Interleukin-21 muteins and methods of treatment

Abstract

Provided herein are IL-21 muteins and fusion proteins comprising the same for use in methods of treating a disease. Related conjugates, nucleic acids, vectors, host cells, pharmaceutical compositions and kits are also provided herein. Methods of making the IL-21 muteins and fusion proteins comprising the same, as well as methods of treating a subject in need thereof, are provided by the present disclosure. Further provided are PD-1 antigen-binding proteins.

Description

[Previous Technology]

[0001] The PD-1 / PD-L1 axis is associated with the suppression of T-cell immune responses in cancer. Antagonists of this pathway have been clinically proven in many solid tumor indications. Nivolumab and pembrolizumab are two such inhibitors targeting the PD-1 pathway, and each has been approved by the US Food and Drug Administration (FDA) for the treatment of metastatic melanoma. Recently, researchers have tested checkpoint inhibition paradigms in other tumor types. Although some progress has been made, checkpoint inhibition therapy remains in the shadow of other cancer treatment options.

[0002] Studies on combinations of checkpoint inhibitors with other agents are ongoing or have recently been completed. For example, a phase III clinical trial tested nivolumab in combination with ipilimumab (a CTLA-4 receptor blocking antibody) in patients with unresectable stage III or IV melanoma. In this study, the highest percentage of patients achieving a complete response was observed in the combination of nivolumab and ipilimumab, outperforming those receiving either drug alone. Other combinations are currently being explored.

[0003] Interleukin-21 (IL-21) is a T-cell-derived pleiotropic cytokine that regulates the activity of innate and adaptive immune cells. IL-21 can enhance T-cell survival and effector function. Because of its key role in antitumor and antiviral responses, in addition to its significant role in inflammatory responses leading to autoimmune and inflammatory diseases, IL-21 has become an attractive target for several therapies.

[0004] However, the development of IL-21-based therapies is complex. Studies demonstrating that enhanced, or confusingly, inhibition of IL-21 action elicits therapeutic effects complicate the research. Additional challenges arise due to the widespread expression of the IL-21 receptor (IL-21R). IL-21R is expressed not only on T cells but also on B cells, NK cells, and bone marrow cells. Therefore, it is essential to carefully limit the widespread activation of IL-21 in leukocytes while avoiding potential toxicity. Limitations to IL-21 signaling must be weighed and selected. Triggering IL-21 action must be designed to occur at the appropriate time and place.

[0005] In fact, there has been no success, especially in clinical practice, of using IL-21 as a monotherapy or in combination with checkpoint inhibitors. Therefore, treatment modalities utilizing IL-21, including combinations of IL-21 with checkpoint inhibitors, remain necessary. IL-21 therapy in combination with immune checkpoint inhibitors also remains needed. [Summary of the Invention]

[0006] The present invention provides an IL-21 mutant protein comprising the amino acid sequence of SEQ ID NO: 2, wherein SEQ ID NO: 2 is QGQDX HMXXM XXXXX XVDXL KNXVN DLVPE FLPAP EDVET NCEWS AFSCF QKAQL KSANT GNNEX XIXXX XXXLX XXXXX TNAGR RQKHR LTCPS CDSYE KKPPK EFLXX FXXLL XXMXX QHXSS RTHGS EDS (SEQ ID NO: 2), and X is any amino acid, and wherein the amino acid sequence of the IL-21 mutant protein differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) in that at least one amino acid is present.

[0007] Therefore, in one embodiment, the present invention also provides an IL-21 mutant protein comprising only one amino acid substitution relative to the wild-type IL-21 amino acid sequence provided herein as SEQ ID NO: 1. In the exemplary embodiment, the amino acid substitution is located at an amino acid position selected from the group consisting of: amino acid position numbers according to SEQ ID NO: 1, 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120, or 123.

[0008] The present invention further provides an IL-21 mutant protein comprising only two amino acid substitutions relative to SEQ ID NO: 1. In the exemplary sample, the amino acid substitutions are located at two amino acid positions selected from the group consisting of: amino acid position numbers according to SEQ ID NO: 1, 5, 9, 15, 70, 71, 72, 73 and 76.

[0009] In exemplary embodiments, the IL-21 mutant protein binds to the IL-21 receptor (IL-21R) with a reduced affinity relative to wild-type IL-21. In exemplary samples, the IL-21 mutant protein binds to human IL-21R with a KD greater than or equal to about 0.04 nM. In exemplary samples, the IL-21 mutant protein binds to cynomolgus monkey IL-21R with a KD greater than or equal to about 0.055 nM.

[0010] The present invention also provides a conjugate comprising the IL-21 mutant protein of the present invention linked to a heterologous portion. In an exemplary sample, the heterologous portion is a polypeptide, thereby making the conjugate a fusion protein. Therefore, the present invention provides a fusion protein comprising the IL-21 mutant protein of the present invention. In an exemplary sample, the fusion protein comprises the IL-21 mutant protein of the present invention linked to an antigen-binding protein such as an antibody or an antigen-binding antibody fragment thereof.

[0011] In a particular embodiment, the fusion protein comprises an IL-21 mutant protein linked to the PD-1 antigen-binding protein of the present invention (e.g., a PD-1 antigen-binding antibody).

[0012] The present invention also provides a PD-1 antigen-binding protein and conjugates and fusion proteins containing the PD-1 antigen-binding protein.

[0013] The present invention further provides nucleic acids comprising a nucleotide sequence encoding the IL-21 mutant protein of the present invention, a PD-1 antigen-binding protein (e.g., a PD-1 antigen-binding antibody), or a fusion protein comprising the IL-21 mutant protein and a PD-1 antigen-binding protein (e.g., a PD-1 antigen-binding antibody). In exemplary samples, the nucleic acid molecule comprises a nucleotide sequence encoding the conjugate or fusion protein of the present invention. Furthermore, this document provides vectors comprising the nucleic acids of the present invention and host cells comprising the nucleic acids of the present invention.

