IL-21 polypeptides and methods of use

Abstract

Disclosed herein are various polypeptides comprising an IL-21 variant. Also provided herein are conjugates comprising the polypeptides disclosed herein and at least one additional moiety. Also provided herein are related nucleic acid molecules, vectors, transformations or host cells, pharmaceutical compositions, and kits. Also provided herein are methods of making the polypeptides or conjugates described herein and methods of treatment using the polypeptides.

Description

Background Art

[0001] 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 4, respectively. IL-21 sometimes refers to a fragment of full-length IL-21 isoform 1, which includes a signal peptide and a mature polypeptide of 133 amino acids (SEQ ID NO:1); or a fragment of full-length IL-21 isoform 2, which includes a signal peptide and a mature polypeptide of 124 amino acids (SEQ ID NO:2). IL-21 is mainly expressed in T follicular helper (Tfh) cells, Th2 and Th17 cell subsets, and NK cells. IL-21 is expressed in NK cells by enhancing CD8 + The low abundance of IL-21 partially explains why the activity of cytotoxic T lymphocytes in the tumor microenvironment is impaired.

[0002] 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 may lead to systemic inflammatory effects in relevant cancer therapies. Summary of the invention

[0003] To address the need for IL-21-related therapeutic agents with better safety and stability profiles, improved pharmacokinetics and maximized pharmacodynamic profiles, and stronger tumor inhibition efficacy, provided herein are polypeptides comprising IL-21 variants having amino acid residue substitutions at position P79 corresponding to wild-type human IL-21. In some embodiments, the wild-type human IL-21 comprises a sequence as set forth 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 comprises one or more amino acid residue substitutions with cysteine ​​in the region corresponding to position 1-10 of the wild-type human IL-21. In some cases, the one or more amino acid residue substitutions are selected from R5C, H6C and R9C. In some cases, the cysteine ​​residues substituted by the one or more amino acid residues in the region of position 1-10 form a disulfide bond with the cysteine ​​amino residue at position P79.

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

[0006] In a specific embodiment, the polypeptide further comprises an amino acid residue substitution at position K73 corresponding to the wild-type human IL-21. In some cases, the amino acid residue substitution at position K73 is an aromatic amino acid. In a specific case, 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 the 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 the wild-type human IL-21. In some cases, the amino acid residue substitution at R76 is R76F.

[0009] In a specific embodiment, the polypeptide further comprises an amino acid residue substitution at position K72 corresponding to the wild-type human IL-21. In some cases, the amino acid residue substitution at K72 is a non-aromatic amino acid comprising a side chain hydroxyl group. 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 the 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 the 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 the 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 the 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 the 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 further comprises an amino acid residue mutation selected from the group consisting of: 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; I16 W, S70L, K73F, P79E; D15L, S70L, K73F, P79E; L13A, S70L, K73F, P79E; D15R, R76F, P78E, P79E; D15L, I 16R, S70L, K73Y, P79E; D15K, I16W, S70L, R76F, P79E; D15R, I16W, S70L, R76F, P79E; STNAGRRQKHR (SEQ ID NO:71), which was replaced by 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:72) 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) 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) / GGGSEGGS(SEQ ID NO:72); S70L, K72S, K73Y, P79E, STNAGRRQKHR(SEQ ID NO:71) / GGGSEGGS(SEQ ID NO:72); D15R, I16W, S70L, K73Y, P79E, STNAGRRQKHR (SEQ ID NO:71) / GGGSEGGS (SEQ ID NO:72); and 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 are polypeptides comprising IL-21 variants having an amino acid residue substitution at position S70 or K73 corresponding to wild-type human IL-21. In some embodiments, the wild-type human IL-21 comprises a sequence as 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 the 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 the wild-type human IL-21 and an amino acid residue substitution at position K73. In a specific embodiment, the polypeptide comprises an amino acid residue substitution with an aromatic amino acid at position K73 corresponding to the wild-type human IL-21. In a particular case, the amino acid residue substitution at position K73 is K73Y or K73F. In other embodiments, the polypeptide further comprises one, two, or three or more amino acid residue substitutions at a position selected from: R5, H6, R9, Q12, L13, D15, I16, K72, R76, P78 and P79. In a specific embodiment, the polypeptide further comprises one, two, or three or four amino acid residue substitutions at a position selected from: Q12, D15, I16 and K72. In some cases, the polypeptide comprises an amino acid residue substitution selected from: 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 replacement (e.g., S70L or S70A) at position S70 corresponding to the wild-type human IL-21 and an amino acid residue replacement at position R76. In some cases, the amino acid residue replacement at position R76 is R76F. In some cases, the polypeptide further comprises one, two, or three or more amino acid residue replacements at a position selected from: R5, H6, R9, Q12, L13, D15, I16, K72, K73, P78 and P79. In some cases, the polypeptide further comprises one, two, or three or four amino acid residue replacements at a position selected from: Q12, D15, I16 and K72. In some cases, the polypeptide comprises an amino acid residue replacement selected from: I16R and I16W. In other cases, the polypeptide comprises an amino acid residue replacement selected from: D15L, D15K and D15R. In still other instances, 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, S7 0L, 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 position 80-90 corresponding to the wild-type human IL-21 is replaced by a short peptide linker. In some embodiments, the length of the short peptide linker is 4 to 10 amino acid residues, such as 4, 5, 6, 7, 8, 9 or 10 amino acid residues. In some embodiments, the length of the short peptide linker is 5 to 9 amino acid residues. In some embodiments, the length of the short peptide linker is 7 to 9 amino acid residues. In further embodiments, the short peptide linker comprises one or more Gly-Ser units and optionally comprises a glutamic acid residue. In a specific embodiment, the short peptide linker comprises the glutamic acid residue, and the glutamic acid residue is located in the middle of the short peptide linker. In a specific embodiment, the short peptide linker is selected from: 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 shows a lower binding affinity for IL-21 receptor (IL-21R) than the wild-type human IL-21. In a specific embodiment, compared with the wild-type human IL-21, the binding affinity shown by the polypeptide for IL-21R is reduced to 1 / 2 to 1 / 1000. In a specific embodiment, compared with the wild-type human IL-21, the polypeptide has a higher K for IL-21R. D value.

[0025] On the other hand, provided herein is a polypeptide comprising an interleukin-21 (IL-21) variant, wherein the amino acid segment STNAGRRQKHR (SEQ ID NO: 71) at position 80-90 corresponding to wild-type human IL-21 is replaced by 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 comprises a glutamic acid residue. In particular cases, the short peptide linker comprises the glutamic acid residue, and the glutamic acid residue is located in the middle of the short peptide linker. In certain cases, the short peptide linker is selected from: 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 (SEQID 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 a specific embodiment, the polypeptide comprises an amino acid residue substitution at position R5, and optionally the substitution is R5C. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position H6, and optionally the substitution is H6C. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position R9, and optionally the substitution is R9C. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position Q12, and optionally the substitution is Q12W. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position L13, and optionally the substitution is L13A. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position D15, and optionally the substitution is D15L, D15K, or D15R. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position I16, and optionally the substitution is I16R or I16W. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position S70, and optionally the substitution is S70L or S70A. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position K72, and optionally the substitution is K72S. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position K73, and optionally the substitution is K73Y or K73F. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position R76, and optionally the substitution is R76F. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position P78, and optionally the substitution is P78G or P78E. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position P79, and optionally the substitution is P79E or P79C.

[0027] In some embodiments, compared with the wild-type human IL-21, the polypeptide shows improved thermal stability. In some embodiments, the polypeptide shows a lower binding affinity than wild-type human IL-21 for IL-21 receptor (IL-21R). In some embodiments, compared with wild-type IL-21, the binding affinity shown by the polypeptide for IL-21R is reduced to 1 / 2 to 1 / 1000. In some embodiments, compared with wild-type IL-21, the polypeptide has a higher K for IL-21R. DValue. In some embodiments, the polypeptide exhibits improved efficacy in inhibiting tumor growth and reducing tumor volume compared to the wild-type human IL-21. In some embodiments, when the polypeptide is fused or administered in combination with at least one additional portion / agent as disclosed herein (such as an anti-PD1 antibody), the polypeptide exhibits improved efficacy and / or achieves synergistic effects in treating tumors.

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

[0029] In some embodiments, the amino acid segment STNAGRRQKHR (SEQ ID NO: 71) at position 80-90 corresponding to the wild-type human IL-21 is replaced by a short peptide linker. In some embodiments, the length of the short peptide linker is 4 to 10 amino acid residues, such as 4, 5, 6, 7, 8, 9 or 10 amino acid residues. In some embodiments, the length of the short peptide linker is 5 to 9 amino acid residues. In some embodiments, the length of the short peptide linker is 7 to 9 amino acid residues. In some embodiments, the short peptide linker comprises one or more Gly-Ser units and optionally comprises a glutamic acid residue. In some embodiments, the short peptide linker comprises the glutamic acid residue, and the glutamic acid residue is located in the middle of the short peptide linker. In some embodiments, the short peptide linker is selected from: 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 as set forth in any one of SEQ ID No. 5-62. In some specific embodiments, the polypeptide consists of a sequence as set forth in any one of SEQ ID No. 5-62.

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

[0032] On the other hand, provided herein are conjugates comprising a polypeptide disclosed herein and at least one additional portion. In some embodiments, the at least one additional portion comprises a crystallizable fragment (Fc) domain of an antibody. In some embodiments, the antibody is IgG, IgA, IgD, IgM or IgE. In some specific embodiments, the IgG is IgG1, IgG2, IgG3 or IgG4.

[0033] In some embodiments, the 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.

[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-associated antigen. In specific embodiments, the antigen participates in the immune checkpoint pathway. In specific embodiments, the antigen is selected from: PD-1, PD-L1, TIGIT, CTLA-4, PD-L2, B7-H3, B7-H4, BTLA, LAG3, CD112, CD112R, CD96, TIM-3, CD47 and CEACAM1. In specific embodiments, the antigen is a co-stimulatory immune checkpoint target. In specific embodiments, the antigen is selected from: 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 the 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] On the other hand, nucleic acid molecules are provided herein, which encode polypeptides disclosed herein or conjugates disclosed herein. In some embodiments, the nucleic acid molecules are DNA molecules. In some embodiments, the nucleic acid molecules are RNA molecules. In some embodiments, the nucleic acid molecules are mRNA molecules. On the other hand, vectors are provided herein, which contain nucleic acid molecules disclosed herein. In some embodiments, the vectors include viral vectors. On the other hand, transformation or host cells are provided herein, which express polypeptides disclosed herein or conjugates disclosed herein. On the other hand, pharmaceutical compositions are provided herein, which contain polypeptides disclosed herein or conjugates disclosed herein and pharmaceutically acceptable carriers. On the other hand, kits are provided herein, which include polypeptides, conjugates, nucleic acid molecules, vectors, transformation or host cells, or pharmaceutical compositions, combinations thereof, and containers.

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

[0038] In another aspect, provided herein is a method of treating a subject in need thereof, 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

[0040] 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. To the extent that publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] This application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0042] The novel features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by referring to the following detailed description and accompanying drawings (also referred to herein as "figures") which set forth illustrative embodiments utilizing the principles of the present disclosure, in which:

[0043] Figure 1 A diagram showing an expression vector containing wild-type IL-21-Fc is shown.

[0044] Figure 2A-2F The graph shows the results of running wild-type IL-21-Fc and IL-21 variant-Fc proteins on an SDS-PAGE protein gel. Figure 2A Shown are the results of running wild-type IL-21-Fc on an SDS-PAGE protein gel. Figure 2B Shown are the results of running m92-Fc on an SDS-PAGE protein gel. Figure 2C Shown are the results of running m98-Fc on an SDS-PAGE protein gel. Figure 2D Shown are the results of running m99-Fc on an SDS-PAGE protein gel. Figure 2E Shown are the results of running ml00-Fc on an SDS-PAGE protein gel. Figure 2F Shown are the results of running m133-Fc on an SDS-PAGE protein gel.

[0045] Figure 3A-Figure 3F The graph shows the results of size exclusion chromatography (SEC) for wild-type IL-21-Fc and IL-21 variant-Fc proteins. Figure 3A The results of detecting wild-type IL-21-Fc on SEC are shown. Figure 3B Results for detection of m92-Fc on SEC are shown. Figure 3C The results of detecting m98-Fc on SEC are shown. Figure 3D Results for detection of m99-Fc on SEC are shown. Figure 3E The results of detecting ml00-Fc on SEC are shown. Figure 3F The results of detecting m133-Fc on SEC are shown.

[0046] Figure 4A-4F The graph shows the results of BLI affinity testing of wild-type IL-21-Fc and IL-21 variant-Fc proteins. Figure 4A Shown are the results of BLI affinity testing of wild-type IL-21-Fc. Figure 4B Shown are the results of BLI affinity testing of m92-Fc. Figure 4C Shown are the results of BLI affinity testing of m98-Fc. Figure 4D Shown are the results of BLI affinity testing of m99-Fc. Figure 4E Shown are the results of BLI affinity testing of ml00-Fc. Figure 4F Shown are the results of BLI affinity testing of m133-Fc.

[0047] Figure 5 Shown is STAT3 phosphorylation in HuT78 cells by wild-type IL-21-Fc or IL-21 mutant-Fc proteins.

[0048] Figure 6 Shown are the results of an in vivo efficacy study investigating the tumor suppressive efficacy of anti-mPD1 antibody_IL21m fusion protein in tumor-bearing mice.

[0049] Figure 7A-7B The graph shows the results of running wild-type IL-21-IgG4 Fc and IL-21 variant-IgG4 Fc proteins on an SDS-PAGE protein gel. Fig. 7A Shown are the results of running wild-type IL-21-IgG4 Fc on an SDS-PAGE protein gel. Figure 7B Shown are the results of running m98-21-IgG4 Fc on an SDS-PAGE protein gel.

[0050] Figure 8A-B The graph shows the results of detecting wild-type IL-21-IgG4 Fc and IL-21 variant-IgG4 Fc proteins on size exclusion chromatography (SEC). Fig. 8A Shown are the results of detecting wild-type IL-21-IgG4 Fc on SEC. Figure 8B Results for detection of m98-IgG4 Fc on SEC are shown.

[0051] Figure 9A-9D The graph shows the results of running wild-type IL-21-His and IL-21 variant-His proteins on an SDS-PAGE protein gel. Fig.9A Shown are the results of running wild-type IL-21-His on an SDS-PAGE protein gel. Fig. 9B Shown are the results of running m98-His on an SDS-PAGE protein gel. Fig. 9C Shown are the results of running m103-His on an SDS-PAGE protein gel. Fig.9D Shown are the results of running m153-His on an SDS-PAGE protein gel.

[0052] Figures 10A-10D The graph shows the results of running wild-type IL-21 or IL-21 variant fusion proteins with anti-mouse PD1 antibody on SDS-PAGE protein gel. Fig. 10A Shown are the results of running anti-mPD1 antibody-wild-type IL21 fusion protein on SDS-PAGE protein gel. Fig. 10B Shown are the results of running anti-mPD1 antibody-IL21m98 fusion protein on SDS-PAGE protein gel. Fig. 10C Shown are the results of running anti-mPD1 antibody-IL21m100 fusion protein on SDS-PAGE protein gel. Fig. 10DShown are the results of running anti-mPD1 antibody-IL21m153 fusion protein on SDS-PAGE protein gel.

[0053] Fig.11 The graph shows the changes in tumor volume over time after tumor-bearing mice were treated with wild-type IL-21-IgG4 Fc or m98-IgG4 Fc protein.

[0054] Fig.12 Shown are changes in tumor volume over time after treatment of tumor-bearing mice with anti-mPD1 antibody alone, m98-IgG4 Fc alone, or a combination of both.

[0055] Fig.13 Shown are the weights of tumors isolated from tumor-bearing mice treated with anti-mPD1 antibody alone, m98-IgG4 Fc alone, or a combination of both. DETAILED DESCRIPTION

[0056] Unless the context clearly indicates otherwise, as used herein and in the appended claims, the singular forms "a", "an" and "the" include plural referents. Thus, for example, reference to "an agent" includes a variety of such agents, and reference to "the cell" includes reference to one or more cells (or cells) known to those skilled in the art and their equivalents, and so on. When ranges are used herein for physical properties (such as molecular weight) or chemical properties (such as chemical formula), all combinations and subcombinations of ranges and specific embodiments thereof are intended to be included. When referring to a number or a numerical range, the term "about" means that the number or numerical range referred to is an approximation within experimental variation (or within statistical experimental error), and therefore, in some cases, the number or numerical range will vary between 1% and 15% of the stated number or numerical range. The term "comprising" (and related terms, such as "having" or "including") is not intended to exclude in certain other embodiments, for example, those of embodiments of any material composition, composition, method or process, etc. described herein that are "consisting of" or "essentially consisting of" the features.

[0057] definition

[0058] As used in the context of the present invention, when describing a specific combination of mutations, 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 combined mutation "S70L, K73Y, P79E" means that mutations S70L, K73Y and P79E are all present in a single IL-21 variant. In addition, as used in the context of 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 by GGGSEGGGS (SEQID NO: 72).

[0059] As used in the specification and appended claims, the following terms have the following indicated meanings unless specified to the contrary.

[0060] "Wild type" or "WT" or "wt" or "native" refers to an amino acid sequence found in nature, including allelic variations or natural isoforms. A wild-type protein or polypeptide has an amino acid sequence that has not been intentionally modified.

[0061] "Variant" or "mutant" of an amino acid sequence (e.g., an amino acid sequence of a peptide, protein or polypeptide) can be used interchangeably to refer to an amino acid insertion variant, an amino acid addition variant, an amino acid deletion variant and / or an amino acid substitution variant. An amino acid insertion variant is characterized in that one or more amino acids are inserted into a specific amino acid sequence (e.g., a wild-type IL-21 sequence or a functional variant thereof). An amino acid addition variant includes an N-terminal and / or C-terminal fusion of one or more amino acids. An amino acid deletion variant is characterized in that one or more amino acids are removed from the sequence. The deletion can be located at any position in the protein sequence. An amino acid deletion variant includes a deletion at the N-terminal and / or C-terminal of a protein to produce an N-terminal and / or C-terminal truncated variant. An amino acid substitution variant is characterized in that one or more amino acids are removed from the sequence and one or more another amino acids are inserted to replace the original amino acids.

