. Two-way ANOVA and Tukey's multiple comparisons were performed. Endpoint analysis is shown. ns, not significant; * , P < 0.05; ** , p < 0.01; *** , p < 0.001; n = 8 mice / group.

[0292] As shown in Figure 6 and Table 6, the anti-mPD1 antibody_IL21m fusion proteins (anti-mPD1 antibody_IL21m98 fusion protein, anti-mPD1 antibody_IL21m100 fusion protein, and anti-mPD1 antibody_IL21m153 fusion protein) significantly reduced tumor growth of MC38 tumors compared to the anti-mPD1 antibody and anti-mPD1 antibody_WT IL21 fusion protein.

[0293] (Example 7) Evaluation of the antitumor activity of IL-21 protein in vivo

[0294] Purified WT IL21-IgG4 Fc and exemplary m98-IgG4 Fc proteins were used in in vivo studies. The full-length amino acid sequence of mature m98-IgG4 Fc is shown below:

Chemical formula

[0295] B16-F10 melanoma cancer cells (5×10 5 cells) were subcutaneously implanted into the flanks of C57BL / 6-Il21r tm1(IL21R) / Bcgen mice (Beijing Biocytogen Co., Ltd, stock number 110766). Approximately 2 days after inoculation, tumor-bearing mice were divided into 3 groups of 10 mice per group by the random block method. Mice bearing B16-F10 cells were treated once every 3 days for 11 days (a total of 4 doses) with either PBS, 1.7 mg / kg of WT IL21-IgG4 Fc protein, or 1.7 mg / kg of m98-IgG4 Fc protein. Body weight and tumor volume were measured every 2 or 3 days throughout the study. Tumor measurements (length (L) and width (W)) were collected 3 times a week using a digital caliper, and tumor volume was calculated as: (L×W×W) / 2.

[0296]

Table 9

[0297] Figure 11 shows tumor growth inhibition (error bars, SEM) in B16-F10 tumor-bearing mice during treatment with the indicated concentrations of WT IL21-IgG4 protein and m98-IgG4 protein. n = 10 mice / group. Two-way ANOVA and Tukey's multiple comparison were performed. Endpoint analysis is shown. ns, not significant; * , P < 0.05; ** , p < 0.01; *** , p < 0.001; n = 10 mice / group.

[0298] As shown in Figure 11 and Table 9, the m98-IgG4 Fc protein significantly further reduced tumor growth of B16-F10 tumors compared to the WT IL21-IgG4 Fc protein. The in vivo study demonstrated the beneficial tumor inhibitory effect of the mutein of the present disclosure.

[0299] (Example 8) Evaluation of the in vivo antitumor activity of IL-21 protein B16-F10 melanoma cancer cells (5×10 5 cells) were subcutaneously implanted into the flanks of C57BL / 6-Il21r tm1(IL21R) / Bcgen mice (Beijing Biocytogen Co., Ltd, stock number 110766). Approximately 2 days after inoculation, the tumor-bearing mice were divided into 4 groups of 10 mice per group by the random block method. Each group of mice bearing B16-F10 cells was treated once every 3 days for 13 days (days 0, 3, 6, 9 and 12, a total of 5 doses) with either PBS, 1.7 mg / kg of m98-IgG4 Fc protein, 3 mg / kg of anti-mPD1 antibody, or 1.7 mg / kg of m98-IgG4 Fc combined with 3 mg / kg of anti-mPD1 antibody. Body weight and tumor volume were measured every 2 or 3 days throughout the study. Tumor measurements (length (L) and width (W)) were collected 3 times a week using digital calipers, and tumor volume was calculated: (L × W × W) / 2.

[0300]

Table 10

[0301] Figure 12 shows the tumor growth curves (error bars, SEM) in B16-F10 tumor-bearing mice during treatment with m98-IgG4 Fc protein, anti-mPD1 antibody, and m98-IgG4 Fc combined with anti-mPD1 antibody at the indicated concentrations. n = 10 mice / group. Two-way ANOVA and Tukey's multiple comparisons were performed. Endpoint analysis is shown. ns, not significant; * , P < 0.05; ** , p < 0.01; *** , p < 0.001; n = 10 mice / group.