[0014] The present invention further provides a kit comprising the IL-21 mutant protein of the present invention, PD-1 antigen-binding protein (e.g., PD-1 antigen-binding antibody), conjugate, fusion protein (e.g., a fusion protein comprising the IL-21 mutant protein and PD-1 antigen-binding protein (e.g., PD-1 antigen-binding antibody), nucleic acid, vector or host cell or a combination thereof.

[0015] This document provides pharmaceutical compositions comprising the IL-21 mutant protein of the present invention, a PD-1 antigen-binding protein (e.g., a PD-1 antigen-binding antibody), a conjugate, a fusion protein (e.g., a fusion protein comprising the IL-21 mutant protein and a PD-1 antigen-binding protein (e.g., a PD-1 antigen-binding antibody), a nucleic acid, a vector or a host cell or a combination thereof.

[0016] This document provides a method for manufacturing IL-21 mutant protein, PD-1 antigen-binding protein (e.g., PD-1 antigen-binding antibody), and fusion proteins comprising IL-21 mutant protein and PD-1 antigen-binding protein (e.g., PD-1 antigen-binding antibody). In an exemplary embodiment, the method includes culturing host cells of the present invention to express IL-21 mutant protein, PD-1 antigen-binding protein (e.g., PD-1 antigen-binding antibody), or fusion proteins comprising IL-21 mutant protein and PD-1 antigen-binding protein (e.g., PD-1 antigen-binding antibody), and harvesting the expressed IL-21 mutant protein, PD-1 antigen-binding protein (e.g., PD-1 antigen-binding antibody), or fusion proteins comprising IL-21 mutant protein and PD-1 antigen-binding protein (e.g., PD-1 antigen-binding antibody).

[0017] The present invention further provides a method of treatment. In an exemplary embodiment, the method is a method of treating an individual in need, comprising administering to the individual in need an amount of the pharmaceutical composition of the present invention that is effective in treating the individual. In an exemplary embodiment, the individual has a tumor (e.g., a solid tumor, a hematologic malignancy, or a lymphoid malignancy) and the pharmaceutical composition is administered to the individual in an amount effective in treating the tumor. In other exemplary embodiments, the tumor is non-small cell lung cancer (NSCLC) (e.g., stage III or IV NSCLC), small cell lung cancer (SCLC), head and neck cancer, kidney cancer, breast cancer, melanoma, ovarian cancer, liver cancer, pancreatic cancer, colon cancer, prostate cancer, stomach cancer, bladder cancer, hepatocellular carcinoma, cancer with high microsatellite instability (i.e., high MSI cancer), lymphoma, or leukemia.

Implementation Method

[0081] Cross-reference to related applications

[0082] The benefits of U.S. Provisional Patent Application No. 62 / 540,692, filed August 3, 2017, and U.S. Provisional Patent Application No. 62 / 616,733, filed January 12, 2018, are claimed pursuant to 35 USC §119(e), the disclosures of which are hereby incorporated by reference. (Materials submitted electronically are incorporated by reference.)

[0083] The computer-readable nucleotide / amino acid sequence list submitted concurrently with this document and marked as follows is incorporated by full reference: a 975,000-byte ASCII (text) file named "51633_Seqlisting.txt" created on July 25, 2018.

[0084] There remains a need for novel immune enhancement methods that can utilize the immune system to fight cancer cells safely and effectively, especially given that current immunotherapies are only effective in a small number of patients and can have significant and often unpredictable toxicity. In one category, a novel class of bifunctional fusion molecules comprising a PD-1-targeting antibody that blocks the PD-1 / PD-L1 interaction and an engineered, low-affinity interleukin-21 mutant protein disclosed herein fulfills this need. The antibody / cytokine fusions described herein overcome significant obstacles associated with cytokine therapeutics, particularly allowing IL-21 cytokines to be delivered and selectively administered like antibodies in a PD-1-targeting manner. When fused with an anti-PD-1 antibody, the IL-21 mutant protein can be selectively activated and amplified in vivo to express PD-1 T cells. Therefore, the antibody / cytokine fusions described herein can improve and prolong the efficacy of currently clinically tested anti-PD-1 therapeutics.

[0085] Combinations of cytokines with co-inhibitory receptor agonists or antagonists remain challenging due to the risk of increased toxicity and the need for complex clinical trial designs (see, for example, Ott et al., J Immunother Cancer 5, 16 (2017); and Hermel et al., Cancer Metastasis Rev 36, 43-50 (2017)). Cytokines may also activate inhibitory feedback pathways, potentially leading to immunosuppression (see, for example, Portielje et al., Clin Cancer Res 9, 76-83 (2003); Wan et al., Immunity 38, 514-527 (2013); and Mooradian et al., Oncoimmunology 7, e1423172 (2018)). Interleukin-21 (IL-21), a type I cytokine and a member of the common cytokine receptor γ-chain (cg-chain) cytokine family, has shown promise as an immunotherapeutic agent for cancer treatment. IL-21 is produced by activated CD4 T cells and natural killer T (NKT) cells, and its signaling occurs via a heterodimeric receptor complex composed of discrete IL-21 receptor (IL-21R) subunits associated with the common γ chain (see, for example, Spolski et al., Nat Rev Drug Discov 13, 379-395 (2014)). Activation of the IL-21R complex leads to activation of the JAK / STAT signaling pathway. IL-21R is widely expressed in hematopoietic cells, including T and B lymphocytes, natural killer (NK) cells, and bone marrow cells. Although not an essential growth or differentiation factor, IL-21 is a potent mitogen and survival factor for NK cells and activated T cells. IL-21 supports the differentiation of CD4(+) T helper 17 (Th17) cells and follicular helper T cells (Tfh) and antagonizes regulatory T cell (Treg) differentiation. In addition, IL-21 can enhance the survival of CD8 T cells and protect less activated but more durable T cell phenotypes, which allows for enhanced tumor and virus control.