[0062] "IL-21 mutant" or "IL-21 mutant protein" or "IL-21 variant" or "IL-21 variant portion" refers to a polypeptide in which one or more amino acids of wild-type human IL-21 or a functional variant thereof are mutated to provide a mutant or variant of wild-type human IL-21. In some embodiments, the sequence of the IL-21 mutant 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 mutant polypeptide provided herein comprises a signal peptide. In some specific embodiments, the signal peptide is a naturally occurring signal peptide of wild-type human IL-21. In some specific embodiments, the signal peptide is any signal peptide known in the art. In some embodiments, the IL-21 mutant polypeptide provided herein does not comprise a signal peptide.

[0063] "Natural amino acids" refer to amino acids from the 20 naturally occurring amino acids found in proteins. Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid); basic amino acids (lysine, arginine, histidine); nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are classified as aromatic amino acids.

[0064] "Unnatural amino acids" refer to amino acids other than the 20 naturally occurring amino acids in proteins.

[0065] "Percent identity" refers to the percentage of identical amino acid residues between two sequences after optimal alignment of the sequences. The optimal alignment of the sequences can be generated manually or by a computer program using a sequence alignment algorithm (e.g., ClustalW, T-coffee, COBALT, BestFit, FASTA, BLASTP, BLASTN, and TFastA). The percent identity can be calculated by determining the number of identical positions between the two sequences compared, dividing this number by the number of positions compared, and then multiplying the result obtained by 100 to obtain the percent identity between the two sequences.

[0066] The term "subject" or "patient" encompasses mammals. Examples of mammals include, but are not limited to, any member of the class mammals: humans, non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs and cats; laboratory animals, including rodents such as rats, mice and guinea pigs, etc. In one aspect, the mammal is a human.

[0067] As used herein, "treatment" or "treatment" are used interchangeably. These terms refer to methods for obtaining beneficial or desired results, including but not limited to therapeutic benefit and / or preventive benefit. "Therapeutic benefit" means eradication or improvement of the underlying condition being treated. In addition, the therapeutic benefit is achieved by eradicating or improving one or more physiological symptoms associated with the underlying condition, so that the improvement is observed in the patient, although the patient is still suffering from the underlying condition. For preventive benefit, in some embodiments, the composition is administered to a patient at risk of developing a particular disease or to a patient reporting one or more physiological symptoms of a disease (even though a diagnosis of the disease may not have been made).

[0068] IL-21

[0069] IL-21 is a member of the cytokine family (IL-2, IL-4, IL-7, IL-9 and IL-15), and its receptor shares a common receptor gamma chain (γc). IL-21 is a pleiotropic type I cytokine, which is mainly produced by T cells and natural killer T (NKT) cells. This cytokine has a variety of effects on the cell types of a wide range, including but not limited to CD4+ and CD8+T cells, B cells, macrophages, monocytes and dendritic cells (DC). In particular, IL-21 drives B cell differentiation into plasma cells, regulates immunoglobulin production, controls proliferation and / or effector function of CD4+ and CD8+ T cells, limits the differentiation of Tregs, and can stimulate epithelial cells and fibroblasts to produce inflammatory mediators (see, e.g., Leonarda and Wan, F1000 Res. 2016;5:F1000 Faculty Rev-224; Stolfi et al., Oncoimmunology. 2012 May 1;1(3):351-354).

[0070] IL-21 receptor (IL-21R) forms heterodimer receptor complex with common γ c, and γ c is also the subunit of the receptor of IL-2, IL-4, IL-7, IL-9 and IL-15.IL-21R part is ligand recognition binding site, and γ c is signal transduction unit.IL-21R subunit mainly expresses on the surface of normal lymphoid tissue, including spleen, thymus, lymph node, peripheral blood lymphocyte, T cell, B cell and NK cell.IL-21R works by JAK / STAT approach, and activates its target gene via JAK1, JAK3 and STAT3 dimer.

[0071] IL-21R transduces 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 on IL-21R knockout mice have shown its important role in regulating the production of immunoglobulins. In addition, it is reported that IL-21 has a powerful anti-tumor effect because it can expand the pool of cytotoxic CD8+T cells, NK cells, and NKT cells.

[0072] Compared with the affinity of wild-type IL-21 protein and IL-21R, the IL-21 variants of the present disclosure have a reduced affinity for IL-21R. Thus, in some embodiments, due to the reduced affinity of IL-21 variants as described herein and the IL-21R in normal lymphoid tissue, polypeptides as described herein produce less systemic cytotoxicity. In some embodiments, polypeptides with less systemic cytotoxicity as described herein therefore have advantages in dosing strategies, as a single drug or in combination therapy when using the therapeutic window and with another functional part (such as antibody, small molecule inhibitor or nucleic acid) to form a more compatible candidate for the bifunctional or multifunctional molecule for therapy.

[0073] In some embodiments, the thermal stability of the IL-21 mutants disclosed herein is improved compared to wild-type IL-21. In some embodiments, the pharmacodynamics of the IL-21 mutants disclosed herein is improved. Therefore, compared to wild-type IL-21, the drugability of the IL-21 mutants is optimized.

[0074] IL-21 variants

[0075] IL-21 variants with substitutions at position P79

[0076] Provided herein are polypeptides comprising IL-21 variants 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 comprises a sequence as set forth in SEQ ID NO: 1 or 2.

[0077] 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.

[0078] 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 corresponding to positions 1-10 of the 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 described cysteine ​​substitutions. In some cases, the one or more amino acid residue substitutions are selected from R5C, H6C and R9C.

[0079] In some cases, the cysteine ​​residue substituted by the one or more amino acid residues in the region of positions 1-10 forms a disulfide bond with the cysteine ​​amino residue at position P79. In some specific cases, the disulfide bond is formed between the cysteine ​​residue at position P79 and the cysteine ​​residue at position 1. In some specific cases, the disulfide bond is formed between the cysteine ​​residue at position P79 and the cysteine ​​residue at position 2. In some specific cases, the disulfide bond is formed between the cysteine ​​residue at position P79 and the cysteine ​​residue at position 3. In some specific cases, the disulfide bond is formed between the cysteine ​​residue at position P79 and the cysteine ​​residue at position 4. In some specific cases, the disulfide bond is formed between the cysteine ​​residue at position P79 and the cysteine ​​residue at position 5. In some specific cases, the disulfide bond is formed between the cysteine ​​residue at position P79 and the cysteine ​​residue at position 6. In some specific instances, the disulfide bond is formed between the cysteine ​​residue at position P79 and the cysteine ​​residue at position 7. In some specific instances, the disulfide bond is formed between the cysteine ​​residue at position P79 and the cysteine ​​residue at position 8. In some specific instances, the disulfide bond is formed between the cysteine ​​residue at position P79 and the cysteine ​​residue at position 9. In some specific instances, the disulfide bond is formed between the cysteine ​​residue at position P79 and the cysteine ​​residue at position 10.

[0080] 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 a position 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 a position selected from the group consisting of S70, K72, K73, and R76.

[0081] In a specific embodiment, the polypeptide further comprises an amino acid residue substitution at position K73 corresponding to the wild-type human IL-21. In some cases, the amino acid residue substitution at position K73 is an aromatic amino acid. In a specific case, the amino acid residue substitution at position K73 is K73Y or K73F.

[0082] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position S70 corresponding to the wild-type human IL-21. In some cases, the amino acid residue substitution at S70 is S70L or S70A.

[0083] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position R76 corresponding to the wild-type human IL-21. In some cases, the amino acid residue substitution at R76 is R76F.

[0084] In a specific embodiment, the polypeptide further comprises an amino acid residue substitution at position K72 corresponding to the wild-type human IL-21. In some cases, the amino acid residue substitution at K72 is a non-aromatic amino acid comprising a side chain hydroxyl group. In some specific cases, the amino acid residue substitution at K72 is K72S.

[0085] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position Q12 corresponding to the wild-type human IL-21. In some cases, the amino acid residue substitution at Q12 is Q12W.

[0086] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position L13 corresponding to the wild-type human IL-21. In some cases, the amino acid residue substitution at L13 is L13A.

[0087] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position D15 corresponding to the wild-type human IL-21. In some cases, the amino acid residue substitution at D15 is D15L, D15K or D15R.

[0088] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position I16 corresponding to the wild-type human IL-21. In some cases, the amino acid residue substitution at I16 is I16R or I16W.

[0089] In certain embodiments, the polypeptide further comprises an amino acid residue substitution at position P78 corresponding to the wild-type human IL-21. In some cases, the amino acid residue substitution at P78 is P78G or P78E.

[0090] In some embodiments, the amino acid segment STNAGRRQKHR (SEQ ID NO: 71) at position 80-90 corresponding to the wild-type human IL-21 is replaced by 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 not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues. In some embodiments, the length of the short peptide linker is 4 to 10 amino acid residues. In some embodiments, the length of the short peptide linker is 5 to 9 amino acid residues. In other embodiments, the short peptide linker comprises one or more Gly-Ser units. In other embodiments, the short peptide linker comprises one or more glutamic acid residues. In other embodiments, the short peptide linker comprises a glutamic acid residue. In a specific embodiment, wherein the short peptide linker comprises a glutamic acid residue, and the glutamic acid residue is located in the middle of the short peptide linker. In some embodiments where the short peptide linker has an odd number (i.e., 2n+1) length, the glutamic acid residue is located in position n+1. In an exemplary embodiment where the length of the short peptide linker is 5 amino acids, the glutamic acid residue is located at position 3. In an exemplary embodiment where the length of the short peptide linker is 7 amino acids, the glutamic acid residue is located at position 4. In some embodiments where the short peptide linker has an even number (i.e., 2n) length, the glutamic acid residue is located at position n or n+1. In an exemplary embodiment where the length of the short peptide linker is 4 amino acids, the glutamic acid residue is located at position 2 or 3. In an exemplary embodiment where the length of the short peptide linker is 6 amino acids, the glutamic acid residue is located at position 3 or 4. In a specific embodiment, the short peptide linker is selected from: 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 shows a lower binding affinity for IL-21 receptor (IL-21R) than the wild-type human IL-21. In a specific embodiment, compared with the wild-type human IL-21, the binding affinity shown by the polypeptide for IL-21R is reduced to 1 / 2 to 1 / 1000. In a specific embodiment, compared with the wild-type human IL-21, the polypeptide has a higher K for IL-21R. D value.

[0092] IL-21 variants with substitutions at position 70

[0093] In another aspect, provided herein are polypeptides comprising IL-21 variants having an amino acid residue substitution at position S70 corresponding to wild-type human IL-21. In some embodiments, the wild-type human IL-21 comprises a sequence as set forth in SEQ ID NO: 1 or 2. In some cases, the amino acid residue substitution at position S70 is S70L or S70A.

[0094] In some embodiments, the polypeptide comprises an amino acid residue substitution (e.g., S70L or S70A) at position S70 corresponding to the wild-type human IL-21 and an amino acid residue substitution at position K73. In a specific embodiment, the polypeptide comprises an amino acid residue substitution with an aromatic amino acid at position K73 corresponding to the wild-type human IL-21. In a particular case, the amino acid residue substitution at position K73 is K73Y or K73F. In other embodiments, the polypeptide further comprises one, two, or three or more amino acid residue substitutions at a position selected from: R5, H6, R9, Q12, L13, D15, I16, K72, R76, P78 and P79. In a specific embodiment, the polypeptide further comprises one, two, or three or four amino acid residue substitutions at a position selected from: Q12, D15, I16 and K72. In some cases, the polypeptide comprises an amino acid residue substitution selected from: 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.

[0095] In some embodiments, the polypeptide comprises an amino acid residue replacement (e.g., S70L or S70A) at position S70 corresponding to the wild-type human IL-21 and an amino acid residue replacement at position R76. In some cases, the amino acid residue replacement at position R76 is R76F. In some cases, the polypeptide further comprises one, two, or three or more amino acid residue replacements at a position selected from: R5, H6, R9, Q12, L13, D15, I16, K72, K73, P78 and P79. In some cases, the polypeptide further comprises one, two, or three or four amino acid residue replacements at a position selected from: Q12, D15, I16 and K72. In some cases, the polypeptide comprises an amino acid residue replacement selected from: I16R and I16W. In other cases, the polypeptide comprises an amino acid residue replacement selected from: D15L, D15K and D15R. In still other instances, the polypeptide comprises an amino acid residue substitution selected from the group consisting of: K72S and Q12W.

[0096] In some embodiments, the amino acid segment STNAGRRQKHR (SEQ ID NO: 71) at position 80-90 corresponding to the wild-type human IL-21 is replaced by 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 not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues. In some embodiments, the length of the short peptide linker is 4 to 10 amino acid residues. In some embodiments, the length of the short peptide linker is 5 to 9 amino acid residues. In other embodiments, the short peptide linker comprises one or more Gly-Ser units. In other embodiments, the short peptide linker comprises one or more glutamic acid residues. In other embodiments, the short peptide linker comprises a glutamic acid residue. In a specific embodiment, wherein the short peptide linker comprises a glutamic acid residue, and the glutamic acid residue is located in the middle of the short peptide linker. In some embodiments where the short peptide linker has an odd number (i.e., 2n+1) length, the glutamic acid residue is located in position n+1. In an exemplary embodiment where the length of the short peptide linker is 5 amino acids, the glutamic acid residue is located at position 3. In an exemplary embodiment where the length of the short peptide linker is 7 amino acids, the glutamic acid residue is located at position 4. In some embodiments where the short peptide linker has an even number (i.e., 2n) length, the glutamic acid residue is located at position n or n+1. In an exemplary embodiment where the length of the short peptide linker is 4 amino acids, the glutamic acid residue is located at position 2 or 3. In an exemplary embodiment where the length of the short peptide linker is 6 amino acids, the glutamic acid residue is located at position 3 or 4. In a specific embodiment, the short peptide linker is selected from: 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).

[0097] In some embodiments, the polypeptide shows a lower binding affinity for IL-21 receptor (IL-21R) than the wild-type human IL-21. In a specific embodiment, compared with the wild-type human IL-21, the binding affinity shown by the polypeptide for IL-21R is reduced to 1 / 2 to 1 / 1000. In a specific embodiment, compared with the wild-type human IL-21, the polypeptide has a higher K for IL-21R. D value.

[0098] IL-21 variants with peptide linkers

[0099] On the other hand, provided herein is a polypeptide comprising an interleukin-21 (IL-21) variant, wherein the amino acid segment STNAGRRQKHR (SEQ ID NO: 71) at position 80-90 corresponding to wild-type human IL-21 is replaced by 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 not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues. In some embodiments, the length of the short peptide linker is 4 to 10 amino acid residues. In some embodiments, the length of the short peptide linker is 5 to 9 amino acid residues. In some embodiments, the length of the short peptide linker is 6 to 9 amino acid residues. In some embodiments, the length of the short peptide linker is 7 to 9 amino acid residues. In other embodiments, the short peptide linker comprises one or more Gly-Ser units. In other embodiments, the short peptide linker comprises one or more glutamic acid residues. In other embodiments, the short peptide linker comprises a glutamic acid residue. In specific embodiments, wherein the short peptide linker comprises a glutamic acid residue, and the glutamic acid residue is located in the middle of the short peptide linker. In some embodiments where the short peptide linker has an odd number (i.e., 2n+1) length, the glutamic acid residue is located in position n+1. In the exemplary embodiment where the length of the short peptide linker is 5 amino acids, the glutamic acid residue is located at position 3. In the exemplary embodiment where the length of the short peptide linker is 7 amino acids, the glutamic acid residue is located at position 4. In some embodiments where the short peptide linker has an even number (i.e., 2n) length, the glutamic acid residue is located in position n or n+1. In the exemplary embodiment where the length of the short peptide linker is 4 amino acids, the glutamic acid residue is located at position 2 or 3. In the exemplary embodiment where the length of the short peptide linker is 6 amino acids, the glutamic acid residue is located at position 3 or 4. In a specific embodiment, the short peptide linker is selected from: 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).

[0100] 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 a specific embodiment, the polypeptide comprises an amino acid residue substitution at position R5, and optionally the substitution is R5C. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position H6, and optionally the substitution is H6C. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position R9, and optionally the substitution is R9C. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position Q12, and optionally the substitution is Q12W. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position L13, and optionally the substitution is L13A. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position D15, and optionally the substitution is D15L, D15K, or D15R. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position I16, and optionally the substitution is I16R or I16W. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position S70, and optionally the substitution is S70L or S70A. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position K72, and optionally the substitution is K72S. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position K73, and optionally the substitution is K73Y or K73F. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position R76, and optionally the substitution is R76F. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position P78, and optionally the substitution is P78G or P78E. In a specific embodiment, the polypeptide comprises an amino acid residue substitution at position P79, and optionally the substitution is P79E or P79C.

[0101] In some embodiments, compared with the wild-type human IL-21, the polypeptide shows improved thermal stability. In some embodiments, the polypeptide shows a lower binding affinity than wild-type human IL-21 for IL-21 receptor (IL-21R). In some embodiments, compared with wild-type IL-21, the binding affinity shown by the polypeptide for IL-21R is reduced to 1 / 2 to 1 / 1000. In some embodiments, compared with wild-type IL-21, the polypeptide has a higher K for IL-21R. D value.

[0102] IL-21 variants with one or more specific substitutions

[0103] In another aspect, provided herein are polypeptides comprising interleukin-21 (IL-21) variants comprising a wild-type human IL-21 amino acid sequence having at least one mutation selected from the group consisting of R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, wherein X1 is L, K, or R, wherein X2 is R or W, wherein X3 is L or A, wherein X4 is Y or F, and X5 is G or E, and wherein X6 is E or C. In some embodiments, the wild-type human IL-21 comprises a sequence as set forth in SEQ ID NO: 1 or 2.