[0302] As shown in Figure 12 and Table 10, administration of 3 mg / kg of anti-mPD1 antibody as a single agent showed no anti-tumor activity, and administration of 1.7 mg / kg of m98-IgG4 Fc as a single agent resulted in partial anti-tumor activity. However, 3 mg / kg of anti-mPD1 antibody in combination with 1.7 mg / kg of m98-IgG4 Fc resulted in statistically significant, synergistic anti-tumor activity compared to treatment with anti-mPD1 antibody alone (P < 0.001) or 1.7 mg / kg of m98-IgG4 Fc (P < 0.05).

[0303] Figure 13 shows the tumor weights (error bars, SEM) in B16-F10 tumor-bearing mice during treatment with m98-IgG4 Fc protein, anti-mPD1 antibody, and m98-IgG4 Fc combined with anti-mPD1 antibody at the indicated concentrations. n = 10 mice / group. Two-way ANOVA and Tukey's multiple comparisons were performed. Endpoint analysis is shown. ns, not significant; * , P < 0.05; ** , p < 0.01; *** , p < 0.001; n = 10 mice / group.

[0304] As shown in FIG. 13, the mean tumor volumes of mice treated with anti-mPD1 antibody (P<0.001) and mice treated with 1.7 mg / kg of m98-IgG4 Fc (P<0.05) were significantly greater than those of mice treated with the combination. No abnormal body weight changes or signs of toxicity were observed. These data suggested that m98-IgG4 Fc and combination administration significantly reduced tumor growth in the B16-F10 model.

[0305] In vivo studies demonstrated the beneficial tumor inhibitory effects of the muteins of the present disclosure, indicating the therapeutic potential of the muteins of the present disclosure as therapeutic agents, either as single agents, in combination, or in multifunctional fusion molecules.

Claims

1. A polypeptide comprising an interleukin-21 (IL-21) variant having one or more amino acid residue substitutions corresponding to wild-type human IL-21, wherein wild-type human IL-21 comprises the sequence described in SEQ ID NO: 1 or 2, and the polypeptide is: (1) P79E or P79C (2) S70L or S70A (3) K73Y or K73F, (4) The amino acid segment of STNAGRRQKHR (SEQ ID NO: 71) at positions 80-90, corresponding to wild-type human IL-21, substituted with a short peptide linker. A polypeptide comprising a short peptide linker which may contain one or more Gly-Ser units.

2. The polypeptide according to claim 3, wherein the polypeptide comprises one or more amino acid residue substitutions with cysteine ​​in the region between positions 1 and 10 corresponding to P79C and wild-type human IL-21, and forms a disulfide bond together with the cysteine ​​residue at position P79.

3. The polypeptide according to claim 2, wherein the one or more amino acid residue substitutions are selected from the group consisting of R5C, H6C, and R9C.

4. The polypeptide according to claim 1, wherein the polypeptide in (1) further comprises one or more amino acid residue substitutions at positions selected from the group consisting of R5, H6, R9, Q12, L13, D15, I16, S70, K72, K73, R76, and P78, and further comprises SEQ ID NO: 71 substituted with a short peptide linker.

5. The polypeptide according to claim 1, wherein in (2), further comprises one or more amino acid residue substitutions at positions selected from the group consisting of R5, H6, R9, Q12, L13, D15, I16, S70, K72, K73, R76, and P78, and further comprises SEQ ID NO: 71 substituted with a short peptide linker.

6. The polypeptide according to claim 1, further comprising, in (2), an amino acid residue substitution at R76 and zero or one or more amino acid residue substitutions at a position selected from the group consisting of R5, H6, R9, Q12, L13, D15, I16, K72, K73, P78, and P79, and further comprising SEQ ID NO: 71 substituted with a short peptide linker.

7. The polypeptide according to claim 1, wherein in (3), further comprises one or more amino acid residue substitutions at positions selected from the group consisting of R5, H6, R9, Q12, L13, D15, I16, K72, K73, P78, and P79, and further comprises SEQ ID NO: 71 substituted with a short peptide linker.

8. The polypeptide according to claim 1, wherein in (4), the short peptide linker is selected from the group consisting of GGSEGGGS (SEQ ID NO: 73), GSEGS (SEQ ID NO: 74), GGSGGGS (SEQ ID NO: 75), GGGSEGGGS (SEQ ID NO: 76), GGSEGGGGS (SEQ ID NO: 77), GSGGS, GGGSGGS (SEQ ID NO: 78), and GGGSEGGGS (SEQ ID NO: 72).