[0086] One challenge of cytokine immunotherapy is that, in addition to activating immune cells to enhance the immune response, the same cytokines can also activate inverse regulatory pathways. For example, IL-2 and IFNγ can activate protective immune responses and regulate T cell responses and inhibitory pathways (such as PD-L1), respectively. In dendritic cells (DCs), IL-21 can inhibit DC maturation and activation, induce apoptosis in known DCs, strongly inhibit T cell activation in mixed cultures, and play a role in inducing tolerability. In humans, IL-21 has been tested as a non-targeted free cytokine in several cancer indications, but despite promising preclinical and early Phase I clinical data, the development of this approach has not progressed further than Phase II testing (see, for example, Thompson et al., J Clin Oncol 26, 2034-2039 (2008); and Davis et al., Clin Cancer Res 15, 2123-2129 (2009)). In more recent preclinical models, the combination of recombinant IL-21 cytokine with co-inhibitory receptor antagonists (e.g., anti-CTLA-4 and anti-PD-1) has demonstrated that IL-21 can prolong the efficacy of these treatments. Such combinations are currently in clinical trials, but their clinical efficacy remains to be confirmed (Lewis et al., Oncoimmunology 7, e1377873 (2017)).

[0087] Unbound by theoretical constraints, the antibody / cytokine fusions described herein are designed to utilize the immunomodulatory activity of IL-21 (essential for mitigating toxicity and off-target immunosuppression), maximize efficacy, and improve clinical feasibility. IL-21 and IL-21 mutant protein

[0088] Interleukin-21 (IL-21) is a cytokine expressed by T cells, B cells, NK cells, and bone marrow cells. It regulates the activity of innate and adaptive immune cells and improves T cell survival and effector function. Several Phase I and II clinical trials have included IL-21 as an investigational drug for the treatment of cancer, inflammatory diseases, and autoimmune diseases, including melanoma, renal cell carcinoma, acute myeloid leukemia, non-Hodgkin's lymphoma, ovarian cancer, colorectal cancer, systemic lupus erythematosus, Crohn's disease, and rheumatoid arthritis.

[0089] IL-21 has a four-helix bundle structure and exists as a monomer. In humans, two isoforms of IL-21 are known, each derived from a precursor molecule. The first IL-21 isoform contains 162 amino acids (aa), of which the first 29 amino acids constitute the signal peptide; and the second IL-21 isoform contains 153 aa, of which, as in the first isoform, the first 29 amino acids constitute the signal peptide. The amino acid sequences (including the signal peptide) of the first and second IL-21 isoforms are provided herein as SEQ ID NO: 258 and SEQ ID NO: 259, respectively.

[0090] IL-21 binds to the heterodimeric IL-21 receptor (IL-21R) expressed on the surface of T cells, B cells, and NK cells. The structure of IL-21R is similar to that of the IL-2 and IL-15 receptors because each of these intercellular receptors contains a common γ chain (γc). In addition to γc, IL-21R contains an α chain, which is important for binding to IL-21. Two isoforms of the human IL-21 receptor α chain exist: isoform 1 and isoform 2. The amino acid sequences of isoform 1 and isoform 2 are provided herein as SEQ ID NO: 256 and 261, respectively. The amino acid sequence of the common human γ chain is provided herein as SEQ ID NO: 257.

[0091] When IL-21 binds to IL-21R, the Jak / STAT signaling pathway is activated, thereby activating the target gene. While inducing IL-21 signaling is therapeutically desirable, the timing and location of signaling should be carefully considered given the broad spectrum of IL-21's manifestations and its ability to enhance CD8 T cell responses and inhibit antigen presentation and T cell activation. The data presented here for the first time support the use of carefully designed IL-21 mutant proteins to achieve IL-21 signaling at the appropriate time and place.

[0092] The present invention provides an IL-21 mutant protein comprising at least one amino acid substitution relative to the wild-type IL-21 amino acid sequence provided herein as SEQ ID NO: 1. For example, the IL-21 mutant protein comprises at least one and at most 34 amino acid substitutions. In an exemplary sample, the IL-21 mutant protein comprises at least one and at most X amino acid substitutions, wherein X is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34. In exemplary embodiments, the IL-21 mutant protein comprises an amino acid sequence that differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by up to 10, 15, 20, or 25 amino acids. In exemplary embodiments, the IL-21 mutant protein comprises an amino acid sequence that differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by up to 7 or up to 5 amino acids. In exemplary embodiments, the IL-21 mutant protein comprises an amino acid sequence that differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 3, 4, 5, or 6 amino acids. In exemplary embodiments, the IL-21 mutant protein comprises an amino acid sequence that differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 3 to 6 amino acids or 1 to 5 amino acids. In an exemplary embodiment, the IL-21 mutant protein comprises an amino acid sequence that differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by one or two amino acids.

[0093] In the exemplary sample, the IL-21 mutant protein contains the amino acid sequence of SEQ ID NO: 2, wherein SEQ ID NO: 2 is QGQDX HMXXM XXXXX XVDXL KNXVN DLVPE FLPAP EDVET NCEWS AFSCF QKAQL KSANT GNNEX XIXXX XXXLX XXXXX TNAGR RQKHR LTCPS CDSYE KKPPK EFLXX FXXLL XXMXX QHXSS RTHGS EDS (SEQ ID NO: 2), wherein X represents any amino acid, and wherein the amino acid sequence of the IL-21 mutant protein differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by at least one amino acid.

[0094] Therefore, in the exemplary sample, the IL-21 mutant protein comprises the sequence of SEQ ID NO: 2, wherein the difference between SEQ ID NO: 2 and SEQ ID NO: 1 is at least one amino acid at the position indicated by X in SEQ ID NO: 2. In the exemplary sample, the IL-21 mutant protein comprising SEQ ID NO: 2 has at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with SEQ ID NO: 1. In the illustrative variant, the IL-21 mutant protein comprises an amino acid sequence that is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) identical to the sequence of SEQ ID NO: 1.