[0104] In some embodiments, the polypeptides described herein comprise only one mutation selected from the group consisting of R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, wherein X1 is L, K, or R, wherein X2 is R or W, wherein X3 is L or A, wherein X4 is Y or F, and X5 is G or E, and wherein X6 is E or C.

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

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

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

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

[0109] In some embodiments, the polypeptide described herein comprises six mutations, each of which is selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5 or P79X6, wherein X1 is L, K or R, wherein X2 is R or W, wherein X3 is L or A, wherein X4 is Y or F, and X5 is G or E, and wherein X6 is E or C.

[0110] In some embodiments, the polypeptide described herein comprises seven mutations, each of which is selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5 or P79X6, wherein X1 is L, K or R, wherein X2 is R or W, wherein X3 is L or A, wherein X4 is Y or F, and X5 is G or E, and wherein X6 is E or C.

[0111] In some embodiments, the polypeptide described herein comprises eight mutations, each of which is selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, wherein X1 is L, K, or R, wherein X2 is R or W, wherein X3 is L or A, wherein X4 is Y or F, and X5 is G or E, and wherein X6 is E or C.

[0112] In some embodiments, the polypeptides described herein comprise nine mutations, each of which is selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, wherein X1 is L, K, or R, wherein X2 is R or W, wherein X3 is L or A, wherein X4 is Y or F, and X5 is G or E, and wherein X6 is E or C.

[0113] In some embodiments, the polypeptide described herein comprises ten mutations, each of which is selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, wherein X1 is L, K, or R, wherein X2 is R or W, wherein X3 is L or A, wherein X4 is Y or F, and X5 is G or E, and wherein X6 is E or C.

[0114] In some embodiments, the polypeptide described herein comprises eleven mutations, each of which is selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, wherein X1 is L, K, or R, wherein X2 is R or W, wherein X3 is L or A, wherein X4 is Y or F, and X5 is G or E, and wherein X6 is E or C.

[0115] In some embodiments, the polypeptide described herein comprises twelve mutations, each of which is selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, wherein X1 is L, K, or R, wherein X2 is R or W, wherein X3 is L or A, wherein X4 is Y or F, and X5 is G or E, and wherein X6 is E or C.

[0116] In some embodiments, the polypeptide described herein comprises thirteen mutations, each of which is selected from R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, wherein X1 is L, K, or R, wherein X2 is R or W, wherein X3 is L or A, wherein X4 is Y or F, and X5 is G or E, and wherein X6 is E or C.

[0117] In some embodiments, the amino acid segment STNAGRRQKHR (SEQ ID NO: 71) at position 80-90 corresponding to the wild-type human IL-21 is replaced by 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 not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues. In some embodiments, the length of the short peptide linker is 4 to 10 amino acid residues. In some embodiments, the length of the short peptide linker is 5 to 9 amino acid residues. In some embodiments, the length of the short peptide linker is 7 to 9 amino acid residues. In other embodiments, the short peptide linker comprises one or more Gly-Ser units. In other embodiments, the short peptide linker comprises one or more glutamic acid residues. In other embodiments, the short peptide linker comprises a glutamic acid residue. In a specific embodiment, wherein the short peptide linker comprises a glutamic acid residue, and the glutamic acid residue is located in the middle of the short peptide linker. In some embodiments where the short peptide linker has an odd number (i.e., 2n+1) length, the glutamic acid residue is located in position n+1. In an exemplary embodiment where the length of the short peptide linker is 5 amino acids, the glutamic acid residue is located at position 3. In an exemplary embodiment where the length of the short peptide linker is 7 amino acids, the glutamic acid residue is located at position 4. In some embodiments where the short peptide linker has an even number (i.e., 2n) length, the glutamic acid residue is located at position n or n+1. In an exemplary embodiment where the length of the short peptide linker is 4 amino acids, the glutamic acid residue is located at position 2 or 3. In an exemplary embodiment where the length of the short peptide linker is 6 amino acids, the glutamic acid residue is located at position 3 or 4. In a specific embodiment, the short peptide linker is selected from: 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).

[0118] On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises an amino acid residue mutation P79E. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations P79E K73Y and P79E. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations K73F and P79E. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations S70L, K73F and P79E. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations S70L, K73F and P79E. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations S70L, K73Y and P79E. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations S70L, K72S, K73F and P79E. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations S70L, K72S, K73Y and P79E. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations D15K, S70L, K73F and P79E. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations D15R, S70L, K73F and P79E. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations I16W, S70L, K73F and P79E. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations D15L, S70L, K73F and P79E. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations L13A, S70L, K73F and P79E. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations D15R, R76F, P78E and P79E. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations D15L, I16R, S70L, K73Y and P79E. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations D15K, I16W, S70L, R76F and P79E. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations D15R, I16W, S70L, R76F and P79E.In another aspect, provided herein is a polypeptide comprising an IL-21 variant, wherein the polypeptide comprises an amino acid residue mutation, i.e., STNAGRRQKHR (SEQ ID NO: 71), which is substituted with GGGSEGGGS (SEQ ID NO: 72) (STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72)). In another aspect, provided herein is a polypeptide comprising an IL-21 variant, wherein the polypeptide comprises an amino acid residue mutation P79E and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). In another aspect, provided herein is a polypeptide comprising an IL-21 variant, wherein the polypeptide comprises an amino acid residue mutation K73Y, P79E and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). On the other hand, provided herein is a polypeptide comprising an IL-21 variant, wherein the polypeptide comprises amino acid residue mutations K73F, P79E and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). On the other hand, provided herein is a polypeptide comprising an IL-21 variant, wherein the polypeptide comprises amino acid residue mutations S70L, K73F, P79E and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). On the other hand, provided herein is a polypeptide comprising an IL-21 variant, wherein the polypeptide comprises amino acid residue mutations S70L, K73Y, P79E and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). On the other hand, provided herein is a polypeptide comprising an IL-21 variant, wherein the polypeptide comprises amino acid residue mutations D15K, S70L, K73F, P79E and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). On the other hand, provided herein is a polypeptide comprising an IL-21 variant, wherein the polypeptide comprises amino acid residue mutations D15R, S70L, K73F, P79E and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). On the other hand, provided herein is a polypeptide comprising an IL-21 variant, wherein the polypeptide comprises amino acid residue mutations I16W, S70L, K73F, P79E and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72).On the other hand, provided herein is a polypeptide comprising an IL-21 variant, wherein the polypeptide comprises amino acid residue mutations S70L, K72S, K73F, P79E and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). On the other hand, provided herein is a polypeptide comprising an IL-21 variant, wherein the polypeptide comprises amino acid residue mutations S70L, K72S, K73Y, P79E and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). On the other hand, provided herein is a polypeptide comprising an IL-21 variant, wherein the polypeptide comprises 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 is a polypeptide comprising an IL-21 variant, wherein the polypeptide comprises amino acid residue mutations D15L, I16R, S70L, K73Y, P79E and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72).

[0119] On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises an amino acid residue mutation P79C. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations R5C, P79C. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations H6C and P79C. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations R9C and P79C. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations R5C, R76F and P79C. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations H6C, R76F and P79C. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations H6C, K73Y and P79C. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations H6C, K73F and P79C. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations H6C, S70L, K73Y and P79C. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations H6C, S70L, K73F, P79C; R9C, D15R, P78G and P79C. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations H6C, D15L, I16R, S70L, K73Y and P79C. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations R9C, D15L, I16R, S70L, K73Y and P79C. On the other hand, provided herein is a polypeptide comprising an IL-21 variant, wherein the polypeptide comprises amino acid residue mutations H6C, P79C and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). On the other hand, provided herein is a polypeptide comprising an IL-21 variant, wherein the polypeptide comprises amino acid residue mutations H6C, K73Y, P79C and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72). On the other hand, provided herein is a polypeptide comprising an IL-21 variant, wherein the polypeptide comprises 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, wherein the polypeptide comprises amino acid residue mutations H6C, S70L, P79C, and STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72).

[0120] On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises an amino acid residue mutation K73Y. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises an amino acid residue mutation K73F. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises an amino acid residue mutation S70L. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises an amino acid residue mutation S70A. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises an amino acid residue mutation S70L and K73Y. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises an amino acid residue mutation S70L and K73F. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises an amino acid residue mutation S70L, K72S and K73Y. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises an amino acid residue mutation S70A, K72S and K73F. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations Q12W, S70A and R76F. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations D15K, S70A and R76F. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations D15L, S70A and R76F. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations D15R, S70A and R76F. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations I16R, S70L and K73Y. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations D15L, I16R, S70L and K73Y. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations D15K, I16W, S70L and K73Y. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations D15K, I16W, S70L and R76F. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations D15R, I16W, S70L and R76F. On the other hand, a polypeptide comprising an IL-21 variant is provided herein, wherein the polypeptide comprises amino acid residue mutations I16W, S70L, K72S and K73F.In another aspect, provided herein is a polypeptide comprising an IL-21 variant, wherein the polypeptide comprises amino acid residue mutations D15R, I16W, S70L and K73Y. In another aspect, provided herein is a polypeptide comprising an IL-21 variant, wherein the polypeptide comprises amino acid residue mutations D15K, I16W, S70L, K72S and K73Y.

[0121] On the other hand, the present invention provides a polypeptide comprising an IL-21 variant, wherein the polypeptide comprises a sequence as set forth in any one of SEQ ID No.5-62. On the other hand, the present invention provides a polypeptide comprising an IL-21 variant, wherein the polypeptide consists of a sequence as set forth in any one of SEQ ID No.5-62. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.5. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.6. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.7. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.8. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.9. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.10. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.11. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.12. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.13. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.14. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.15. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.16. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.17. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.18. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.19. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.20. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.21. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No. 22. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No. 23. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No. 24. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No. 25. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No. 26.In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.27. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.28. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.29. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.30. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.31. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.32. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.33. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.34. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.35. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.36. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.37. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.38. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.39. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.40. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.41. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.42. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.43. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.44. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.45. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.46. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No. 47. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No. 48. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No. 49. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No. 50. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No. 51.In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.52. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.53. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.54. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.55. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.56. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.57. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.58. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.59. In some embodiments, the polypeptide comprises or consists of a sequence as set forth in SEQ ID No.60. In some embodiments, the polypeptide comprises or consists of a sequence as 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.

[0122] In some embodiments, the polypeptide exhibits a lower binding affinity for IL-21 receptor (IL-21R) than the wild-type human IL-21. In some embodiments, compared with the wild-type human IL-21, the binding affinity exhibited by the polypeptide for IL-21R is reduced to 1 / 2 to 1 / 1000. In some embodiments, compared with the wild-type human IL-21, the polypeptide has a higher K for IL-21R. D value.

[0123] In some embodiments, the wild-type human IL-21 comprises the amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the wild-type human IL-21 consists of the amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, 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.

[0124] 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 include non-natural amino acids, wherein the cytokine is conjugated to a protein, wherein the attachment point is not a non-natural amino acid. In some embodiments, the polypeptides described herein include non-natural amino acids, wherein the cytokine is conjugated to a protein, wherein the attachment point is a non-natural amino acid. In some embodiments, the amino acid residues of the IL-21 variants described herein are mutated before being combined (or reacted) with an additional part. In some embodiments, mutation to non-natural amino acids reduces the possibility of the autoantigen response of the immune system or minimizes it.

[0125] Non-limiting examples of unnatural amino acids include p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, p-methoxyphenylalanine, O-methyl-L-tyrosine, p-propargyloxyphenylalanine, p-propargyl-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-azidophenylalanine, acyl-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-borylphenylalanine, O-propargyltyrosine, 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 analogs; unnatural analogs of threonine amino acids; alkyl, aryl, acyl, azido, cyano, halogen, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phosphate, phosphono, phosphine, heterocycle, enone, imine, aldehyde, hydroxylamine, ketone or amino substituted amino acids or combinations thereof; amino acids with photoactivatable crosslinkers; spin labeled amino acids; fluorescent amino acids; metal binding amino acids; metal containing amino acids; radioactive amino acids; photocaged and / or photoisomerizable amino acids; amino acids containing biotin or biotin analogs; ketone containing amino acids; amino acids containing polyethylene glycol or polyethers; heavy atom substituted amino acids; chemically cleavable or photocleavable amino acids; amino acids with elongated side chains; amino acids containing toxic groups; sugar substituted amino acids; amino acids containing carbon-linked sugars; redox active amino acids; α-hydroxy amino acids; amino thioacids; 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-propargylethoxy-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyl tetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-poly , fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyltyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)seleno)propionic acid, 2-amino-3-(phenylseleno)propionic acid and selenocysteine. ;

[0126] In some embodiments, the unnatural amino acid comprises p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, O-methyl-L-tyrosine, p-methoxyphenylalanine, p-propargyloxyphenylalanine, p-propargyl-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 phenylalanine derivative containing benzophenone, ketone, iodide, methoxy, acetyl, benzoyl or azide. In some embodiments, the non-natural amino acid is a lysine derivative containing benzophenone, ketone, iodide, methoxy, acetyl, benzoyl or azide.

[0127] In some embodiments, non-natural amino acids include selective reactive groups, or reactive groups for site-selective labeling of target polypeptides. In some embodiments, the chemical reaction is a bioorthogonal reaction (e.g., biocompatibility and selective reaction). In some cases, the chemical reaction is Cu (I)-catalyzed or "copper-free" alkyne-azide triazole formation reaction, Staudinger connection (Staudinger ligation), inverse electron demand Diels-Alder (IEDDA) reaction, "light click" chemistry or metal-mediated process, such as olefin metathesis reaction and Suzuki-Miyaura or Sonogashira cross coupling. In some embodiments, non-natural amino acids include photoreactive groups, which are cross-linked when irradiated with, for example, ultraviolet light. In some embodiments, non-natural amino acids include light cage amino acids.

[0128] In some embodiments, the unnatural amino acid is a para-substituted, meta-substituted, or ortho-substituted amino acid derivative.

[0129] IL-21 binding and activity

[0130] In some embodiments, polypeptides disclosed herein exhibit reduced affinity for IL-21R relative to wild-type human IL-21. In some embodiments, the reduced affinity is relative to wild-type human IL-21, and the binding affinity for IL-21R is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% or more.

[0131] The binding affinity of the polypeptides provided herein for the target receptor or its subunits can be measured by measuring the dissociation constant (K D ) to assess. In some embodiments, the K of the polypeptide provided herein combined with IL-21R D is the K of wild-type IL-21 D In some embodiments, the K of polypeptide provided herein is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1000 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, or at least 1000 times higher than that of IL-21R. D is the K of wild-type IL-21 DThe amount of the peptide or peptide composition may be about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times, about 200 times, about 300 times, about 400 times, about 500 times, about 600 times, about 700 times, about 800 times, about 900 times, about 1000 times, about 200 times, about 300 times, about 400 times, about 500 times, about 600 times, about 700 times, about 800 times, about 900 times, or about 1000 times higher than the amount of the peptide or peptide composition.

[0132] In some embodiments, the polypeptides provided herein are present in an amount of at least 10 -10 M. At least 10 -9 M. At least 10 -8 M. At least 10 - 7 M. At least 10 -6 M. At least 10 -5 M. At least 10 -4 M. At least 10 -3 M. At least 10 -2 M. At least 10 -1 M or larger K D In some embodiments, the polypeptides provided herein are present in an amount of about 10 -10 M, about 10 -9 M, about 10 -8 M, about 10 -7 M, about 10 -6 M, about 10 -5 M, about 10 -4 M, about 10 -3 M, about 10 -2 M or about 10 -1 M of K D In some embodiments, the polypeptides provided herein are in the form of 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 M of K D Binds to IL-21R.

[0133] IL-21 signaling activity by the IL-21 variants provided herein can be assessed by IL21-mediated STAT3 phosphorylation. In some embodiments, HuT78 cells can be treated with the IL-21 polypeptides provided herein, and then STAT3 phosphorylation can be analyzed. STAT3 phosphorylation can be used to calculate the half maximal effect concentration or EC 50 The relative activity of the IL-21 variants provided herein can be determined by comparing the EC 50 value to determine.

[0134] In some embodiments, the relative activity of the IL-21 variants provided herein is at least 1 / 1, at least 1 / 1.5, 1 / 2, at least 1 / 2.5, at least 1 / 3, at least 1 / 3.5, at least 1 / 4, at least 1 / 4.5, at least 1 / 5, at least 1 / 5.5, at least 1 / 6, at least 1 / 7, at least 1 / 8, at least 1 / 9, at least 1 / 10, at least 1 / 15, at least 1 / 20, at least 1 / 25, at least 1 / 30, at least 1 / 35, at least 1 / 40, at least 1 / 45, at least 1 / 50 lower than the STAT3 phosphorylation activity of wild-type IL-2. In some embodiments, the EC of the STAT3 phosphorylation activity of the IL-21 variants provided herein is at least 1 / 2, at least 1 / 2.5, at least 1 / 3, at least 1 / 3.5, at least 1 / 4, at least 1 / 4.5, at least 1 / 5, at least 1 / 5.5, at least 1 / 6, at least 1 / 7, at least 1 / 8, at least 1 / 9, at least 1 / 10, at least 1 / 15, at least 1 / 20, at least 1 / 25, at least 1 / 30, at least 1 / 35, at least 1 / 40, at least 1 / 45, at least 1 / 50 lower than the STAT3 phosphorylation activity of wild-type IL-2. 50 is the STAT3 phosphorylation activity of wild-type IL-2 EC 50 at least 1 times, at least 1.5 times, 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 5.5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, at least 45 times, at least 50 times higher.

[0135] Conjugate

[0136] In another aspect, provided herein are conjugates comprising 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.