9. The polypeptide according to claim 1, wherein in (4), the polypeptide further comprises one or more amino acid residue substitutions at positions selected from the group consisting of R5, H6, R9, Q12, L13, D15, I16, S70, K72, K73, R76, P78, and P79.

10. The amino acid residue substitution at position R5 is R5C, the amino acid residue substitution at position H6 is H6C, the amino acid residue substitution at position R9 is R9C, the amino acid residue substitution at position Q12 is Q12W, the amino acid residue substitution at position L13 is L13A, the amino acid residue substitution at position D15 is D15L, D15K, or D15R, the amino acid residue substitution at position I16 is I16R or I16W, the amino acid residue substitution at position S70 is S70L or S70A, the amino acid residue substitution at position K72 is K72S, the amino acid residue substitution at position K73 is K73Y, or The polypeptide according to any one of claims 1 to 6 and 8, wherein the molecule is K73F, the amino acid residue substitution at position R76 is R76F, the amino acid residue substitution at position P78 is P78G or R78E, the amino acid residue substitution at position K79 is K79E or P79C, and the short peptide linker substituting SEQ ID NO: 71 is selected from the group consisting of GGSEGGS (SEQ ID NO: 73), GSEGS (SEQ ID NO: 74), GGSGGS (SEQ ID NO: 75), GGGSEGGGS (SEQ ID NO: 76), GGSEGGGS (SEQ ID NO: 77), GSGGS, GGGSGGS (SEQ ID NO: 78), and GGGSEGGGS (SEQ ID NO: 72).

11. R5C, H6C, R9C, Q12W, L13A, D15X １ , I16X ２ , S70X ３ , K72S, K73X ４ , R76F, P78X ５ , or P79X ６ A polypeptide comprising an interleukin-21 (IL-21) variant comprising a wild-type human IL-21 amino acid sequence having at least one mutation selected from １ where X ２ is L, K, or R, and X ３ is L or A, and X ４ is Y or F, and X ５ is G or E, and X ６ is E or C, and the wild-type human IL-21 comprises the sequence set forth in SEQ ID NO: 1 or 2.

12. The polypeptide according to claim 11, wherein the amino acid segment of STNAGRRQKHR (SEQ ID NO: 71) at positions 80-90, corresponding to wild-type human IL-21, is substituted with a short peptide linker.

13. The polypeptide according to claim 12, wherein the short peptide linker is selected from the group consisting of GGSEGGGS (SEQ ID NO: 73), GSEGS (SEQ ID NO: 74), GGSGGGS (SEQ ID NO: 75), GGGSEGGGS (SEQ ID NO: 76), GGGSEGGGS (SEQ ID NO: 77), GSGGS, GGGSGGS (SEQ ID NO: 78), and GGGSEGGGS (SEQ ID NO: 72).

14. A polypeptide comprising an interleukin-21 (IL-21) variant, 1) P79E; 2) K73Y, P79E; 3) K73F, P79E; 4) S70L, K73F, P79E; 5) S70L, K73Y, P79E; 6) S70L, K72S, K73F, P79E; 7) S70L, K72S, K73Y, P79E; 8) D15K, S70L, K73F, P79E; 9) D15R, S70L, K73F, P79E; 10) I16W, S70L, K73F, P79E; 11) D15L, S70L, K73F, P79E; 12) L13A, S70L, K73F, P79E; 13) D15R, R76F, P78E, P79E; 14) D15L, I16R, S70L, K73Y, P79E; 15) D15K, I16W, S70L, R76F, P79E; 16) D15R, I16W, S70L, R76F, P79E; 17) STNAGRRQKHR (SEQ ID NO: 71) (STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72)) which is replaced by GGGSEGGGS (SEQ ID NO: 72); 18) P79E, STNAGRRQKHR (Sequence ID 71) / GGGSEGGGS (Sequence ID 72); 19) K73Y, P79E, STNAGRRQKHR (Sequence ID 71) / GGGSEGGGGS (Sequence ID 72); 20) K73F, P79E, STNAGRRQKHR (Sequence ID 71) / GGGSEGGGS (Sequence ID 72); 21) S70L, K73F, P79E, STNAGRRQKHR (Sequence ID 71) / GGGSEGGGS (Sequence ID 72); 22) S70L, K73Y, P79E, STNAGRRQKHR (Sequence ID 71) / GGGSEGGGS (Sequence ID 72); 23) D15K, S70L, K73F, P79E, STNAGRRQKHR (Sequence ID 71) / GGGSEGGGS (Sequence ID 72); 24) D15R, S70L, K73F, P79E, STNAGRRQKHR (Sequence ID 71) / GGGSEGGGS (Sequence ID 72); 25) I16W, S70L, K73F, P79E, STNAGRRQKHR (Sequence ID 71) / GGGSEGGGS (Sequence ID 72); 26) S70L, K72S, K73F, P79E, STNAGRRQKHR (Sequence ID 71) / GGGSEGGGGS (Sequence ID 72); 27) S70L, K72S, K73Y, P79E, STNAGRRQKHR (Sequence ID 71) / GGGSEGGGS (Sequence ID 72); 28) D15R, I16W, S70L, K73Y, P79E, STNAGRRQKHR (Sequence ID 71) / GGGSEGGGS (Sequence ID 72); and 29) D15L, I16R, S70L, K73Y, P79E, STNAGRRQKHR (Sequence ID 71) / GGGSEGGGGS (Sequence ID 72) A polypeptide containing amino acid residue mutations selected from the group consisting of the following.