[0095] In an exemplary embodiment, the IL-21 mutant protein comprises an amino acid sequence that includes at least one amino acid substitution relative to the wild-type IL-21 amino acid sequence, and the amino acid substitution occurs within the N-terminal half of the amino acid sequence. For example, the amino acid substitution occurs within positions 5-25 or 8-23 (inclusive) of the amino acid position number according to SEQ ID NO: 1.

[0096] In an exemplary embodiment, the IL-21 mutant protein comprises an amino acid sequence that includes at least one amino acid substitution relative to the wild-type IL-21 amino acid sequence, and the amino acid substitution occurs within the C-terminal half of the amino acid sequence. For example, the amino acid substitution occurs within positions 100-133 or 109-123 (inclusive) of the amino acid position number according to SEQ ID NO: 1.

[0097] In an exemplary embodiment, the IL-21 mutant protein comprises an amino acid sequence that includes at least one amino acid substitution relative to the wild-type IL-21 amino acid sequence, and the amino acid substitution occurs within the middle third of the amino acid sequence. For example, the amino acid substitution occurs within positions 55-85 or 65-80 (inclusive) of the amino acid position number according to SEQ ID NO: 1.

[0098] The present invention also provides an IL-21 mutant protein comprising only one amino acid substitution relative to the wild-type IL-21 amino acid sequence provided herein as SEQ ID NO: 1. In the exemplary sample, the amino acid substitution is located at an amino acid position selected from the group consisting of: amino acid position numbers according to SEQ ID NO: 1, 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120, or 123. In other exemplary samples, the amino acid substitutions are located at positions selected from the group consisting of: amino acid positions numbered according to SEQ ID NO: 1, 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120, or 123. In other exemplary samples, the IL-21 mutant protein comprises any of the amino acid sequences of SEQ ID NO: 3-21 and 23-37.

[0099] The present invention further provides an IL-21 mutant protein comprising only two amino acid substitutions relative to SEQ ID NO: 1. In exemplary samples, the amino acid substitutions are located at two amino acid positions selected from the group consisting of: amino acid position numbers according to SEQ ID NO: 1, 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120, or 123. In other exemplary samples, the amino acid substitutions are located at two amino acid positions selected from the group consisting of: amino acid position numbers according to SEQ ID NO: 1, 5, 9, 15, 70, 71, 72, 73, and 76. In other exemplary samples, amino acid substitutions are located at two amino acid positions selected from the group consisting of: amino acid positions 5, 9, 73, and 76 according to SEQ ID NO: 1. In exemplary samples, at least one of the two amino acid substitutions is located at position 76 according to amino acid position number 76 according to SEQ ID NO: 1. In exemplary samples, the IL-21 mutant protein comprises any of the amino acid sequences of SEQ ID NO: 199-208 and 210-212.

[0100] In an exemplary embodiment, the IL-21 mutant protein comprises an amino acid sequence that includes at least one amino acid substitution relative to the wild-type IL-21 amino acid, and the amino acid substitution is a conserved amino acid substitution. As used herein, the term "conservative amino acid substitution" refers to the substitution of one amino acid with another amino acid having similar properties, such as size, charge, hydrophobicity, hydrophilicity, and / or aromaticity, and includes an exchange within one of the following five groups: I. Small aliphatic, nonpolar, or micropolar residues: Ala, Ser, Thr, Pro, Gly; II. Polar, negatively charged residues and their acetylamines and esters: Asp, Asn, Glu, Gln, sulfonylanamine, and homosulfonylanamine; III. Polar, positively charged residues: His, Arg, Lys; ornithine (Orn); IV. Large aliphatic, nonpolar residues: Met, Leu, Ile, Val, Cys, leucine (Nle), homocysteine; V. Large aromatic residues: Phe, Tyr, Trp, acetylated phenylalanine.

[0101] In exemplary embodiments, the IL-21 mutant protein comprises an amino acid sequence that includes at least one amino acid substitution relative to the wild-type IL-21 amino acid, and the amino acid substitution is a non-conservative amino acid substitution. As used herein, the term "non-conservative amino acid substitution" is defined herein as the substitution of one amino acid with another amino acid having different properties, such as size, charge, hydrophobicity, hydrophilicity, and / or aromaticity, and includes exchanges outside the above five groups.

[0102] In the exemplary sample, the IL-21 mutant protein comprises an amino acid sequence containing at least one amino acid substitution relative to the wild-type IL-21 amino acid sequence, and the substituted amino acid is a naturally occurring amino acid. "Naturally occurring amino acid," "standard amino acid," or "typical amino acid" means one of the 20 α-amino acids (Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp, Ser, Thr, Asn, Gln, Cys, Gly, Pro, Arg, His, Lys, Asp, Glu) found directly encoded by codons of the universal genetic code in eukaryotes. In the exemplary sample, the IL-21 mutant protein comprises an amino acid sequence containing at least one amino acid substitution relative to the wild-type IL-21 amino acid sequence, and the substituted amino acid is a non-standard amino acid or an amino acid not incorporated into the protein during translation. Non-standard amino acids include (but are not limited to): selenocysteine, pyrrolizidine, ornithine, leucine, β-amino acids (e.g., β-alanine, β-aminoisobutyric acid, β-phenylalanine, β-homophylline, β-glutamic acid, β-glutamic acid, β-homotrylamine, β-leucine, β-lysine), homoamino acids (e.g., homophenylalanine, homoserine, homoarginine, monocysteine, homocysteine), N-methylamino acids (e.g., L-caryophylline, N-methylalanine, N-methylisoleucine, N-methylleucine), 2-aminooctanoic acid, 7-aminocephalosporanic acid, 4-aminocinnamic acid, α-aminocyclohexanepropionic acid, amino-(4-hydroxyphenyl)acetic acid, 4-aminonicotinic acid, 3-aminophenylacetic acid and their analogues.