[0137] In some embodiments, the at least one additional part is attached to the non-natural or natural amino acid in the polypeptide. In some embodiments, the polypeptide comprises at least one additional part attached to the natural amino acid in the polypeptide. In some embodiments, the polypeptide comprises at least one additional part attached to the non-natural amino acid in the polypeptide. In some embodiments, the at least one additional part is attached to the N-terminal or C-terminal amino acid of the polypeptide. In some embodiments, various combination sites are disclosed herein, for example, the first additional part is attached to the amino acid in the polypeptide, and the second additional part is attached to the N-terminal or C-terminal of the polypeptide. In some embodiments, a single additional part is attached to multiple residues (for example, staples) of a polypeptide. In some embodiments, the at least one additional part is attached to both the N-terminal and C-terminal amino acids of a polypeptide.

[0138] In some embodiments, the at least one additional moiety may be attached to the N-terminus of the IL-21 variant moiety. In some embodiments, the at least one additional moiety may be attached to the C-terminus of the IL-21 variant moiety.

[0139] Water soluble polymer

[0140] In some embodiments, the at least one additional part is a water-soluble polymer. In some embodiments, the water-soluble polymer is non-peptide, non-toxic and biocompatible. If the beneficial effects associated with using a substance alone or with another substance (e.g., an active agent, such as IL-21 or a polypeptide disclosed herein) in conjunction with living tissue (e.g., administered to a subject) exceed any harmful effects evaluated by a clinician (e.g., a physician, a toxicologist, or a clinical development expert), the substance can be considered to be biocompatible.

[0141] In some embodiments, the water-soluble polymer is non-immunogenic. A substance can be considered non-immunogenic if its intended use in vivo does not produce an undesirable immune response (e.g., antibody formation), or if an immune response is produced, such response is not considered clinically significant or important when evaluated by a clinician (e.g., physician, toxicologist, or clinical development specialist).

[0142] In some cases, the water-soluble polymer is characterized by having about 2 to about 300 ends. Non-limiting examples of water-soluble polymers include poly(alkylene glycols) such as polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, etc., poly(oxyethylated polyols), poly(enols), poly(vinyl pyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(sugar), poly(a-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.

[0143] In some embodiments, the water-soluble polymer is not limited to a particular structure. In some cases, the water-soluble polymer is linear (for example, end-capped, such as alkoxy PEG or difunctional PEG), branched or multi-armed (for example, bifurcated PEG or PEG attached to a polyol core), tree-like (or star-shaped) framework, each with or without one or more degradable bonds. In addition, the internal structure of the water-soluble polymer can be organized in any number of different repeating patterns, such as homopolymers, alternating copolymers, random copolymers, block copolymers, alternating trimers, random trimers or block trimers.

[0144] 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 weight average molecular weight ranges 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.

[0145] Non-limiting examples of weight average molecular weights of water-soluble polymers 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. The water-soluble polymer of any of the foregoing total molecular weights may be branched versions (e.g., a branched 40,000 Da water-soluble polymer comprising two 20,000 Da polymers). In some embodiments, the conjugate does not have any PEG moieties attached directly or indirectly to a PEG having a weight average molecular weight of less than about 6,000 Da.

[0146] PEG can comprise a plurality of (OCH2CH2) monomers or (CH2CH2O) monomers. The number of repeating units can be identified by the subscript "n" in "(OCH2CH2)n". Thus, the value of "n" can generally 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 whose molecular weight is known, 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 monomers.

[0147] In some cases, the water-soluble polymer is an end-capped polymer, i.e., having at least one end capped by a relatively inert group (such as a low C1-C6 alkoxy group or a hydroxyl group). For example, the water-soluble polymer can be methoxy-PEG (mPEG), which is a linear form of PEG in which one end of the PEG is a methoxy (-OCH3) group and the other end is a hydroxyl or other functional group that can be optionally chemically modified. Non-limiting examples of water-soluble polymers include linear or branched discrete PEG (dPEG); linear, branched or bifurcated PEG; and Y-shaped PEG derivatives.

[0148] In some embodiments, the polypeptides described herein are conjugated to a water-soluble polymer selected from the group consisting of poly(alkylene glycols) such as polyethylene glycol (PEG), poly(propylene glycol) (PPG), copolymers of ethylene glycol and propylene glycol, etc., poly(oxyethylated polyols), poly(enols), poly(vinyl pyrrolidone), poly(hydroxyalkyl methacrylamide), poly(hydroxyalkyl methacrylate), poly(sugar), poly(a-hydroxy acid), poly(vinyl alcohol) (PVA), polyacrylamide (PAAm), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), polydimethylacrylamide (PDAAm), polyphosphazenes, polyoxazolines (POZ), poly(N-acryloylmorpholine), and combinations thereof. For example, the IL-21 polypeptide is conjugated to PEG (e.g., PEGylated).

[0149] In some embodiments, the water-soluble polymer comprises polyglycerol (PG), for example, HPG, LPG, mid-functional PG, linear block hyperbranched PG or side chain functional PG. In some cases, the polyglycerol is hyperbranched PG (HPG).

[0150] 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 generated by polymerization of telechelic or di-terminal functionalized PEG-based macromonomers; PEG derivatives containing comonomers containing degradable bonds, such as poly[(ethylene oxide)-co-(methylene ethylene oxide)][P(EO-co-MEO)], cyclic ketene acetals such as 5,6-benzo-2-methylene-1,3-dioxepane (BMDO), 2-methylene-1,3-dioxepane (MDO), and 2-methylene-4-phenyl-1,3-dioxepane (MPDL) copolymerized with OEGMA or poly-(s-caprolactone)-graft-poly(ethylene oxide) (PCL-g-PEO).

[0151] 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-methacryloyloxyethylphosphatidylcholine) (PMPC).

[0152] In some embodiments, the water-soluble polymer comprises a polycarbonate. Non-limiting examples of polycarbonates include pentafluorophenyl-5-methyl-2-oxo-1,3-dioxane-5-carboxylate (MTC-OC6F5).

[0153] In some embodiments, the water-soluble polymer comprises a polymer hybrid, such as, for example, a polycarbonate / PEG polymer hybrid, a peptide / protein-polymer conjugate, or a hydroxyl-containing and / or zwitterion-derivatized polymer (e.g., a hydroxyl-containing and / or zwitterion-derivatized PEG polymer).

[0154] In some embodiments, the water-soluble polymer comprises a polysaccharide. Non-limiting examples of polysaccharides include dextran, polysialic acid (PSA), hyaluronic acid (HA), amylose, heparin, heparan sulfate (HS), dextrin, or hydroxyethyl starch (HES).

[0155] 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.

[0156] In some embodiments, water-soluble polymer comprises polyoxazoline polymer.Polyoxazoline polymer is a kind of linear synthetic polymer, and has low polydispersity similar to PEG.In some cases, polyoxazoline polymer is polydispersity polyoxazoline polymer, characterizes by mean molecular weight.In some cases, the mean molecular weight of polyoxazoline polymer comprises 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 500,000Da. In some cases, the polyoxazoline polymer comprises poly(2-methyl 2-oxazoline) (PMOZ), poly(2-ethyl 2-oxazoline) (PEOZ), or poly(2-propyl 2-oxazoline) (PPOZ). In some cases, the cytokine (e.g., interleukin, IFN, or TNF) polypeptide is conjugated to the polyoxazoline polymer.

[0157] In some embodiments, the water soluble polymer is a polyacrylic acid polymer.

[0158] In some embodiments, the water-soluble polymer comprises a polyamine. A polyamine is an organic polymer comprising two or more primary amino groups. In some embodiments, the polyamine comprises a branched polyamine, a straight chain 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.

[0159] Lipids

[0160] In some embodiments, the at least one additional part is a lipid. The lipid can be a fatty acid, such as a saturated fatty acid or an unsaturated fatty acid. Such fatty acids can have a length of 6 to 26 carbon atoms, 6 to 24 carbon atoms, 6 to 22 carbon atoms, 6 to 20 carbon atoms, 6 to 18 carbon atoms, 20 to 26 carbon atoms, 12 to 26 carbon atoms, 12 to 24 carbon atoms, 12 to 22 carbon atoms, 12 to 20 carbon atoms or 12 to 18 carbon atoms. In some cases, the fatty acid has a length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 carbon atoms. The non-limiting examples of fatty acids include caproic acid, heptanoic acid, caprylic acid, nonanoic acid, capric acid, undecanoic acid, lauric acid (dodecanoic acid), tridecanoic acid, myristic acid (tetradecanoic acid), pentadecanoic acid, palmitic acid (hexadecanoic acid), pearlic acid (heptadecanoic acid), stearic acid (octadecanoic acid), nonadecanoic acid, arachidic acid (eicosanoic acid), heneicosanoic acid, behenic acid (behenic acid), tricosanoic acid, lignoceric acid (tetracosanoic acid), pentacosanoic acid and cerotic acid (hexacosanoic acid). In some embodiments, lipids are bound to one or more serum proteins to increase serum stability and / or serum half-life.

[0161] protein

[0162] 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 thyroxine transporter. In some embodiments, the protein or its binding fragment comprises an antibody or its binding fragment.

[0163] In some embodiments, at least one additional part described herein is a tag that can be used for functions such as purification or detection, which extends at the N-terminus, C-terminus, or both the N-terminus and the C-terminus of the polypeptide disclosed herein. In some embodiments, the tag can be a polyglutamic acid tag, a FLAG tag, a HA tag, a poly-His tag (with about 5-10 histidines) (SEQ ID NO: 80), a hexahistidine tag (HHHHHH) (SEQ ID NO: 81), an 8X-His tag (HHHHHHHH) (SEQ ID NO: 82), a Myc tag, a glutathione-S-transferase tag, a green fluorescent protein tag, a maltose binding protein tag, a thioredoxin tag, or an Fc tag. In some embodiments, the at least one additional part is an Fc tag. In some specific embodiments, the Fc fragment is IgG, IgA, IgD, IgM, or IgE. In some specific embodiments, the IgG is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the extension or additional part can be an N-terminal signal peptide fused to a protein to enhance expression. Although such signal peptides are often cleaved during expression in cells, some polypeptides may contain cytokines with intact signal peptides.

[0164] In some embodiments, the at least one additional moiety comprises an antigen binding molecule. In a specific embodiment, the antigen binding molecule is an antibody or an antigen binding fragment thereof. In a specific embodiment, the antigen binding molecule is a multispecific antigen binding molecule. In a specific case, the antigen binding molecule is a bispecific antibody or a bispecific antigen binding fragment thereof. In other specific cases, the antigen binding molecule is an antibody-drug conjugate (ADC). In other embodiments, the at least one additional portion is selected from: 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), triabody, tetrabody, V-NAR domain, Fcab, IgGACH2, DVD-Ig, antibody prodrug (probody), DARPin, Centyrin, affinity body, affilin, affitin, anticalin, high affinity multimer (avimer), Fynomer, Kunitz domain peptide, monomer (monobody) (or adnectin), tribody and nanofitin.

[0165] In other embodiments, the at least one additional moiety 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 portion of a light chain and a heavy chain bridged by disulfide bonds); Fab' (e.g., an antibody fragment comprising a single antigen binding domain comprising Fab and an additional portion of a heavy chain through a hinge region); F(ab')2 (e.g., two Fab' molecules joined by an interchain disulfide bond in the hinge region of a heavy chain; the Fab' molecules can be oriented toward the same or different epitopes); bispecific Fab (e.g., a Fab molecule with two antigen binding domains, each of which can be oriented to a different epitope); a single chain comprising a variable region, also called scFv (e.g., a single light chain and a variable antigen binding determining region of a heavy chain of an antibody linked together by a chain of 10-25 amino acids); disulfide-linked Fv or dsFv (e.g., a single light chain and a variable antigen binding determining region of a heavy chain of an antibody linked together by disulfide bonds); camelized VH (e.g., a variable antigen binding determining region of a single heavy chain of an antibody, wherein some of the amino acids at the VH interface are those present in the heavy chain of a naturally occurring camelid antibody); bispecific scFv (e.g., an scFv or dsFv molecule having two antigen binding domains, each of which can be directed to a different epitope); diabody (e.g., a dimeric scFv formed when the VH domain of a first scFv is assembled with the VL domain of a second scFv and the VL domain of the first scFv is assembled with the VH domain of a second scFv; the two antigen binding regions of the diabody can be directed toward the same or different epitopes); triabody (e.g., a trimerized scFv, formed in a manner similar to a diabody, but wherein three antigen binding domains are produced in a single complex; the three antigen binding domains can be directed toward the same or different epitopes); and tetrabody (e.g., a tetrameric scFv, formed in a manner similar to a diabody, but wherein four antigen binding domains are produced in a single complex; the four antigen binding domains can be directed toward the same or different epitopes).

[0166] 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 a specific embodiment, the tumor-associated antigen is selected from human epidermal growth factor receptor 2 (HER2), CD20, CD33, B cell maturation antigen (BCMA), prostate-specific membrane antigen (PSMA), DLL3, ganglioside GD2 (GD2), CD 123, anoctamin-1 (Ano1), mesothelin, carbonic anhydrase IX (CAIX), tumor-associated calcium signaling protein 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-α), pregnancy-associated plasma protein A (PAPP-A), CD37, epithelial cell adhesion molecule (EpCAM), CD2, CD 19, CD30, CD38, CD40, CD52, CD70, CD79b, fms-like tyrosine kinase 3 (FLT3), glypican 3 (GPC3), B7 homolog 6 (B7H6), CC chemokine receptor type 4 (CCR4), CXC 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 receptor 3.

[0167] In some embodiments, the antigen binding molecules conjugated to the polypeptides described herein target tumor cells. In some embodiments, the antigen is an immune checkpoint antigen or an immune checkpoint-associated antigen. In a specific embodiment, the antigen participates in the immune checkpoint pathway. In a specific embodiment, the antigen is selected from: PD-1, PD-L1, TIGIT, CTLA-4, PD-L2, B7-H3, B7-H4, BTLA, LAG3, CD112, CD112R, CD96, TIM-3, CD47 and CEACAM1. In a specific embodiment, the antigen is a co-stimulatory immune checkpoint target. In a specific embodiment, the antigen is selected from: CD155, ICOS, OX40, CD137, CD137L, CD27, CD28 and GITR.

[0168] In some embodiments, the at least one additional moiety is attached to the C-terminus and / or N-terminus of the IL-21 variant. In some embodiments, the at least one additional moiety is attached to the IL-21 variant directly or via a linker.

[0169] In some cases, a plurality of additional moieties disclosed herein can be attached to a single polypeptide disclosed herein. In some cases, a plurality of polypeptides disclosed herein can be attached to a single additional moiety disclosed herein. In some cases, a single additional moiety disclosed herein is attached to a single polypeptide disclosed herein. In some cases, the ratio of polypeptide to additional moiety is 1:1. In some cases, the ratio of polypeptide to additional moiety 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 polypeptide to additional moiety is about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0170] In some embodiments, the at least one additional part is albumin or a functional fragment thereof. Albumin is a family of water-soluble globular proteins. Albumin is commonly found in plasma, accounting for about 55%-60% of all plasma proteins. Human serum albumin (HSA) is a 585 amino acid polypeptide comprising 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 also comprises a binding site that can reversibly or irreversibly interact with endogenous ligands (such as fatty acids, bilirubin or heme) or exogenous compounds (such as heterocyclic or aromatic compounds).

[0171] In some embodiments, the at least one additional moiety is transferrin. Transferrin is a 679 amino acid polypeptide with a size of about 80 kDa and comprising two F 3+ Binding sites, one located in the N-terminal domain and the other located in the C-terminal domain. Human transferrin has a half-life of about 7-12 days.

[0172] In some embodiments, the at least one additional moiety is transthyretin (TTR). Transthyretin is a transporter protein located in serum and cerebrospinal fluid that transports the thyroid hormone thyroxine (T4) and retinol binding protein, which binds to retinol.

[0173] In some embodiments, a conjugate comprising a polypeptide described herein and an antibody or antigen-binding fragment thereof described herein is enriched in relevant tumor tissues via the action of the antibody or antigen-binding fragment thereof described herein. Therefore, the anti-tumor activity of the polypeptide described herein can be enhanced.

[0174] In some embodiments, the at least one additional moiety comprises a bioconjugate (e.g., a toll-like receptor (TLR) agonist, such as a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 agonist; or a synthetic ligand, such as Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, Poly I:C, Poly A:U, AGP, MPLA, RC-529, MDF2p, CFA, or flagellin).

[0175] Sections categorized by function

[0176] In some cases, the at least one additional portion reduces the interaction of IL-21 with one or more IL-21 receptor domains or subunits. In some embodiments, the at least one additional portion blocks the interaction of IL-21 with one or more IL-21 domains or subunits through one or more cognate receptors. In some cases, the at least one additional portion increases serum half-life and / or improves in vivo stability.

[0177] Masking part

[0178] In some embodiments, the at least one additional part is a shielding part. The shielding part can block, block, suppress, reduce or otherwise reduce (i.e., shield) IL-21 for the activity or combination of its cognate receptor or protein. For example, polypeptide disclosed herein can be activated by protease at the target site (such as in a tumor microenvironment) by including a proteolytic cleavable joint. In some embodiments, a proteolytic cleavable joint connects the polypeptide to the shielding part. When the cleavable joint is proteolytically cleaved at the target site, the polypeptide disclosed herein can be activated so that the polypeptide can be combined with its cognate receptor or protein.

[0179] The masking portion may include a cleavable peptide and / or a linker. In some embodiments, the cleavable linker comprises a cleavable peptide. A cleavable peptide is a polypeptide comprising a cleavage site, such as a protease cleavage site. The cleavage site is an identifiable site for a portion of a cleavable peptide of a cleavable linker as described herein. In some embodiments, the cleavage peptide comprises 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, DNA enzymes, RNA enzymes, metals, acids, and bases.

[0180] In some embodiments, the protease cleavage site is a matrix metalloproteinase (MMP) cleavage site, a metalloproteinase cleavage site containing a disintegrin and metalloproteinase domain (ADAM), 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 leguminous protein (legumain) protease cleavage site. In embodiments, the matrix metalloproteinase (MMP) cleavage site is an MMP9 cleavage site, an MMP13 cleavage site or an MMP2 cleavage site. In embodiments, the metalloproteinase cleavage site containing a disintegrin and metalloproteinase domain (ADAM) is an ADAM9 metalloproteinase cleavage site, an ADAM10 metalloproteinase cleavage site or an ADAM17 metalloproteinase cleavage site. The protease cleavage site can be indicated by a specific amino acid sequence.