15. A polypeptide comprising an interleukin-21 (IL-21) variant, 1) P79C; 2) R5C, P79C; 3) H6C, P79C; 4) R9C, P79C; 5) R5C, R76F, P79C; 6) H6C, R76F, P79C; 7) H6C, K73Y, P79C; 8) H6C, K73F, P79C; 9) H6C, S70L, K73Y, P79C; 10) H6C, S70L, K73F, P79C; 11) R9C, D15R, P78G, P79C; 12) H6C, D15L, I16R, S70L, K73Y, P79C; 13) R9C, D15L, I16R, S70L, K73Y, P79C; 14) H6C, P79C, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGGS (SEQ ID NO: 72); 15) H6C, K73Y, P79C, STNAGRRQKHR (Sequence ID 71) / GGGSEGGGS (Sequence ID 72); 16) H6C, K73F, P79C, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGGS (SEQ ID NO: 72); and 17) H6C, S70L, P79C, STNAGRRQKHR (Sequence ID 71) / GGGSEGGGGS (Sequence ID 72) A polypeptide containing amino acid residue mutations selected from the group consisting of the following.

16. A polypeptide comprising an interleukin-21 (IL-21) variant, 1) K73Y; 2) K73F; 3) S70L, K73Y; 4) S70L, K73F; 5) S70L, K72S, K73Y; 6) S70A, K72S, K73F; 7) Q12W, S70A, R76F; 8) D15K, S70A, R76F; 9) D15L, S70A, R76F; 10) D15R, S70A, R76F; 11) I16R, S70L, K73Y; 12) D15L, I16R, S70L, K73Y; 13) D15K, I16W, S70L, K73Y; 14) D15K, I16W, S70L, R76F; 15) D15R, I16W, S70L, R76F; 16) I16W, S70L, K72S, K73F; 17) D15R, I16W, S70L, K73Y; and 18) D15K, I16W, S70L, K72S, K73Y A polypeptide containing amino acid residue mutations selected from the group consisting of the following.

17. A polypeptide comprising an interleukin-21 (IL-21) variant, (1) S70L, K73F, and P79E; (2) S70L, K72S, K73F, and P79E; (3) S70L, K73Y, and P79E; (4) S70L, K72S, K73Y, and P79E A polypeptide containing amino acid residue mutations selected from the group consisting of the following.

18. A polypeptide comprising an interleukin-21 (IL-21) variant, comprising the sequence shown in any one of SEQ ID NOs. 5 to 62, or consisting of the sequence shown in any one of SEQ ID NOs. 5 to 62.

19. A conjugate comprising a polypeptide according to any one of claims 1 to 9 and 11 to 17 and at least one additional portion, wherein the at least one additional portion comprises a crystallizable fragment (Fc) domain of an antibody or an antigen-binding molecule.

20. The conjugate according to claim 19, wherein the antigen is a tumor-associated antigen, an immune checkpoint antigen, or an immune checkpoint-associated antigen.

21. A composition comprising a polypeptide according to any one of claims 1 to 9 and 11 to 17, or a conjugate according to claim 20, for use in the treatment of subjects having or suspected of having solid tumors.

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