[0103] In an exemplary embodiment, the IL-21 mutant protein of the present invention comprises an amino acid sequence having at least one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), wherein the amino acid substitution is at one or more of positions 5, 8, 9, 12, 14, 15, 65, 66, 69, 70, 72, 73, 75, 76, 77, 80, 116 and 119 of SEQ ID NO: 1, and the substituted amino acid is an aliphatic amino acid. In the illustrative example, the IL-21 mutant protein of the present invention comprises an amino acid sequence having only one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is located at one or more of positions 5, 8, 9, 12, 14, 15, 65, 66, 69, 70, 72, 73, 75, 76, 77, 80, 116 or 119 of SEQ ID NO: 1, and the substituted amino acid is an aliphatic amino acid.

[0104] In an exemplary embodiment, the IL-21 mutant protein of the present invention comprises an amino acid sequence having at least one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), wherein the amino acid substitution is at one or more of positions 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 69, 70, 72, 73, 75, 76, 77, 78, 79, 110, 112, 116, 117, 119, 120 or 123 of SEQ ID NO: 1, and the substituted amino acid is an acidic amino acid. In the illustrative example, the IL-21 mutant protein of the present invention comprises an amino acid sequence having only one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is located at one or more of positions 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 69, 70, 72, 73, 75, 76, 77, 78, 79, 110, 112, 116, 117, 119, 120, or 123 of SEQ ID NO: 1, and the substituted amino acid is an acidic amino acid.

[0105] In an exemplary embodiment, the IL-21 mutant protein of the present invention comprises an amino acid sequence having at least one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), wherein the amino acid substitution is at one or more of positions 5, 9, 73, 76, 109, 113, or 116 of SEQ ID NO: 1, and the substituted amino acid is a basic amino acid. In an exemplary embodiment, the IL-21 mutant protein of the present invention comprises an amino acid sequence having only one amino acid substitution relative to SEQ ID NO: 1, and the amino acid at positions 5, 9, 73, 76, 109, 113, or 116 of SEQ ID NO: 1 is a basic amino acid.

[0106] In an exemplary embodiment, the IL-21 mutant protein of the present invention comprises an amino acid sequence having at least one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), wherein the amino acid substitution is at one or more of positions 5, 8, 9, 70, or 76 of SEQ ID NO: 1, and the substituted amino acid is an aromatic amino acid. In an exemplary embodiment, the IL-21 mutant protein of the present invention comprises an amino acid sequence having only one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is at positions 5, 8, 9, 70, or 76 of SEQ ID NO: 1, and the substituted amino acid is an aromatic amino acid.

[0107] In an exemplary embodiment, the IL-21 mutant protein of the present invention comprises an amino acid sequence having at least one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), wherein the amino acid substitution is at one or more of positions 5, 8, 9, 12, 15, 73, 76, 116, or 119 of SEQ ID NO: 1, and the substituted amino acid is an amino acid containing a side-chain amide. In an exemplary embodiment, the IL-21 mutant protein of the present invention comprises an amino acid sequence having only one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is at one or more of positions 5, 8, 9, 12, 15, 73, 76, 116, or 119 of SEQ ID NO: 1, and the substituted amino acid is an amino acid containing a side-chain amide.

[0108] In an exemplary embodiment, the IL-21 mutant protein of the present invention comprises an amino acid sequence having at least one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), wherein the amino acid substitution is at one or more of positions 5, 8, 9, 11, 12, 14, 15, 73, 76, 116, or 119 of SEQ ID NO: 1, and the substituted amino acid is an amino acid containing a side-chain hydroxyl group. In an exemplary embodiment, the IL-21 mutant protein of the present invention comprises an amino acid sequence having only one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is at one or more of positions 5, 8, 9, 11, 12, 14, 15, 73, 76, 116, or 119 of SEQ ID NO: 1, and the substituted amino acid is an amino acid containing a side-chain hydroxyl group.

[0109] In an exemplary embodiment, the IL-21 mutant protein of the present invention comprises an amino acid sequence having at least one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), wherein the amino acid substitution is at one or more of positions 65, 66, 69, 70, 72, 73, 75, 76, 77, or 80 of SEQ ID NO: 1, and the substituted amino acid is an imine. In an exemplary embodiment, the IL-21 mutant protein of the present invention comprises an amino acid sequence having only one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is at one or more of positions 65, 66, 69, 70, 72, 73, 75, 76, 77, or 80 of SEQ ID NO: 1, and the substituted amino acid is an amino acid comprising an imine.

[0110] In an exemplary embodiment, the IL-21 mutant protein of the present invention comprises an amino acid sequence having at least one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), wherein the amino acid substitution is at one or more of positions 5, 9, 15, 76, 116, or 119 of SEQ ID NO: 1, and the substituted amino acid is an amino acid containing a sulfur-containing side chain. In an exemplary embodiment, the IL-21 mutant protein of the present invention comprises an amino acid sequence having only one amino acid substitution relative to SEQ ID NO: 1, wherein the amino acid substitution is at positions 5, 9, 15, 76, 116, or 119 of SEQ ID NO: 1, and the substituted amino acid is an amino acid containing a sulfur-containing side chain.

[0111] In the exemplary sample, the IL-21 mutant protein of the present invention comprises an amino acid sequence having at least one amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1), wherein the at least one amino acid substitution is shown in Table A. Table A amino acid position of SEQ ID NO: 1 Examples of substituted amino acids (by single-letter code) Exemplary SEQ ID NO: 5 A, D, E, G, H, I, K, L, M, N, Q, S, T, V, Y 38 8 A, D, E, G, N, S 39 9 A, D, E, G, H, I, K, L, M, N, Q, S, T, V, Y 40 11 D, S 41 12 A, D, E, N, S, T, V 42 13 D 43 14 A, D, S 44 15 A,E,I,M,N,Q,S,T,V 45 16 D, E 46 19 D 47 23 D 48 65 D、G、P 49 66 D、G、P 50 68 Q 51 69 D、G、P 52 70 E、G、P、Y、T 53 71 L 54 72 A、D、G、P 55 73 A、D、E、G、H、I、N、P、Q、S、V 56 75 D、G、P 58 76 A、D、E、G、H、I、K、L、M、N、P、Q、S、T、V、Y 59 77 D、G、P 60 78 D 61 79 D 62 80 G、P 63 109 K 64 110 D 65 112 D 66 113 K 67 116 A、D、E、I、K、L、M、N、S、T、V 68 117 D 69 119 A, D, E, M, N, Q, S, T 70 120 D 71 123 D 72

[0112] In an exemplary embodiment, the IL-21 mutant protein of the present invention comprises an amino acid sequence having only one amino acid substitution relative to SEQ ID NO: 1, and the amino acid substitution is one of the amino acid substitutions shown in Table A. In other embodiments, the IL-21 mutant protein of the present invention comprises an amino acid sequence having two amino acid substitutions relative to SEQ ID NO: 1, and the amino acid substitution is one of two of the amino acid substitutions shown in Table A.