[0181] Half-life extension domain

[0182] In some embodiments, the at least one additional portion is a half-life extension domain that increases the serum half-life and / or improves stability of the polypeptide disclosed herein. The half-life extension domain can be fused to the N-terminus or C-terminus of the polypeptide disclosed herein. In some embodiments, a proteolytically cleavable linker connects the polypeptide disclosed herein to the half-life extension domain.

[0183] 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 about three weeks due to its size and sensitivity to FcRn-mediated circulation, thereby reducing the possibility of intracellular degradation. Therefore, linking the IL-21 polypeptide to albumin can significantly extend the serum half-life of the IL-21 polypeptide.

[0184] In some embodiments, the IL-21 polypeptide herein comprises an IL-21 mutant protein fused to a heterologous polypeptide (i.e., a polypeptide that is not IL-21 and preferably is not a variant of IL-21, e.g., a conjugated moiety described herein). The heterologous polypeptide can increase the circulating half-life of the IL-21 polypeptide. For example, the polypeptide that increases the circulating half-life can be serum albumin, such as HSA.

[0185] In some embodiments, the IL-21 polypeptide provided herein comprises an amino acid sequence heterologous to wild-type human IL-21 or a functional variant thereof. The amino acid sequence may be a half-life extension portion that increases the stability of the polypeptide by increasing the in vivo half-life of the polypeptide, for example, when administered to a subject. The half-life extension portion may be a protein, an antibody, an albumin, an immunoglobulin or a fragment thereof, transferrin or PEG. The antibody may be an anti-albumin antibody. The albumin may be a serum albumin, such as mouse serum albumin or human serum albumin. In some embodiments, the immunoglobulin fragment is an Fc domain of a human immunoglobulin, for example, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM.

[0186] In some embodiments, the polypeptides disclosed herein comprise a half-life extension domain comprising 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.

[0187] In some embodiments, the albumin polypeptide is connected to a masking moiety. In some embodiments, the masking moiety is connected to the N-terminus or C-terminus of an albumin polypeptide. In some embodiments, the albumin polypeptide is connected to a masking moiety via a linker. In some embodiments, the linker is connected to the amino-terminus or carboxyl-terminus of an albumin polypeptide. In some embodiments, the N-terminal or C-terminal spacer domain of the linker is connected to the N-terminus or C-terminus of an albumin polypeptide. In some embodiments, the cleavable peptide of the linker is connected to the N-terminus or C-terminus of an albumin polypeptide. In some embodiments, the albumin polypeptide is connected to a cytokine or a functional fragment thereof (e.g., IL-21 or a mutant protein thereof). In some embodiments, a cytokine or a functional fragment thereof is connected to the N-terminus or C-terminus of an albumin polypeptide. In some embodiments, the albumin polypeptide is connected to a cytokine or a functional fragment thereof via a linker. In some embodiments, the linker is connected to the amino-terminus or carboxyl-terminus of an albumin polypeptide.

[0188] In some embodiments, the conjugate comprises a polypeptide disclosed herein and a combination of two or more of the moieties described above.

[0189] Synergy

[0190] 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 specific embodiments, the conjugates described herein exhibit stronger anti-tumor activity than the polypeptides described herein. In some embodiments, the conjugates described herein exhibit stronger anti-tumor activity than at least one additional moiety. In specific embodiments, the conjugates described herein cause fewer side effects than the polypeptides described herein. In some embodiments, the conjugates described herein cause fewer side effects than at least one additional moiety.

[0191] Thermal stability

[0192] In some embodiments, compared with wild-type human IL-21, the polypeptides described herein or conjugates show improved thermal stability. In other embodiments, compared with wild-type human IL-21, the polypeptides described herein or conjugates are more stable in vitro. In other embodiments, compared with wild-type human IL-21, the polypeptides described herein or conjugates are more stable in vivo.

[0193] In some embodiments, the thermal stability of polypeptides or conjugates described herein is assessed by NanoTemper assay (Prometheus). In some embodiments, the thermal stability of polypeptides or conjugates described herein is assessed by qPCR (Thermo Fisher) equipped with a protein thermal shift (PTS) research solution. In some embodiments, the thermal stability of polypeptides or conjugates described herein is assessed by differential scanning calorimetry (DSC). In some embodiments, the thermal stability of polypeptides or conjugates described herein is assessed by differential scanning fluorimetry (DSF). In some specific embodiments, the melting temperature of mutant IL21 polypeptides described herein is higher than the melting temperature of wild-type IL21 equivalents. In some specific embodiments, the melting temperature of mutant IL21 conjugates described herein is higher than the melting temperature of wild-type IL21 equivalents.

[0194] Features and Benefits

[0195] In some embodiments, the polypeptides or conjugates described herein exhibit any one or more of the following, such as one, two, three, four, five, six or seven: (1) better pharmacodynamic properties than wild-type IL-21 or a wild-type IL-21 conjugate; (2) better pharmacodynamic properties than at least one additional moiety; (3) stronger anti-tumor activity than wild-type IL-21 or a wild-type IL-21 conjugate; (4) stronger anti-tumor activity than at least one additional moiety; (5) fewer side effects than wild-type IL-21 or a wild-type IL-21 conjugate; (6) synergistic effect in combination with at least one additional agent (compared to the administration of any of them alone); and (7) improved thermal stability compared to wild-type IL-21 or a wild-type IL-21 conjugate.

[0196] Nucleic acids, vectors and transformed or host cells

[0197] In another aspect, provided herein are nucleic acid molecules encoding a polypeptide disclosed herein or a conjugate disclosed herein. In another aspect, provided herein are vectors comprising nucleic acid molecules disclosed herein. In some embodiments, the vectors include viral vectors. In another aspect, provided herein are transformed or host cells expressing a polypeptide disclosed herein or a conjugate disclosed herein.

[0198] Nucleic acid molecules can encode polypeptides as described herein. Nucleic acid molecules can be codon optimized to encode polypeptides as described herein. Such nucleic acid molecules can be inserted into expression vectors, which can then be transformed or incorporated into host cells. The expression vector can be a plasmid. Host cells comprising expression vectors can be used to express IL-21 mutants as described herein. The host cell can be Escherichia coli (E.coli) or other suitable bacterial cells.

[0199] Unless otherwise indicated, the present invention can be performed using standard procedures known to those skilled in the art, such as Michael R. Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 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., eds., John Wiley and Sons, Inc.); Current Protocols in Immunology (CPI) (John E. Coligan et al., eds., John Wiley and Sons, Inc.); Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et al., eds., John Wiley and Sons, Inc.); Culture of Animal Cells: A Manual of Basic Technique, 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, eds., Academic Press, 1st edition, 1998); Methods in Molecular biology, Vol. 180, Transgenesis Techniques, Alan R. Clark, ed., 2nd edition, 2002, Humana Press; and Methods in Meolcular Biology, Vol. 203, 2003, Transgenic Mouse, Marten H. Hofker and Jan van Deursen, eds., all of which are incorporated herein by reference in their entirety.

[0200] Reagent test kit

[0201] 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, a combination thereof, and a container.

[0202] In some embodiments, kits or articles are provided herein, comprising polypeptides as described herein, including pharmaceutical compositions thereof. The kit may include instructions for use of the polypeptides, such as in the methods provided herein. Thus, in some embodiments, the kit includes instructions for use of the polypeptides in methods for treating a disorder described herein (e.g., cancer) of the subject by administering a therapeutically effective amount of the polypeptides to a subject in need thereof. In some embodiments, the subject is a human. In some embodiments, the disorder is cancer.

[0203] The kit may also include a container. Non-limiting examples of suitable containers include bottles, vials (e.g., double-chamber vials), syringes (such as single-chamber or double-chamber syringes), test tubes, and intravenous infusion (IV) bags. The container may be formed of a variety of materials such as glass or plastic. The container may hold a preparation of the IL-21 polypeptide. In some embodiments, the preparation is a lyophilized preparation. In some embodiments, the preparation is a frozen preparation. In some embodiments, the preparation is a liquid preparation.

[0204] The kit can also include a label or package insert, which is located on the container or is associated with the container, and can indicate instructions for reconstructing and / or using the preparation. The label or package insert can also indicate that the preparation can be used for or is intended to be used for intravenous, subcutaneous or other modes of administration, for the treatment of the disease (for example, cancer) of the object. The container holding the preparation can be a disposable bottle or a multiple use bottle, which can allow repeated administration of the reconstructed preparation. The kit can also include a second container comprising a suitable diluent. The kit can also include other materials desired from a commercial, treatment and user perspective, including other buffers, diluents, filters, needles, syringes and package inserts printed with instructions for use.

[0205] In some embodiments, a kit for a single-dose administration unit is provided herein. Such a kit includes a container of an aqueous formulation of a therapeutic IL-21 polypeptide, including a single-chamber or multi-chamber prefilled syringe.

[0206] In some embodiments, provided herein are articles or kits comprising the formulations described herein for administration in an automatic injector device. Automatic injectors can be described as injection devices that deliver their contents after activation without requiring additional action from the patient or the user. They are particularly suitable for self-medication of therapeutic formulations when the delivery rate must be constant and the delivery time is greater than a few moments.

[0207] Pharmaceutical composition

[0208] On the other hand, provided herein is a pharmaceutical composition comprising a polypeptide disclosed herein or a conjugate disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition of the present disclosure can be a combination of a compound (e.g., a polypeptide as described herein) and other chemical components (such as a carrier, a stabilizer, a diluent, a dispersant, a suspending agent, a thickening agent and / or an excipient). The pharmaceutical composition facilitates administration of the compound to an organism.

[0209] The pharmaceutical preparation for administration can include an aqueous solution of the active compound in a water-soluble form. The suspension of the active compound can be prepared into 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. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. The suspension can also contain a suitable stabilizer or increase the solubility of the compound to allow the preparation of a highly concentrated solution. The active ingredient can be in powder form for use before use with a suitable vehicle (e.g., sterile pyrogen-free water) to form.

[0210] In practicing the methods of treatment or uses provided herein, a therapeutically effective amount of a compound described herein is administered in the form of a pharmaceutical composition to a subject suffering from 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, and other factors.

[0211] Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers (including excipients and adjuvants) that aid in processing the active compound into a pharmaceutically usable formulation. The formulation can be modified according to the selected route of administration. Pharmaceutical compositions comprising the compounds described herein can be manufactured, for example, by mixing, dissolving, emulsifying, encapsulating, embedding or compressing processes.

[0212] The pharmaceutical composition may comprise at least one pharmaceutically acceptable carrier, diluent or excipient and a compound described herein as a free base or a pharmaceutically acceptable salt form. The pharmaceutical composition may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

[0213] Methods for preparing compositions comprising compounds described herein include formulating the compounds with one or more inert, pharmaceutically acceptable excipients or carriers to form solid, semisolid or liquid compositions. Solid compositions include, for example, powders, tablets, dispersible granules, capsules and cachets. Liquid compositions include, for example, solutions in which the compounds are dissolved, emulsions containing the compounds, or solutions containing liposomes, micelles or nanoparticles containing compounds as disclosed herein. Semisolid compositions include, for example, gels, suspensions and creams. The compositions may be in the form of liquid solutions or suspensions, solid forms suitable for dissolution or suspension in liquids prior to use, or in the form of emulsions. These compositions may also contain small amounts of non-toxic auxiliary substances, such as wetting agents or emulsifiers, pH buffers and other pharmaceutically acceptable additives.

[0214] Non-limiting examples of dosage forms suitable for use in the present disclosure include liquids, powders, gels, nanosuspensions, nanoparticles, microgels, aqueous or oily suspensions, emulsions, and any combination thereof.

[0215] Non-limiting examples of pharmaceutically acceptable excipients suitable for use in the present disclosure include binders, disintegrants, anti-adherents, antistatic agents, surfactants, antioxidants, coating agents, colorants, plasticizers, preservatives, suspending agents, emulsifiers, antimicrobial agents, spheronization agents, and any combination thereof.

[0216] Compositions of the present disclosure can be, for example, immediate release forms or controlled release formulations. Immediate release formulations can be formulated to allow compounds to act quickly after administration. Non-limiting examples of immediate release formulations include easily soluble formulations. Controlled release formulations can be pharmaceutical formulations that are adapted to allow the release rate and release profile of active agents to match physiological and time treatment needs, or are formulated to achieve the release of active agents at a programmed rate. Non-limiting examples of controlled release formulations include granules, delayed release granules, hydrogels (e.g., hydrogels of synthetic or natural origin), other gelling agents (e.g., dietary fibers forming gels), matrix-based formulations (e.g., formulations comprising a polymer material in which at least one active ingredient is dispersed), particles in the matrix, polymer mixtures, and granular masses.

[0217] In some embodiments, the controlled release formulation is a delayed release form. The delayed release form can be formulated to delay the effect of the compound for a prolonged period of time. The delayed release form can be formulated to delay the release of one or more compounds of an effective dose, delaying for example about 4, about 8, about 12, about 16 or about 24 hours.

[0218] The controlled release formulation can be a sustained release form. The sustained release form can be formulated to continue the effect of, for example, the compound over an extended period of time. The sustained release form can be formulated to provide an effective dose of any compound described herein (e.g., to provide a physiologically effective blood profile) over about 4, about 8, about 12, about 16, or about 24 hours.

[0219] The compounds described herein can be conveniently formulated into pharmaceutical compositions consisting of one or more pharmaceutically acceptable carriers. See, for example, Remington's Pharmaceutical Sciences, latest edition, EW Martin Mack Pub. Co., Easton, PA, incorporated by reference in its entirety, which discloses pharmaceutically acceptable excipients and carriers and methods for preparing pharmaceutical compositions. Such carriers can be carriers for administering the composition to humans and non-humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The pharmaceutical composition may also contain one or more additional active ingredients, such as antimicrobial agents, anti-inflammatory agents, and anesthetics.

[0220] In addition to the agents disclosed herein, pharmaceutical formulations may contain additional carriers as well as thickeners, diluents, buffers, preservatives and surfactants.

[0221] The pharmaceutical compositions disclosed herein can be administered in a therapeutically effective amount by a variety of 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, intraocular administration, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, intraarterial administration, intrathecal administration, inhalation, intralesional administration, intradermal administration, epidural administration, absorption through epithelial or mucosal and skin linings (e.g., oral mucosa, rectal and intestinal mucosa), intracapsular administration, subcapsular administration, intracardiac administration, transtracheal administration, subcutaneous administration, subarachnoid administration, subcapsular administration, intraspinal administration, or intrasternal administration.

[0222] The pharmaceutical composition can be applied in a local manner, for example, via direct injection of the compound into an organ, optionally in the form of a reservoir or sustained release formulation or implant. The pharmaceutical composition can be provided in the form of a quick release formulation, in the form of an extended release formulation, or in the form of an intermediate release formulation. Quick release forms can provide immediate release. Extended release formulations can provide controlled release or sustained delayed release.

[0223] 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" encompasses any agent administered to treat a symptom or disease in an animal in need of such treatment. The compounds can also be administered as a component of a vaccine, i.e., in combination with essentially any formulation intended for active immunoprophylaxis.

[0224] The toxicity and therapeutic efficacy of the compounds herein can be determined in cell culture and experimental animals by standard pharmaceutical procedures. Cell culture assays and animal studies can be used to determine LD50 (a dose lethal to 50% of the population) and ED50 (a dose effective for 50% of the population treatment). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as a ratio of LD50 / ED50. Compounds herein that exhibit a larger therapeutic index are preferred. The data obtained from these cell culture assays and animal studies can be used in a series of doses suitable for use in humans. The dosage of such compounds is preferably within a series of circulating concentrations including ED50 with little or no toxicity. The dosage can vary within this range depending on a variety of factors, for example, the dosage form employed, the route of administration utilized, the condition of the subject, etc.

[0225] By determining IC50, a preliminary estimate of the therapeutically effective dose can be made from a cell culture assay. The dosage can then be formulated in an animal model to achieve a circulating plasma concentration range including the IC50 as determined in cell culture. Such information can be used to more accurately determine the dosage that can be used for humans. The level in plasma can be measured, for example, by HPLC. Exact preparation, route of administration, and dosage can be selected by individual physicians according to the patient's condition.

[0226] The attending physician of the patient treated with the compounds herein will know how and when to terminate, interrupt or adjust the administration due to toxicity, organ dysfunction, etc. Conversely, if the clinical response is insufficient (excluding toxicity), the attending physician also knows to adjust the treatment to a higher level. The size of the administered dose in the management of the disease of interest will vary with the severity of the condition to be treated, the route of administration, etc. For example, the severity of the condition can be partially evaluated by standard prognostic evaluation methods. In addition, the dosage and possible dosage frequency will also vary according to the age, weight and response of the individual patient.

[0227] The compounds herein can be administered to an individual as a pharmaceutical formulation appropriately formulated for the route of delivery and the condition being treated. Suitable routes may include oral, rectal, transdermal, vaginal, transmucosal or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injection, and intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal or intraocular injection, etc. For transmucosal administration, penetrants suitable for the barrier to be permeated may be used in the formulation.

[0228] The compounds herein can be formulated as liquids with a carrier, which may include a buffer and / or salt, such as phosphate buffered saline. Alternatively, the compounds herein can be formulated as solids with 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.

[0229] For oral delivery, the final product formulated can be tablets, pills, capsules, dragees, liquids, gels, syrups, slurries, suspensions, etc. In addition, push-fit capsules made of gelatin and soft sealed capsules made of gelatin and a plasticizer (such as glycerol or sorbitol) can be used. Push-fit capsules can contain the active ingredients mixed with fillers as above, while in soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin or liquid polyethylene glycols.

[0230] Preparations for oral delivery can involve conventional mixing, dissolving, granulation, dragee manufacturing, grinding, emulsification, encapsulation, embedding, freeze-drying processes, etc. The compounds herein can also be mixed with solid excipients, optionally grinding the resulting mixture, and treating the granular mixture (if necessary) to obtain tablets or dragee cores after adding suitable adjuvants. Suitable excipients are particularly fillers such as sugars, including lactose, sucrose, mannitol, sorbitol, etc.; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, polyvinyl pyrrolidone (PVP), etc., and any two or more mixtures thereof. If necessary, disintegrants such as cross-linked polyvinyl pyrrolidone, agar, alginic acid or its salts, such as sodium alginate, etc. can be added.

[0231] If injection is required, 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 physiological saline buffer. In addition, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. 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, dextran, etc. Optionally, the suspension may also contain a suitable stabilizer or an agent that increases the solubility of the compound to allow the preparation of a highly concentrated solution.

[0232] method

[0233] In another aspect, provided herein is a method for preparing a polypeptide described herein or a conjugate described herein, comprising: (a) constructing a nucleic acid molecule and a vector described herein; (b) culturing a transformed or host cell described herein; and (c) harvesting the polypeptide from the transformed or host cell.

[0234] In another aspect, provided herein is a method of treating a subject in need thereof, 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.

[0235] 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 a metastatic cancer. In some cases, the cancer is a recurrent or refractory cancer. In some embodiments, the cancer is untreated. Cancer. An untreated cancer may be a cancer that has not been treated with therapy.

[0236] In some embodiments, the cancer is a leukemia, a lymphoma, a sarcoma, a myeloma, a glioma, a glioblastoma, a glioblastoma multiforme, a glioma, a head and neck cancer, a colorectal cancer, a colon cancer, a prostate cancer, a castration-resistant prostate cancer, a pancreatic cancer, a melanoma, a 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), a neuroblastoma, a lung cancer (e.g., non-small cell lung cancer, squamous cell lung cancer (e.g., head, neck, or esophageal), adenocarcinoma, large cell lung cancer, small cell lung cancer, carcinoid, a sarcoma), an ovarian cancer, a bone cancer (e.g., osteosarcoma, chondrosarcoma, Ewing sarcoma), a bladder cancer, a cervical cancer, a 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's lymphoma, neuroblastoma, rhabdomyosarcoma, primary The invention relates to a type of cancer, for example, thrombocythemia, primary macroglobulinemia, primary brain tumor, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, precancerous skin lesions, neuroblastoma, genitourinary tract cancer, malignant hypercalcemia, adrenocortical carcinoma, endocrine or exocrine pancreatic tumor, medullary thyroid cancer, medullary thyroid cancer, 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 cancer include thyroid cancer, cancer of the endocrine system, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, esophageal cancer, liver cancer, kidney cancer, ovarian cancer, stomach cancer or uterine cancer.

[0237] 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.

[0238] In some embodiments, the cancer is a hematological malignancy, such as a 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), Waldenstrom's macroglobulinemia, multiple myeloma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, Burkitt's lymphoma, non-Burkitt's 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, plasma cell myeloma, plasmacytoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or lymphomatoid granulomatosis.

[0239] In some embodiments, the subject described herein has or is suspected of having an inflammatory disease or an autoimmune disease. In some embodiments, the inflammatory disease or autoimmune disease is atherosclerosis, obesity, inflammatory bowel disease (IBD), rheumatoid arthritis, allergic encephalitis, psoriasis, atopic skin disease, osteoporosis, peritonitis, hepatitis, lupus, celiac disease, Sjögren's syndrome ( 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 diseases, cartilage inflammation, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile enteropathic arthritis, juvenile recurrent Allergic arthritis, juvenile Reiter's syndrome, seronegative endarteropathy and arthropathy (SEA) syndrome, juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile systemic lupus erythematosus, juvenile vasculitis, oligoarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, dermatomyositis, psoriatic arthritis, scleroderma, vasculitis, myositis, polymyositis, dermatomyositis, polyarteritis nodosa, Wegener's granulomatosis 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, sinusitis, sinusitis with polyps, eosinophilic esophagitis, eosinophilic bronchitis, Guillain-Barre disease, thyroiditis (e.g., Graves' disease), Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, transplant rejection, renal injury, or hepatitis C-induced vasculitis.

[0240] Example

[0241] Example 1: Recombinant expression and preparation of IL-21 variants.

[0242] 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.

[0243] Table 1 Full-length amino acid sequences of IL-21 variants

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260] Note: In the examples and figures disclosed herein, IL-21 variants with an Fc tag are labeled as "m(ID)-Fc" and IL-21 variants with a His tag are labeled as "m(ID)-His".

[0261]

[0262]

[0263]

[0264]

[0265]

[0266] Exemplary WT IL-21 and IL-21 variants are shown in Tables 1 and 2. They were generated with Fc tags by adding human IgG1 Fc (SEQ ID NO. 63) directly to the C-terminus of each WT IL-21 and IL-21 variant, and adding an 18-aa signal peptide (SEQ ID NO. 64) directly at the N-terminus of each mature WT IL-21 and IL-21 variant (the signal peptide was added to facilitate expression in mammalian cell lines and subsequently cleaved in the expression product).

[0267] IgG1 The sequence of Fc is (from N-terminus to C-terminus): EPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0268] (SEQ ID NO.63)

[0269] The sequence of the 18-aa signal peptide is (from N-terminus to C-terminus):

[0270] MHSSALLCCLVLLTGVRA

[0271] (SEQ ID NO.64)

[0272] An exemplary amino acid sequence of the encoded IL-21-Fc construct is (from N-terminus to C-terminus):

[0273]

[0274] (Amino acid sequence of WT IL-21-human IgG1 Fc construct (SEQ ID NO. 65), in which the bold segment represents the 18-aa signal peptide; the underlined segment represents the mature IL-21 sequence; the italic portion represents the human IgG1-Fc tag)

[0275] All variants share the above construct structure with the same signal peptide and Fc tag sequence, but each variant has its own unique mature IL-21 sequence as shown in Tables 1 and 2.

[0276] 1) Construction of expression plasmid.

[0277] 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.

[0278] An exemplary expression sequence for an IL-21-Fc construct is (from N-terminus to C-terminus):

[0279]

[0280]

[0281] (WT IL-21-Fc DNA expression sequence (SEQ ID NO.66), wherein segment (i) represents the NotI endonuclease cleavable site; segment (ii) represents the Okazaki fragment element; the bold segment (iii) represents the 18-aa signal peptide coding sequence; the underlined segment (iv) represents the mature IL-21 coding sequence; the italic segment (v) represents the human IgG1-Fc tag coding sequence; segment (vi) represents the stop codon; and segment (vii) represents the Xbal endonuclease cleavable site).

[0282] The confirmed DNA sequence was constructed into the expression vector pcDNA3.1 (Thermo Fisher, catalog number V79020). The plasmid containing the IL-21-Fc gene ( Figure 1 The exemplary WT IL-21 recombinant expression vector shown in Figure 1 was transformed into E. coli DH5α cells. A large amount of wild-type-Fc plasmid and variant-Fc plasmid were obtained by culturing E. coli DH5α cells for amplification and plasmid purification. The preparation of plasmid constructs was operated according to the following protocol: Michael R. Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (4th edition, 2012), which is also incorporated herein by reference in its entirety.

[0283] 2) Expression of wild-type IL-21-Fc and IL-21 variant-Fc proteins in HEK293F cells.

[0284] The cells were plated at 0.5 × 10 6 The inoculum volume was 10 cells / ml in 300 mL of medium (Gibco TM FreeStyle TM 293 expression medium, catalog number 12338018). The cells were incubated in a shaking incubator at 37°C, 120 rpm and 5% carbon dioxide concentration for 24 hours until the cell density reached 1×10 6Cells / ml. Then, 300 μg of wild-type-Fc or variant-Fc expression plasmids were added to 30 ml of culture medium. The mixture was vortexed for 3 s to mix thoroughly. About 1.2 ml (0.5 mg / ml) of transfection reagent PEI MAX 40K (Polysciences, catalog number 24765) was added to the transfection reagent / plasmid mixture. The mixture was kept static for 20 min and then added to HEK293F cells. After transfection, the cells were incubated in a shaking incubator at 37 ° C, 120 rpm and 5% carbon dioxide concentration for 48-54 hours. After incubation, the supernatant of the culture medium was separated by centrifugation and prepared for protein purification.

[0285] 3) Purification and detection of wild-type IL-21-Fc and IL-21 variant-Fc proteins.

[0286] Wild-type IL-21-Fc and IL-21 variant-Fc proteins are purified using protein A agarose microspheres. Appropriate amount of protein A agarose microspheres are added to the supernatant in the previous step, and incubated for 10min at 4 ° C. Wild-type IL-21-Fc and IL-21 variant-Fc proteins attached to protein A agarose microspheres are collected by centrifuging the mixture for 3 minutes at a low speed (1000rpm). Then appropriate volume of washing buffer (50mM PBS, pH 7.4) is added to remove non-specific proteins. Finally, appropriate volume of elution buffer (glycine-HCl, pH 3) is added to elute wild-type-Fc or variant-Fc from microspheres, and pH is adjusted to 7.4, so as to obtain purified wild-type-Fc or variant-Fc.

[0287] The wild-type IL-21 and IL-21 variants designed in Table 1 were prepared in the form of IL-21-Fc. The purified wild-type IL-21-Fc and IL-21 variant-Fc proteins were detected by SDS-PAGE protein gel. 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 show that these IL-21 variant-Fc proteins have higher expression than WT IL21-Fc. The SDS-PAGE protein gel detection results show that the size of the reduced and non-reduced samples of WT IL-21-Fc, m92-Fc, m98-Fc, m99-Fc, m100-Fc and m133-Fc is consistent with the theoretical molecular weight (see Figure 2A-2F ).

[0288] The purity and protein aggregation state of wild-type IL-21-Fc and IL-21 variant-Fc proteins were also detected by size exclusion chromatography (SEC). The SEC results at 280 nm showed that the peaks of wild-type IL-21-Fc, m92-Fc, m98-Fc, m99-Fc, m100-Fc and m133-Fc were all around 8.5 to 9 min. The data indicate that these proteins are monomeric proteins and do not produce aggregated proteins (see Figure 3A-Figure 3F ).

[0289] Table 3 Yields of purified wild-type IL-21-Fc and IL-21 variant-Fc

[0290]

[0291] Alternative preparation 1

[0292] WT IL-21 or the IL-21 variants of the disclosure were also prepared and purified using human IgG4 Fc.

[0293] The full-length amino acid sequences of the IL-21 variants are shown in Table 1. Alignments between the variants and the wild type are shown in Table 2. Exemplary WT IL-21 and IL-21 variants are shown in Tables 1 and 2. They were generated with Fc tags by fusing human IgG4 Fc (SEQ ID NO. 68) to the C-terminus of each WT IL-21 and IL-21 variant, and additionally adding an 18-aa signal peptide (SEQ ID NO. 64) directly at the N-terminus of each mature WT IL-21 and IL-21 variant (the signal peptide was added to facilitate expression in mammalian cell lines and subsequently cleaved in the expression product).

[0294] IgG4 Fc sequence (from N-terminus to C-terminus):

[0295]

[0296]

[0297] (SEQ ID NO.68)

[0298] The sequence of the 18-aa signal peptide is (from N-terminus to C-terminus): MHSSALLCCLVLLTGVRA (SEQ ID NO. 64)

[0299] An exemplary amino acid sequence of the encoded IL-21-IgG4 Fc construct is (from N-terminus to C-terminus):

[0300]

[0301] (Amino acid sequence of WT IL-21-IgG4 Fc construct (SEQ ID NO. 69), wherein the bold segment represents the 18-aa signal peptide; the underlined segment represents the mature IL-21 sequence; and the italic portion represents the human IgG4 Fc tag).

[0302] All variants share the above construct structure with the same signal peptide and IgG4 Fc tag sequence, but each variant has its own unique mature IL-21 sequence as shown in Tables 1 and 2.

[0303] 1) Construction of expression plasmid.

[0304] Based on the amino acid sequence of wild-type IL-21-IgG4 Fc and IL-21 variant-IgG4 Fc, the 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). The plasmid containing the IL-21-IgG4Fc gene was transformed into Escherichia coli DH5α cells. Amplification and plasmid purification were performed by culturing Escherichia coli DH5α cells to obtain a large amount of wild-type plasmids and variant plasmids. 2) Expression of wild-type IL-21-IgG4 Fc and IL-21 variant-IgG4 Fc proteins in CHO cells.

[0305] CHO cells (CHO-K1, Taizhou Biointron Biological, Inc.) were cultured at 0.5×10 6 The cells were inoculated in 30 mL of culture medium (Taizhou Biointron Biological, Inc.) in a 100 mL shake flask at an inoculation volume of 10 cells / ml. The cells were incubated in a shaking incubator at 37°C, 120 rpm, and 5% carbon dioxide concentration for 24 hours until the cell density reached 1.5 × 10 6 Cells / ml. Then, 60 μg wild-type-IgG4 Fc or variant-IgG4 Fc expression plasmids were added to 1 ml culture medium. The mixture was vortexed for 3 s to mix thoroughly. About 15 μl transfection reagent (Taizhou BiointronBiological, Inc.) was added to 1 ml culture medium. The plasmid mixture was added to the transfection reagent mixture. The transfection reagent / plasmid mixture was kept static for 20 min, and then added to the CHO cells. After transfection, the cells were incubated in a shaking incubator at 37 ° C, 120 rpm and 5% carbon dioxide concentration for 48-54 hours. After incubation, the supernatant of the culture medium was centrifuged and prepared for protein purification.

[0306] 3) Purification and detection of wild-type IL-21-IgG4 Fc and IL-21 variant-IgG4 Fc proteins.

[0307] Wild-type IL-21-IgG4 Fc and IL-21 variant-IgG4 Fc protein are purified using protein A agarose microspheres. Appropriate amount of protein A agarose microspheres are added to the supernatant in the previous step, and incubated for 10min at 4 ° C. Wild-type IL-21-IgG4 Fc and IL-21 variant-IgG4 Fc proteins attached to protein A agarose microspheres are collected by centrifuging the mixture for 3 minutes at a low speed (1000rpm). Then appropriate volume of washing buffer (50mM PBS, pH 7.4) is added to remove non-specific proteins. Finally, appropriate volume of elution buffer (glycine-HCl, pH 3) is added to elute wild-type-IgG4 Fc or variant-IgG4 Fc from microspheres, and pH is adjusted to 7.4, so as to obtain the wild-type-IgG4 Fc or variant-IgG4 Fc of purification.

[0308] The wild-type IL-21 and IL-21 variants designed in Table 1 were prepared in the form of IL-21-IgG4 Fc. The purified wild-type IL-21-IgG4 Fc and IL-21 variant-IgG4 Fc proteins were detected by SDS-PAGE protein gel. Quantification is shown in Table 7. The data show that the m98-IgG4 Fc protein has a higher expression than the WT IL-21-IgG4 Fc. The SDS-PAGE protein gel detection results show that the size of the reduced sample and the non-reduced sample of WT IL-21-IgG4 Fc and m98-IgG4 Fc is consistent with the theoretical molecular weight (see Figure 7A-7B ).

[0309] The purity of WT IL-21 IgG4 Fc and m98-IgG4 Fc proteins was also detected by size exclusion chromatography (SEC). The SEC results at 280 nm showed that the peak of WT IL-21 IgG4 Fc was at 8.6 min and the peak of m98-IgG4 Fc was at 8 min. The data indicated that these proteins were monomeric proteins without aggregation (see Figure 8A-B ).

[0310] Table 7 Yields of purified WT IL-21-IgG4 Fc and m98-IgG4 Fc

[0311]

[0312] Alternative preparation 2

[0313] Other well-known tags, such as a His tag, can also be used to prepare and purify WT IL-21 or the IL-21 variants of the disclosure.

[0314] The IL-21 variant was designed to be linked to the C-terminus with a GGGGS linker (SEQ ID NO: 91) and a His6 tag (SEQ ID NO: 81).

[0315] An exemplary sequence for an IL-21-His tag is:

[0316]

[0317] (SEQ ID NO:67; WT IL-21-His, wherein the underlined portion represents the IL-21 sequence; the italic portion represents the G4S linker (SEQ ID NO:91) and the His6 tag (SEQ ID NO:81))

[0318] 1) Construction of expression plasmid.

[0319] Based on the amino acid sequence of wild-type IL-21-His and IL-21 variant-His (including GGGGS joint (SEQ ID NO:91) and His tag), the DNA sequence is synthesized and confirmed by gene sequencing. The confirmed DNA sequence is constructed into the expression vector pcDNA3.1 (Thermo Fisher, catalog number V79020). The plasmid containing the IL-21-His gene is transformed into Escherichia coli DH5α cells. Amplification and plasmid purification are carried out by cultivating Escherichia coli DH5α cells to obtain a large amount of wild-type plasmids and variant plasmids.

[0320] 2) Expression of wild-type IL-21-His and IL-21 variant-His in HEK293F cells.

[0321] The cells were plated at 0.5 × 10 6 The inoculum volume was 10 cells / ml in 300 mL of medium (Gibco TM FreeStyle TM 293 expression medium, catalog number 12338018). The cells were incubated in a shaking incubator at 37°C, 120 rpm and 5% carbon dioxide concentration for 24 hours until the cell density reached 1×10 6Cells / ml. Then, 300 μg of wild-type or mutant expression plasmids were added to 30 ml PBS. The mixture was vortexed for 3 s to mix thoroughly. About 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 kept static for 20 min and then added to HEK293F cells. After transfection, the cells were incubated in a shaking incubator at 37 ° C, 120 rpm and 5% carbon dioxide concentration for 48-54 hours. After incubation, the supernatant of the culture medium was separated by centrifugation and prepared for protein purification.

[0322] 3) Purification and detection of wild-type IL-21-His and IL-21 variant-His.