[0113] In the exemplary sample, the IL-21 mutant protein of the present invention comprises the amino acid sequences shown in Table B. Table B Amino acid substitution amino acid position of SEQ ID NO: 1 Substituted amino acids SEQ ID NO: Amino acid substitution amino acid position of SEQ ID NO: 1 Substituted amino acids SEQ ID NO: R5A 5 A 73 S70E 70 E 136 R5D 5 D 74 S70G 70 G 137 R5E 5 E 75 S70P 70 P 138 R5G 5 G 76 S70Y 70 Y 139 R5H 5 H 77 S70T 70 T 140 R5I 5 I 78 K72D 72 D 141 R5K 5 K 79 K72G 72 G 142 R5L 5 L 80 K72P 72 P 143 R5M 5 M 81 K72A 72 A 144 R5N 5 N 82 K73A 73 A 145 R5Q 5 Q 83 K73D 73 D 146 R5S 5 S 84 K73E 73 E 147 R5T 5 T 85 K73G 73 G 148 R5V 5 V 86 K73H 73 H 149 R5Y 5 Y 87 K73I 73 I 150 I8A 8 A 88 K73N 73 N 151 I8D 8 D 89 K73P 73 P 152 I8E 8 E 90 K73Q 73 Q 153 I8G 8 G 91 K73S 73 S 154 I8N 8 N 92 K73V 73 V 155 I8S 8 S 93 K75D 75 D 156 R9A 9 A 94 K75G 75 G 157 R9D 9 D 95 K75P 75 P 158 R9E 9 E 96 R76A 76 A 159 R9G 9 G 97 R76D 76 D 160 R9H 9 H 98 R76E 76 E 161 R9I 9 I 99 R76G 76 G 162 R9K 9 K 100 R76H 76 H 163 R9L 9 L 101 R76I 76 I 164 R9M 9 M 102 R76K 76 K 165 R9N 9 N 103 R76L 76 L 166 R9Q 9 Q 104 R76M 76 M 167 R9S 9 S 105 R76N 76 N 168 R9T 9 T 106 R76P 76 P 169 R9V 9 V 107 R76Q 76 Q 170 R9Y 9 Y 108 R76S 76 S 171 R11D 11 D 109 R76T 76 T 172 R11S 11 S 110 R76V 76 V 173 Q12A 12 A 111 R76Y 76 Y 174 Q12D 12 D 249 K77D 77 D 175 Q12E 12 E 250 K77G 77 G 176 Q12N 12 N 251 K77P 77 P 177 Q12S 12 S 252 P78D 78 D 61 Q12T 12 T 253 P79D 79 D 62 Q12V 12 V 254 S80G 80 G 178 L13D 13 D 112 S80P 80 P 179 I14A 14 A 114 E109K 109 K 64 I14D 14 D 115 R110D 110 D 65 I14S 14 S 116 K112D 112 D 66 D15A 15 A 117 S113K 113 K 67 D15E 15 E 118 Q116A 116 A 180 D15I 15 I 119 Q116D 116 D 181 D15M 15 M 120 Q116E 116 E 182 D15N 15 N 121 Q116I 116 I 183 D15Q 15 Q 122 Q116K 116 K 184 D15S 15 S 123 Q116L 116 L 185 D15T 15 T 283 Q116M 116 M 186 D15V 15 V 124 Q116N 116 N 187 I16D 16 D 125 Q116S 116 S 188 I16E 16 E 126 Q116T 116 T 189 Q19D 19 D 47 Q116V 116 V 190 Y23D 23 D 48 K117D 117 D 69 R65D 65 D 127 I119A 119 A 191 R65G 65 G 128 I119D 119 D 192 R65P 65 P 129 I119E 119 E 193 I66D 66 D 130 I119M 119 M 194 I66G 66 G 131 I119N 119 N 195 I66P 66 P 132 I119Q 119 Q 196 N68Q 68 Q 51 I119S 119 S 197 V69D 69 D 133 I119T 119 T 198 V69G 69 G 134 H120D 120 D 71 V69P 69 P 135 L123D 123 D 72

[0114] In an exemplary embodiment, the IL-21 mutant protein of the present invention comprises the amino acid sequence of any one of SEQ ID NO: 47, 48, 51, 61, 62, 64-67, 69, 71-112, 114-198, 249-254 or 283. In the illustrative phenotype, the IL-21 mutant protein comprises an amino acid sequence that is at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identical to one of SEQ ID NO: 47, 48, 51, 61, 62, 64-67, 69, 71-112, 114-198, 249-254, or 283.

[0115] The present invention further provides an IL-21 mutant protein comprising only two amino acid substitutions relative to SEQ ID NO: 1, wherein the two amino acid substitutions occur at two of positions 5, 9, 15, 70, 71, 72, 73, and 76 of SEQ ID NO: 1. In an exemplary embodiment, the IL-21 mutant protein comprises only two amino acid substitutions relative to SEQ ID NO: 1, wherein the two substitutions occur at a pair of amino acid positions selected from the group consisting of: 5 and 76; 9 and 76; 15 and 70; 15 and 71; 15 and 72; 15 and 73; 70 and 73; 70 and 76; 71 and 73; 71 and 76; 72 and 73; 72 and 76; and 73 and 76. In an exemplary embodiment, the IL-21 mutant protein comprises any one of the amino acid sequences of SEQ ID NO: 199-208 and 210-212. In other embodiments, the present invention further provides an IL-21 mutant protein having only two amino acid substitutions relative to SEQ ID NO: 1, wherein the two amino acid substitutions occur at positions 5, 9, 73, and 76 of SEQ ID NO: 1. In an exemplary embodiment, one of the substitutions occurs at position 76 of SEQ ID NO: 1. In an exemplary embodiment, the substituted amino acid at position 76 of SEQ ID NO: 1 is an aliphatic amino acid or an acidic amino acid. In an exemplary embodiment, the aliphatic amino acid is alanine. In an exemplary embodiment, the acidic amino acid is aspartic acid or glutamic acid. In an exemplary embodiment, the acidic amino acid is glutamic acid. In an exemplary embodiment, the IL-21 mutant protein comprises the substituted amino acid at position 76 of SEQ ID NO: 1 and the aliphatic amino acid or acidic amino acid at positions 5, 9, or 73 of SEQ ID NO: 1. In the exemplary samples, the substituted amino acid at positions 5, 9, or 73 is an aliphatic amino acid, an acidic amino acid, or an amino acid with a side-chain amide. In the exemplary samples, the aliphatic amino acid is alanine, the acidic amino acid is glutamic acid, and the amino acid with a side-chain amide is glutamic acid. In the exemplary samples, the IL-21 mutant protein includes the substituted amino acid (aliphatic or acidic, as appropriate) at position 76 of SEQ ID NO: 1 and the substituted amino acid at position 5 or 9 (according to SEQ ID NO: 1).

[0116] In the exemplary samples, the IL-21 mutant protein of the present invention comprises the amino acid sequence of any of the SEQ ID NO: shown in Table C. Table C Amino acid substitution The position of the first amino acid in SEQ ID NO: 1 First-substituted amino acid The position of the second amino acid in SEQ ID NO: 1 Second-substituted amino acid SEQ ID NO: R5E, R76E 5 E 76 E 239 R5E, R76A 5 E 76 A 238 R5A, R76A 5 A 76 A 236 R5Q, R76A 5 Q 76 A 240 R5A, R76E 5 A 76 E 237 R5Q, R76E 5 Q 76 E 241 R9E, R76E 9 E 76 E 245 R9A, R76E 9 A 76 E 243 R9E, R76A 9 E 76 A 244 R9A, R76A 9 A 76 A 242 D15N、S70T 15 N 70 T 213 D15N、I71L 15 N 71 L 214 D15N、K72A 15 N 72 A 215 D15N、K73A 15 N 73 A 216 S70T、K73Q 70 T 73 Q 219 S70T、R76A 70 T 76 A 246 S70T、R76D 70 T 76 D 247 S70T、R76E 70 T 76 E 248 I71L、K73Q 71 L 73 Q 217 I71L、R76A 71 L 76 A 227 I71L、R76D 71 L 76 D 228 I71L、R76E 71 L 76 E 229 K72A、K73Q 72 A 73 Q 218 K72A、R76A 72 A 76 A 230 K72A、R76D 72 A 76 D 231 K72A、R76E 72 A 76 E 232 K73A、R76A 73 A 76 A 233 K73A、R76D 73 A 76 D 234 K73A、R76E 73 A 76 E 235

[0117] In the exemplary sample, the IL-21 mutant protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 213-219 and 227-248. In the exemplary sample, IL-21 comprises an amino acid sequence having at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with one of SEQ ID NO: 213-219 and 227-248. Peptide length

[0118] The IL-21 mutant protein described herein may comprise a peptide backbone having any number of amino acids, i.e., any peptide length. In some embodiments, the peptide length described herein is nearly the same as that of SEQ ID NO: 1, i.e., 133 (± about 1 to about 20, ± about 1 to about 15, ± about 1 to about 10, or ± about 1 to about 5) amino acids. In some embodiments, the peptide length of the present invention is longer than 133 amino acids due to fusion with another polypeptide chain, such as an antibody heavy chain comprising about 400 to about 600 amino acids or an antibody light chain comprising about 150 to about 300 amino acids, as further described herein. Additional peptide modifications

[0119] In alternative or additional embodiments of the invention, the IL-21 mutant protein is lipidized (e.g., myristylated, palmitylated), glycosylated, amylated, carboxylated, phosphorylated, esterified, amylated, acetylated, cyclized, or converted to an acid addition salt and / or, where appropriate, dimerized, polymerized, or conjugated, as further described herein. Pharmaceutically acceptable salts.

[0120] In the exemplary samples, the IL-21 mutant protein is presented as a salt, such as a pharmaceutically acceptable salt. Such salts can be prepared on-site during the final isolation and purification of the IL-21 mutant protein, or separately prepared by reacting a free basic functional group with a suitable acid. Examples of acids that can be used to form pharmaceutically acceptable acid addition salts include, for example, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid; and organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid.

[0121] Representative acid addition salts include (but are not limited to) acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, diglucose, glyceryl phosphate, hemisulfate, heptanate, hexanoate, transbutenedioate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (hydroxyethanesulfonate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmitate, pectinate, persulfate, 3-phenylpropionate, picrate, pentanoate, propionate, succinate, tartrate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate, and undecanoate.

[0122] Base addition salts can also be prepared on-site during the final isolation and purification of the IL-21 mutant protein, or by reacting the carboxylic acid-containing portion with a suitable base, such as a hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, or with ammonia or an organic primary, secondary, or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, alkali metal or alkaline earth metal-based cations, such as lithium, sodium, potassium, calcium, magnesium, and aluminum salts and their analogues; and non-toxic quaternary ammonia and amine cations, including ammonium, tetramethylammonium, tetraethylammonium, methylammonium, dimethylammonium, trimethylammonium, triethylammonium, diethylammonium, and ethylammonium. Other representative organic amines that can be used to form base addition salts include, for example, ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and their analogues.