[0323] Use Ni-NTA agarose microspheres (Thermo Fisher, catalog number R901) to purify wild-type IL-21 and IL-21 variants containing His tags. Appropriate amount of Ni-NTA agarose microspheres are added to the supernatant in the previous step, and incubated for 30min at 4 ° C. The wild-type IL-21 and IL-21 variants attached to the Ni-NTA agarose microspheres are collected by centrifuging the mixture for 3 minutes at a low speed (1000rpm). Then add appropriate volume of washing buffer (50mM PBS, pH 7.4, 10mM imidazoles) to remove non-specific proteins. Finally, add appropriate volume of elution buffer (50mM PBS, pH 7.4, 250mM imidazoles) to wash out wild type or variant from microspheres, so as to obtain purified wild type or variants.

[0324] The wild-type IL-21 and some IL-21 variants designed in Table 1 were prepared in the form of IL-21-His. The purified wild-type IL-21-His and IL-21 variant-His proteins were detected by SDS-PAGE protein gel. The results of SDS-PAGE protein gel detection showed that the sizes of the reduced samples and non-reduced samples of WT IL-21-His, m98-his, m103-his and m153-his were consistent with the theoretical molecular weight (see Figure 9A-9D ).

[0325] Example 2: Recombinant expression and preparation of mutant IL-21-antibody fusion protein.

[0326] IL-21 or IL-21 variants are shown in Table 1 or Table 2. IL-21 or IL-21 variants and antibodies (e.g., anti-PD-1 antibodies) can be fused. In particular, 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.

[0327] 1) Construction of expression plasmid.

[0328] Based on the amino acid sequence of wild-type IL-21 and IL-21 variants, the DNA sequence is synthesized and designed to contain the heavy chain or light chain sequence of the antibody. The sequence is confirmed by gene sequencing. The confirmed DNA sequence is constructed into the expression vector pcDNA3.1 (Thermo Fisher, catalog number V79020). The plasmid containing the IL-21 gene is transformed into Escherichia coli DH5α cells. Amplification and plasmid purification are carried out by cultivating Escherichia coli DH5α cells to obtain a large amount of wild-type-antibody and variant-antibody plasmids.

[0329] 2) Expression of wild-type IL-21-antibody and IL-21 variant-antibody fusion proteins in HEK293F cells.

[0330] The cells were plated at 0.5 × 10 6 The inoculum volume was 10 cells / ml in 300 mL of medium (Gibco TM FreeStyle TM 293 expression medium, catalog number 12338018). The cells were incubated in a shaking 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 PBS. The mixture was vortexed for 3 s to mix thoroughly. About 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 kept static for 20 min and then added to HEK293F cells. After transfection, the cells were incubated in a shaking incubator at 37 ° C, 120 rpm and 5% carbon dioxide concentration for 48-54 hours. After incubation, the supernatant of the culture medium was separated by centrifugation and prepared for protein purification.

[0331] 3) Purification of wild-type IL-21-antibody and IL-21 variant-antibody.

[0332] Use protein A agarose microspheres to purify wild-type IL-21-antibody and IL-21 variant-antibody fusion protein. Appropriate amount of protein A agarose microspheres are added to the supernatant in the previous step, and incubated for 10min at 4 ° C. The wild-type IL-21-antibody and IL-21 variant-antibody fusion protein attached to the protein A agarose microspheres are collected by centrifuging the mixture for 3 minutes at a low speed (1000rpm). Then add the washing buffer (50mM PBS, pH 7.4) of an appropriate volume to remove non-specific protein. Finally, add the elution buffer (glycine-HCl, pH 3) of an appropriate volume to wash out wild-type-antibody or variant-antibody fusion protein from microspheres, so as to obtain the wild-type-antibody or variant-antibody fusion protein of purification.

[0333] The PD-1 antibody-IL-21 fusion protein was constructed so that IL-21 was attached to the C-terminus of the heavy chain of the mouse PD-1 antibody via a linker (GGGGS (SEQ ID NO: 91)). 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 mutants (m98, m100, and m153) and the following anti-mPD1 antibody sequences:

[0334] Anti-mPD1 antibody heavy chain sequence:

[0335] EVQLQESGPGLVKPSQSLSLTCSVTGYSITSSYRWNWIRKFPGNRL

[0336] EWMGYINSAGISNYNPSLKRRISITRDTSKNQFFLQVNSVTTEDAA

[0337] TYYCARSDNMGTTPFTYWGQGTLVTVSSAKTTPPSVYPLAPGSAA

[0338] QTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLY

[0339] TLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCIC

[0340] TVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVAISKDDPEVQFSWFV

[0341] DDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVN

[0342] SAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITD

[0343] FFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG(SEQ ID NO:92)

[0344] Anti-mPD1 antibody light chain sequence:

[0345] DIVMTQGTLPNPVPSGESVSITCRSSKSLLYSDGKTYLNWYLQRPG

[0346] QSPQLLIYWMSTRASGVSDRFSGSGSGTDFTLKISGVEAEDVGIYY

[0347] CQQGLEFPTFGGGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCF

[0348] LNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ ID NO:93)

[0349] In the IL-21_PD-1 antibody fusion protein thus prepared, taking the anti-mPD1 antibody-IL21m98 fusion protein as an example, the full-length sequence of the anti-mPD-1 heavy chain fused with IL-21 is described:

[0350]

[0351]

[0352] (The underlined segment represents the mature IL-21 sequence; the italicized portion represents the flexible linker; and the rest is anti-mPD1 Ab)

[0353] 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 sizes of the reduced and non-reduced samples of the fusion were consistent with the theoretical molecular weight (see Figures 10A-10D ).

[0354] Example 3: Determination of thermal stability of IL-21 mutant proteins.

[0355] To evaluate conformational stability, the melting point Tm (°C) values ​​indicating the structural stability of the samples were obtained by monitoring endogenous tryptophan and tyrosine fluorescence at emission wavelengths of 330 nm and 350 nm.

[0356] The stability of IL-21 mutant proteins was assessed by detecting small changes in tryptophan and tyrosine fluorescence in mutant IL-21 proteins using label-free nanoDSF technology (Prometheus, PR NT.48). In short, wild-type IL-21 or mutant IL-21 proteins were diluted to 0.4 mg / ml in PBS. 10 μL wild-type IL-21 or mutant IL-21 proteins were filled in the capillary and then placed on the sample holder. A temperature gradient of 1 ° C / min was applied from 25 ° C to 95 ° C, and the endogenous protein fluorescence at 330 and 350 nm was recorded. The data were analyzed using the data analysis software provided by the NT.48 instrument.

[0357] As shown in Table 8, m98-His and m153-His exhibited high thermal stability, with Tm of 62.02°C and 63.12°C, respectively, which were significantly higher than WT IL-21-His (Tm 48.47°C).

[0358] Table 8: Melting temperatures (Tm) of wild-type IL-21 and mutant IL-21 determined by label-free nanoDSF technology.

[0359]

[0360] Example 4: Determination of the affinity of mutant IL-21 proteins for human IL-21R.

[0361] By label-free biolayer interferometry ( R8, BLI) to determine the affinity of mutant IL-21 for human IL-21R (ACRO, catalog number: ILR-H5226):

[0362] 1: Wild-type and mutant IL-21-Fc proteins were prepared and diluted with buffer (10 mM PBS + 0.02% TW-20 + 0.1% BSA, pH 7.4) to a final concentration of 5 μg / ml.

[0363] 2: The IL-21R protein was diluted with 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, resulting in a total of 5 concentrations: 1000 nM, 250 nM, 62.5 nM, 15.6 nM and 3.9 nM.

[0364] 3: The protein A sensor was immersed in a buffer solution (10 mM PBS + 0.02% tween-20 + 0.1% BSA, pH 7.4) for 10 minutes for equilibrium.

[0365] 4: IL-21 mutant-Fc protein is loaded onto the protein A sensor. The loading time is 90s. Then the mutant-Fc protein is eluted, and then the balance is 30s. Then the mutant-Fc protein is merged with 4 kinds of IL-21R proteins of different concentrations. The binding time is 120s. Finally, the IL-21R protein bound by buffer elution is used. The dissociation time is 180s.

[0366] 5: Use The Octet Data Acquisition software of R8 was used for data processing and data fitting.

[0367] The affinity data in Table 4 show that the affinity of WT IL-21-Fc for IL-21R (K D ) is 4.57E-10, and the affinity of IL-21 mutant-Fc for IL-21R is lower than that of wild-type IL-21-Fc for IL-21R.

[0368] The interaction of WT IL-21-Fc with IL-21R is "fast Kon, slow Koff". However, the interaction of mutants (such as m92-Fc, m98-Fc, m99-Fc, m100-Fc and m133-Fc) with IL-21R is fast Kon, fast Koff (see Figure 4A-4F ).

[0369] Table 4 Affinity between IL-21-Fc (wild type or mutant) and IL-21R

[0370]

[0371]

[0372] Note: "Very Weak" in Table 4 indicates that the assay data could not be fitted because the affinity was too low to be detected.

[0373] Example 5: Determination of p-STAT3 phosphorylation by IL-21 mutants in HuT78 cells.

[0374] IL-21R is expressed on the surface of HuT78 cell.IL-21 can be combined with IL-21R, thereby stimulate the phosphorylation of Tyr705 of STAT3 protein, and STAT3 protein is the downstream signal transduction protein of IL-21R in HuT78 cell.Utilize phosphorus-STAT3 (Tyr705) test kit / 62AT3PET (Cisbio) to measure the phosphorylation of Tyr705 phosphorylation of STAT3 protein, as the representative of the ability of the downstream signal transduction pathway of evaluation IL-21 mutant activation IL-21R.According to the suggestion of manufacturers, use phosphorus-STAT3 (Tyr705) assay kit to detect STAT3 phosphorylation (Cisbio, catalog number: 64NT3PEH).

[0375] STAT3 phosphorylation assay

[0376] 1) The IL-21-Fc fusion protein was diluted with IMDM complete medium in 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.

[0377] 2) HuT78 cells were seeded at a density of 33,000 cells / well (8 μl) in HTRF 96-well low volume white plates (Cisbio, catalog number 66PL96100), then HuT78 cells were stimulated with 12uL / well IL-21-Fc fusion for 30 minutes at 37°C, and then 4 μl of supplemented lysis buffer (4X) (Cisbio, catalog number 62AT3PET) was added per well and incubated at room temperature for 30 min under shaking. Finally, 4 μl of premix antibody solution (Cisbio, catalog number 62AT3PET) was added and incubated overnight at room temperature.

[0378] 3) FRET signals from the assay were detected using an EnVision Multilable plate reader (Perkin Elmer). Data were analyzed by first determining the HTRF ratio as recommended by Cisbio and then calculating the multiple relative to background using data from unstimulated cells. EC50 values ​​were calculated by fitting the dependent data to a four-parameter logistic model using dotmatics software.

[0379] The results showed that WT IL-21-Fc activated the phosphorylation of STAT3 in HuT78 cells. 50 The affinity of IL-21 mutant-Fc for IL-21R protein was reduced to varying degrees, so IL-21 mutant-Fc activated STAT3 phosphorylation EC in HuT78 cells. 50 The values ​​are relatively large. (See Figure 5 and Table 5).

[0380] Table 5 STAT3 phosphorylation by wild-type IL-21-Fc or IL-21 mutant-Fc in HuT78 cells

[0381]

[0382]

[0383] Example 6: Evaluation of the in vivo anti-tumor activity of IL-21-antibody fusion protein

[0384] The in vivo efficacy of the IL-21-antibody fusion proteins prepared in Example 2 (anti-mPD1 antibody-IL21m98 fusion protein, anti-mPD1 antibody-IL21m100 fusion protein, and anti-mPD1 antibody-IL21m153 fusion protein) was also evaluated and compared with the anti-mPD1 antibody_WT IL21 fusion protein.

[0385] Establish hIL21R KI mouse model, and tumor-bearing mice are divided into control group and experimental group. Control group receives vehicle, and experimental group is further divided into (1) WT IL21_antibody fusion protein; (2) single antibody; and (3) IL21 mutant_antibody fusion protein. In each of the three subgroups, different concentrations and / or dosing regimens are optimized. In order to easily monitor the change of tumor load over time without killing animals, subcutaneous tumors are preferred. Before and after injection of IL21 mutant_antibody conjugates, multiple time points are measured for efficacy and safety: the test / control tumor weight percentage (%T / C) calculated every day for measuring tumors, tumor growth delay, net logarithmic cell killing, median days to tumor weight defined or tumor doubling of a specified number, tumor regression, animal survival rate and basic health status (e.g., body weight and exercise). For non-subcutaneous tumors, tumors are excised and physically weighed, and then the same standard is used to determine tumor boundaries. Alternatively, tumors can be monitored with imaging techniques (e.g., bioluminescence, ultrasound, magnetic resonance imaging).

[0386] Specifically, MC38 mouse colon cancer cells (1×10 6cells) were implanted subcutaneously into C57BL / 6-Il21r tm1(IL21R) / Bcgen mice (Beijing Biocytogen Co., Ltd, stock number: 110766) in the flank. Mice carrying MC38 cells were treated with PBS, 3 mg / kg anti-mPD1 antibody, 3.65 mg / kg anti-mPD1 antibody_WT IL21 fusion protein, 3.65 mg / kg anti-mPD1 antibody_IL21m98 fusion protein, 3.65 mg / kg anti-mPD1 antibody_IL21m100 fusion protein or 3.65 mg / kg anti-mPD1 antibody_IL21m153 fusion protein, twice a week for 2 weeks (4 doses in total). Throughout the study, body weight and tumor volume were measured every 3 or 4 days. Tumor measurements (length (L) and width (W)) were collected three times a week using a digital caliper, and tumor volume (L×W×W) / 2 was calculated. The results are shown in Table 6 below.

[0387] Table 6. Tumor growth inhibition

[0388]

[0389] Figure 6 Figure 2 shows the inhibition of tumor growth in MC38 tumor-bearing mice after treatment with the indicated concentrations of anti-mPD1 antibody_IL21m fusion protein and anti-mPD1 antibody_WT IL21 fusion protein (error bars, SEM). n = 8 mice / group. Two-way ANOVA and Tukey multiple comparisons were performed, and endpoint analysis is shown; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 8 mice / group.

[0390] like Figure 6 As shown in Table 6, 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 with anti-mPD1 antibody and anti-mPD1 antibody_WT IL21 fusion protein.

[0391] Example 7: Evaluation of the in vivo anti-tumor activity of IL-21 protein

[0392] Purified WT IL21-IgG4 Fc and exemplary m98-IgG4 Fc proteins were used in the in vivo studies. The full-length amino acid sequence of mature m98-IgG4 Fc is shown below:

[0393]

[0394]

[0395] (Amino acid sequence of m98-IgG4 Fc construct (SEQ ID NO. 70), wherein the underlined segment represents the mature m98 sequence; the italic part represents the human IgG4 Fc tag.) In other words, SEQ ID NO. 70 does not contain the 18aa signal peptide.

[0396] B16-F10 melanoma cancer cells (5×10 5 cells) were implanted subcutaneously into C57BL / 6-Il21r tm1(IL21R) / Bcgen mice (Beijing Biocytogen Co., Ltd, stock number: 110766) in the flank. Around the 2nd day after inoculation, tumor-bearing mice were divided into 3 groups by random grouping, with 10 mice in each group. Mice carrying B16-F10 cells were treated with PBS, 1.7mg / kg WT IL21-IgG4 Fc protein or 1.7mg / kg m98-IgG4 Fc protein, once every three days for 11 days (a total of 4 doses). Throughout the study, body weight and tumor volume were measured every 2 or 3 days. Tumor measurements (length (L) and width (W)) were collected three times a week using a digital caliper, and tumor volume (L×W×W) / 2 was calculated.

[0397] Table 9. Tumor Growth Inhibition

[0398]

[0399] Fig.11 Figure 1 shows the inhibition of tumor growth in mice bearing B16-F10 tumors after treatment with the indicated concentrations of WT IL21-IgG4 Fc protein and m98-IgG4 Fc protein (error bars, SEM). n = 10 mice / group. Two-way ANOVA and Tukey's multiple comparisons were performed, and endpoint analysis is shown; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 10 mice / group.

[0400] like Fig.11 As shown in Table 9, m98-IgG4 Fc protein significantly further reduced tumor growth of B16-F10 tumors compared to WT IL21-IgG4 Fc protein. In vivo studies showed that the mutant proteins of the present disclosure have favorable tumor inhibitory effects.

[0401] Example 8: Evaluation of the in vivo anti-tumor activity of IL-21 protein

[0402] B16-F10 melanoma cancer cells (5×10 5cells) were implanted subcutaneously into C57BL / 6-Il21r tm1(IL21R) / Bcgen mice (Beijing Biocytogen Co., Ltd, stock number: 110766) in the flank. Around the second day after inoculation, the tumor-bearing mice were divided into 4 groups by random grouping, with 10 mice in each group. Each group of mice carrying B16-F10 cells was treated with PBS, 1.7 mg / kg m98-IgG4 Fc protein, 3 mg / kg anti-mPD1 antibody, or a combination of 1.7 mg / kg m98-IgG4 Fc and 3 mg / kg anti-mPD1 antibody, once every three days for 13 days (on days 0, 3, 6, 9 and 12, a total of 5 doses). Throughout the study, body weight and tumor volume were measured every 2 or 3 days. Tumor measurements (length (L) and width (W)) were collected three times a week using a digital caliper, and tumor volume (L×W×W) / 2 was calculated.

[0403] Table 10. Tumor growth inhibition

[0404]

[0405] Fig.12 Figures show tumor growth curves (error bars, SEM) of mice bearing B16-F10 tumors after treatment with the indicated concentrations of m98-IgG4 Fc protein, anti-mPD1 antibody, and a combination of m98-IgG4 Fc and anti-mPD1 antibody. n = 10 mice / group. Two-way ANOVA and Tukey multiple comparisons were performed, and endpoint analysis is shown; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 10 mice / group.

[0406] like Fig.12 As shown in Table 10, 3 mg / kg anti-mPD1 antibody administered as a single agent showed no anti-tumor activity, and 1.7 mg / kg m98-IgG4 Fc administered as a single agent produced partial anti-tumor activity. However, 3 mg / kg anti-mPD1 antibody combined with 1.7 mg / kg m98-IgG4 Fc produced statistically significant and synergistic anti-tumor activity compared to treatment with anti-mPD1 antibody alone (P<0.001) or 1.7 mg / kg m98-IgG4 Fc (P<0.05).

[0407] Fig.13Figures show tumor weights (error bars, SEM) of mice bearing B16-F10 tumors after treatment with the indicated concentrations of m98-IgG4 Fc protein, anti-mPD1 antibody, and a combination of m98-IgG4 Fc and anti-mPD1 antibody. n = 10 mice / group. Two-way ANOVA and Tukey multiple comparisons were performed, and endpoint analysis is shown; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 10 mice / group.

[0408] like Fig.13 As shown in the figure, the average tumor mass of mice treated with anti-mPD1 antibody (P<0.001) and mice treated with 1.7 mg / kg m98-IgG4 Fc (P<0.05) was significantly greater than that of mice treated with the combination. No abnormal body weight changes or signs of toxicity were observed. These data indicate that m98-IgG4 Fc and combination administration significantly reduced tumor growth in the B16-F10 model.

[0409] In vivo studies show that the muteins of the present disclosure have favorable tumor inhibitory effects, indicating their therapeutic potential as therapeutic agents when used as single agents, in combination, or in multifunctional fusion molecules.

Claims

1. A polypeptide comprising an interleukin-21 (IL-21) variant having an amino acid residue substitution at position P79 corresponding to wild-type human IL-21.

2. The polypeptide of claim 1, wherein the wild-type human IL-21 comprises the sequence set forth in SEQ ID NO: 1 or 2.

3. The polypeptide of claim 1 or 2, wherein the amino acid residue substitution at position P79 is P79E or P79C.

4. The polypeptide of claim 3, wherein the amino acid residue substitution at position P79 is P79E.

5. The polypeptide of claim 3, wherein the amino acid residue substitution at position P79 is P79C, and wherein the polypeptide further comprises one or more amino acid residue substitutions with cysteine ​​in the region corresponding to positions 1-10 of the wild-type human IL-21.

6. The polypeptide of claim 5, wherein the one or more amino acid residue substitutions are selected from the group consisting of R5C, H6C and R9C.

7. The polypeptide according to any one of claims 5 to 6, wherein the cysteine ​​residue substituted by the one or more amino acid residues in the region of positions 1 to 10 forms a disulfide bond together with the cysteine ​​residue at position P79.

8. The polypeptide of any one of claims 1-7, wherein 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, and P78.

9. The polypeptide of claim 8, wherein the polypeptide comprises one or more amino acid residue substitutions at positions selected from the group consisting of S70, K72, K73 and R76.

10. The polypeptide of claim 8, wherein the polypeptide further comprises an amino acid residue substitution at position K73 corresponding to the wild-type human IL-21.

11. The polypeptide of claim 10, wherein the amino acid residue substitution at position K73 is an aromatic amino acid.

12. The polypeptide of claim 10, wherein the amino acid residue substitution at position K73 is K73Y or K73F.

13. The polypeptide of any one of claims 8 to 12, wherein the polypeptide further comprises an amino acid residue substitution at position S70 corresponding to the wild-type human IL-21.

14. The polypeptide of claim 13, wherein the amino acid residue substitution at S70 is S70L or S70A.

15. The polypeptide of any one of claims 8 to 14, wherein the polypeptide further comprises an amino acid residue substitution at position R76 corresponding to the wild-type human IL-21.

16. The polypeptide of claim 15, wherein the amino acid residue substitution at R76 is R76F.

17. The polypeptide of any one of claims 8-16, wherein the polypeptide further comprises an amino acid residue substitution at position K72 corresponding to the wild-type human IL-21.

18. The polypeptide of claim 17, wherein the amino acid residue substitution at K72 is a non-aromatic amino acid comprising a side chain hydroxyl group, and optionally the amino acid residue substitution at K72 is K72S.

19. The polypeptide of claim 8, wherein the polypeptide further comprises an amino acid residue substitution at position Q12 corresponding to the wild-type human IL-21.

20. The polypeptide of claim 19, wherein the amino acid residue substitution at Q12 is Q12W.

21. The polypeptide of claim 8, wherein the polypeptide further comprises an amino acid residue substitution at position L13 corresponding to the wild-type human IL-21.

22. The polypeptide of claim 21, wherein the amino acid residue substitution at L13 is L13A.

23. The polypeptide of claim 8, wherein the polypeptide further comprises an amino acid residue substitution at position D15 corresponding to the wild-type human IL-21.

24. The polypeptide of claim 23, wherein the amino acid residue substitution at D15 is D15L, D15K or D15R.

25. The polypeptide of claim 8, wherein the polypeptide further comprises an amino acid residue substitution at position I16 corresponding to the wild-type human IL-21.

26. The polypeptide of claim 25, wherein the amino acid residue substitution at I16 is I16R or I16W.

27. The polypeptide of claim 8, wherein the polypeptide further comprises an amino acid residue substitution at position P78 corresponding to the wild-type human IL-21.

28. The polypeptide of claim 27, wherein the amino acid residue substitution at P78 is P78G or P78E.

29. A polypeptide comprising an interleukin-21 (IL-21) variant having an amino acid residue substitution at position S70 corresponding to wild-type human IL-21.

30. The polypeptide of claim 29, wherein the wild-type human IL-21 comprises the sequence set forth in SEQ ID NO: 1 or 2.

31. The polypeptide of claim 29 or 30, wherein the amino acid residue substitution at position S70 is S70L or S70A.

32. A polypeptide comprising an interleukin-21 (IL-21) variant having an amino acid residue substitution at position K73 corresponding to wild-type human IL-21.

33. The polypeptide of any one of claims 29-31, wherein the polypeptide further comprises an amino acid residue substitution at position K73 corresponding to the wild-type human IL-21.

34. The polypeptide of claim 32 or 33, wherein the polypeptide comprises a substitution of an amino acid residue with an aromatic amino acid at position K73 corresponding to the wild-type human IL-21.

35. The polypeptide of claim 34, wherein the amino acid residue substitution at position K73 is K73Y or K73F.

36. The polypeptide of any one of claims 32-35, wherein the polypeptide further comprises one, two, or 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.

37. The polypeptide of any one of claims 32-35, wherein the polypeptide further comprises one, two, or three or four amino acid residue substitutions at positions selected from the group consisting of: Q12, D15, I16 and K72.

38. The polypeptide of claim 36, wherein the polypeptide comprises an amino acid residue substitution selected from the group consisting of I16R and I16W.

39. The polypeptide of claim 36, wherein the polypeptide comprises an amino acid residue substitution selected from the group consisting of: D15L, D15K, and D15R.

40. The polypeptide of claim 36, wherein the polypeptide comprises an amino acid residue substitution selected from the group consisting of: K72S and Q12W.

41. The polypeptide of any one of claims 29-31, wherein the polypeptide further comprises an amino acid residue substitution at position R76 corresponding to the wild-type human IL-21.

42. The polypeptide of claim 41, wherein the amino acid residue substitution at position R76 is R76F.

43. The polypeptide of claim 41 or 42, wherein the polypeptide further comprises one, two, or 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.

44. The polypeptide of claim 41 or 42, wherein the polypeptide further comprises one, two, or three or four amino acid residue substitutions at positions selected from the group consisting of: Q12, D15, I16 and K72.

45. The polypeptide of claim 43, wherein the polypeptide comprises an amino acid residue substitution selected from the group consisting of I16R and I16W.

46. ​​The polypeptide of claim 43, wherein the polypeptide comprises an amino acid residue substitution selected from the group consisting of: D15L, D15K, and D15R.

47. The polypeptide of claim 43, wherein the polypeptide comprises an amino acid residue substitution selected from the group consisting of: K72S and Q12W.

48. The polypeptide of any one of the preceding claims, wherein the amino acid stretch STNAGRRQKHR (SEQ ID NO: 71) at positions 80-90 corresponding to the wild-type human IL-21 is substituted with a short peptide linker.

49. The polypeptide of claim 48, wherein the short peptide linker is 4 to 10 or 5 to 9 amino acid residues in length.

50. The polypeptide of claim 48 or 49, wherein the short peptide linker comprises one or more Gly-Ser units and optionally comprises a glutamic acid residue.

51. The polypeptide of claim 50, wherein the short peptide linker comprises the glutamic acid residue, and the glutamic acid residue is located in the middle of the short peptide linker.

52. The polypeptide of claim 49, wherein 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, GGGSGGS (SEQ ID NO: 78) and GGGSEGGGS (SEQ ID NO: 72).

53. 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 substituted with a short peptide linker.

54. The polypeptide of claim 53, wherein the short peptide linker is 4 to 10 or 5 to 9 amino acid residues in length.

55. The polypeptide of claim 53 or 54, wherein the short peptide linker comprises one or more Gly-Ser units and optionally comprises a glutamic acid residue.

56. The polypeptide of claim 55, wherein the short peptide linker comprises the glutamic acid residue, and the glutamic acid residue is located in the middle of the short peptide linker.

57. The polypeptide of claim 54, wherein 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, GGGSGGS (SEQ ID NO: 78) and GGGSEGGGS (SEQ ID NO: 72).

58. The polypeptide of any one of claims 53-57, wherein 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.

59. The polypeptide of claim 58, wherein the polypeptide comprises the amino acid residue substitution at position R5, and optionally the substitution is R5C.

60. The polypeptide of claim 58, wherein the polypeptide comprises the amino acid residue substitution at position H6, and optionally the substitution is H6C.

61. The polypeptide of claim 58, wherein the polypeptide comprises the amino acid residue substitution at position R9, and optionally the substitution is R9C.

62. The polypeptide of claim 58, wherein the polypeptide comprises the amino acid residue substitution at position Q12, and optionally the substitution is Q12W.

63. The polypeptide of claim 58, wherein the polypeptide comprises the amino acid residue substitution at position L13, and optionally the substitution is L13A.

64. The polypeptide of claim 58, wherein the polypeptide comprises the amino acid residue substitution at position D15, and optionally the substitution is D15L, D15K or D15R.

65. The polypeptide of claim 58, wherein the polypeptide comprises the amino acid residue substitution at position I16, and optionally the substitution is I16R or I16W.

66. The polypeptide of claim 58, wherein the polypeptide comprises the amino acid residue substitution at position S70, and optionally the substitution is S70L or S70A.

67. The polypeptide of claim 58, wherein the polypeptide comprises the amino acid residue substitution at position K72, and optionally the substitution is K72S.

68. The polypeptide of claim 58, wherein the polypeptide comprises the amino acid residue substitution at position K73, and optionally the substitution is K73Y or K73F.

69. The polypeptide of claim 58, wherein the polypeptide comprises the amino acid residue substitution at position R76, and optionally the substitution is R76F.

70. The polypeptide of claim 58, wherein the polypeptide comprises the amino acid residue substitution at position P78, and optionally the substitution is P78G or P78E.

71. The polypeptide of claim 58, wherein the polypeptide comprises the amino acid residue substitution at position P79, and optionally the substitution is P79E or P79C.

72. A polypeptide comprising an interleukin-21 (IL-21) variant, wherein the interleukin-21 (IL-21) variant comprises a wild-type human IL-21 amino acid sequence having at least one mutation selected from the group consisting of R5C, H6C, R9C, Q12W, L13A, D15X1, I16X2, S70X3, K72S, K73X4, R76F, P78X5, or P79X6, wherein X1 is L, K, or R, wherein X2 is R or W, wherein X3 is L or A, wherein X4 is Y or F, and X5 is G or E, and wherein X6 is E or C.

73. The polypeptide of claim 72, wherein the wild-type human IL-21 comprises the sequence set forth in SEQ ID NO: 1 or 2.

74. The polypeptide of any one of claims 72-73, wherein the amino acid stretch STNAGRRQKHR (SEQ ID NO: 71) at positions 80-90 corresponding to said wild-type human IL-21 is substituted with a short peptide linker.

75. The polypeptide of claim 74, wherein the short peptide linker is 4 to 10 or 5 to 9 amino acid residues in length.

76. The polypeptide of claim 74 or 75, wherein the short peptide linker comprises one or more Gly-Ser units and optionally comprises a glutamic acid residue.

77. The polypeptide of claim 76, wherein the short peptide linker comprises the glutamic acid residue, and the glutamic acid residue is located in the middle of the short peptide linker.

78. The polypeptide of claim 75, wherein 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).

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

80. A polypeptide comprising an interleukin-21 (IL-21) variant, wherein the polypeptide comprises an amino acid residue mutation selected from the group consisting of: 2) P79C; 3) R5C, P79C; 4) H6C, P79C; 5) R9C, P79C; 6) R5C, R76F, P79C; 7) H6C, R76F, P79C; 8) H6C, K73Y, P79C; 9) H6C, K73F, P79C; 10)H6C, S70L, K73Y, P79C; 11)H6C, S70L, K73F, P79C; 12)R9C, D15R, P78G, P79C; 13)H6C, D15L, I16R, S70L, K73Y, P79C; 14)R9C, D15L, I16R, S70L, K73Y, P79C; 15) H6C, P79C, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72); 16) H6C, K73Y, P79C, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGS (SEQ ID NO: 72); 17) H6C, K73F, P79C, STNAGRRQKHR (SEQ ID NO: 71) / GGGSEGGGS (SEQ ID NO: 72); and 18) H6C, S70L, P79C, STNAGRRQKHR (SEQ ID NO:71) / GGGSEGGS (SEQ ID NO:72).

81. A polypeptide comprising an interleukin-21 (IL-21) variant, wherein the polypeptide comprises an amino acid residue mutation selected from the group consisting of: 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.

82. A polypeptide comprising an interleukin-21 (IL-21) variant, wherein the polypeptide comprises amino acid residue mutations selected from the group consisting of: S70L, K73F, P79E; S70L, K72S, K73F, P79E; S70L, K73Y, P79E; and S70L, K72S, K73Y, P79E.

83. A polypeptide comprising an interleukin-21 (IL-21) variant, wherein the polypeptide comprises a sequence as set forth in any one of SEQ ID Nos. 5-62.

84. A polypeptide comprising an interleukin-21 (IL-21) variant, wherein the polypeptide consists of a sequence as set forth in any one of SEQ ID Nos. 5-62.

85. The polypeptide of any one of the preceding claims, wherein the polypeptide exhibits lower binding affinity for the IL-21 receptor (IL-21R) than the wild-type human IL-21.

86. The polypeptide of claim 85, wherein the binding affinity exhibited by the polypeptide for IL-21R is reduced to 1 / 2 to 1 / 1000 compared to the wild-type human IL-21.

87. The polypeptide of claim 86, wherein the polypeptide has a higher K for IL-21R than the wild-type human IL-21. D value.

88. A conjugate comprising the polypeptide of any preceding claim and at least one additional moiety.

89. The conjugate of claim 88, wherein the at least one additional moiety comprises a crystallizable fragment (Fc) domain of an antibody.

90. The conjugate of claim 89, wherein the antibody is IgG, IgA, IgD, IgM or IgE.

91. The conjugate of claim 90, wherein the IgG is IgG1, IgG2, IgG3 or IgG4.

92. The conjugate of any one of claims 88-91, wherein the at least one additional moiety comprises an antigen binding molecule.

93. The conjugate of claim 92, wherein the antigen binding molecule is an antibody or an antigen binding fragment thereof.

94. The conjugate of claim 93, wherein the antigen binding molecule is a multispecific antigen binding molecule.

95. The conjugate of any one of claims 92-94, wherein the antigen binding molecule targets a tumor cell or an immune cell.

96. The conjugate of claim 95, wherein the antigen is a tumor-associated antigen.

97. The conjugate of claim 95, wherein the antigen is an immune checkpoint antigen or an immune checkpoint-associated antigen.

98. The conjugate of claim 97, wherein the antigen is involved in an immune checkpoint pathway, and optionally the antigen is selected from: PD-1, PD-L1, TIGIT, CTLA-4, PD-L2, B7-H3, B7-H4, BTLA, LAG3, CD112, CD112R, CD96, TIM-3, CD47 and CEACAM1.

99. The conjugate of claim 97, wherein the antigen is a co-stimulatory immune checkpoint target, and optionally the antigen is selected from: CD155, ICOS, OX40, CD137, CD137L, CD27, CD28 and GITR.

100. The conjugate of any one of claims 88-99, wherein the at least one additional moiety is attached to the C-terminus and / or N-terminus of the IL-21 variant.

101. The conjugate of claim 100, wherein the at least one additional moiety is attached to the IL-21 variant directly or via a linker.

102. The polypeptide or conjugate of any one of the preceding claims, wherein the polypeptide exhibits improved thermal stability compared to the wild-type human IL-21.

103. A nucleic acid molecule encoding the polypeptide of any one of claims 1-87 or the conjugate of any one of claims 88-101.

104. A vector comprising the nucleic acid molecule of claim 103.

105. The vector of claim 104, wherein the vector comprises a viral vector.

106. A transformed or host cell expressing a polypeptide as described in any one of claims 1-87 or a conjugate as described in any one of claims 88-101.

107. A pharmaceutical composition comprising the polypeptide of any one of claims 1-87 or the conjugate of any one of claims 88-101 and a pharmaceutically acceptable carrier.

108. A kit comprising the polypeptide, conjugate, nucleic acid molecule, vector, transformed or host cell, or pharmaceutical composition of any of the preceding claims, a combination thereof, and a container.

109. A method for preparing a polypeptide according to claims 1-87 or a conjugate according to claims 88-101, comprising: a) constructing the nucleic acid molecule according to claim 103 and the vector according to claim 104 or 105; b) culturing the transformed or host cell according to claim 106; and c) harvesting the polypeptide from the transformed or host cell.

110. A method of treating a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 107 in an amount effective to treat the subject.

111. The method of claim 110, wherein the subject has or is suspected of having a solid tumor.

112. The nucleic acid molecule of claim 103, wherein the nucleic acid molecule is a DNA molecule.

113. The nucleic acid molecule of claim 103, wherein the nucleic acid molecule is an RNA molecule.

114. The nucleic acid molecule of claim 113, wherein the nucleic acid molecule is an mRNA molecule.

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