[0123] Furthermore, the basic nitrogen-containing group can be quaternarily ammonized using surfactants such as: low-carbon alkyl halides, such as methyl, ethyl, propyl, and butyl chlorides, methyl, ethyl, propyl, and butyl bromides, and methyl, ethyl, propyl, and butyl iodides; long-chain halides, such as decyl chloride, lauryl chloride, myristyl chloride, and stearyl chloride, decyl bromide, lauryl bromide, myristyl bromide, and stearyl bromide, as well as decyl iodide, lauryl iodide, myristyl iodide, and stearyl iodide; aryl alkyl halides, such as benzyl bromide and phenethyl bromide, and others. This yields water-soluble or oil-soluble or water-dispersible or oil-dispersible products. Purification

[0124] The IL-21 mutant protein of the present invention can be purified. As used herein, the term "purification" means an increase in purity, where "purity" is a relative term and not necessarily absolute purity. In illustrative samples, the purity of the compound (e.g., in the composition) is at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 95%, or at least or about 98%, or about 100%. Peptide mimics

[0125] In some forms, the IL-21 mutant protein is a peptide mimic, or at least a portion thereof is a peptide mimic. Peptide mimics and methods of their manufacture are known in the art. See, for example, Advances in Amino Acid Mimetics and Peptidomimetics, Vol. 1 & 2, edited by Abell, A., JAI Press Inc., Greenwich, CT, 2006. In some forms, the peptide mimic is a D-peptide mimic containing a D-isomer amino acid. In some forms, the peptide mimic is a peptide-like substance in which the side chain of the amino acid is attached to the α-nitrogen atom of the peptide backbone. Methods of manufacturing peptide-like substances are known in the art. See, for example, Zuckermann et al., JACS 114(26): 10646-10647 (1992) and Design, Synthesis, and Evaluation of Novel Peptoids, Fowler, Sarah, University of Wisconsin-Madison, 2008. In some forms, the peptide mimic is a β-peptide containing a β-amino acid with an amino group bonded to the β-carbon rather than the α-carbon. Methods for manufacturing β-peptides are known in this art. See, for example, Seebach et al., Helvetica Chimica Acta 79(4): 913-941 (1996). Binding characteristics

[0126] In an exemplary embodiment, the IL-21 mutant protein binds to the IL-21 receptor (IL-21R) with a reduced affinity relative to wild-type IL-21 for IL-21R. In an exemplary embodiment, the IL-21 mutant protein binds to human IL-21R with a reduced affinity relative to wild-type human IL-21 for the α chain of human IL-21R. In an exemplary embodiment, the IL-21 mutant protein binds to the α chain of human IL-21R with a reduced affinity relative to wild-type human IL-21 for the α chain of human IL-21R. In certain embodiments, the IL-21 mutant protein, which binds to the α chain of human IL-21R with a reduced affinity relative to wild-type human IL-21, contains one, two, or more substitutions at amino acid positions selected from the group consisting of: amino acid positions numbered according to SEQ ID NO: 1, 5, 8, 9, 12, 13, 16, 19, 23, 65, 66, 69, 70, 72, 73, 75, 76, 77, 78, 79, and 80. Specific amino acid substitutions that may be made at such positions are discussed herein (see, for example, Tables A, B, and C).

[0127] In exemplary embodiments, the IL-21 mutant protein binds to the γ chain of human IL-21R with a reduced affinity relative to that of wild-type human IL-21. In specific embodiments, the IL-21 mutant protein binding to the γ chain of human IL-21R with a reduced affinity relative to that of wild-type human IL-21 contains one, two, or more substitutions at amino acid positions selected from the group consisting of: amino acid position numbers according to SEQ ID NO: 1, 11, 14, 15, 109, 110, 112, 113, 116, 117, 119, 120, and 123. Specific amino acid substitutions that may be made at such positions are discussed herein (see, for example, Tables A, B, and C).

[0128] In an exemplary embodiment, the IL-21 mutant protein binds to the γ chain of human IL-21R with a reduced affinity relative to the α chain of wild-type IL-21. In an exemplary embodiment, the IL-21 mutant protein binds to cynomolgus monkey IL-21R with a reduced affinity relative to the α chain of wild-type cynomolgus monkey IL-21R. In an exemplary embodiment, the IL-21 mutant protein binds to the α chain of cynomolgus monkey IL-21R with a reduced affinity relative to the α chain of wild-type cynomolgus monkey IL-21R. In an exemplary embodiment, the IL-21 mutant protein binds to the γ chain of cynomolgus monkey IL-21R with a reduced affinity relative to the γ chain of wild-type cynomolgus monkey IL-21R. In an exemplary embodiment, the IL-21 mutant protein binds to the γ chain of cynomolgus monkey IL-21R with a reduced affinity relative to the α chain of wild-type cynomolgus monkey IL-21.

[0129] The IL-21 mutant protein provided herein binds to IL-21R in a non-covalent and reversible manner. In exemplary embodiments, the binding strength of the mutant protein to IL-21R can be described by its affinity, which is a measure of the strength of the interaction between the binding site of the mutant protein and IL-21R. In exemplary samples, the IL-21 mutant protein provided herein has a high affinity for IL-21R and therefore binds a larger amount of IL-21R in a shorter time than the low-affinity IL-21 mutant protein. In exemplary samples, the IL-21 mutant protein provided herein has a low affinity for IL-21R and therefore binds a smaller amount of IL-21R in a longer time than the high-affinity IL-21 mutant protein. In the exemplary samples, the IL-21 mutant protein has an equilibrium association constant KA of at least 10⁵ M⁻¹, at least 10⁶ M⁻¹, at least 10⁷ M⁻¹, at least 10⁸ M⁻¹, at least 10⁹ M⁻¹, or at least 10¹⁰ M⁻¹. As will be known to those skilled in the art, KA can be affected by factors including pH, temperature, and buffer composition.

[0130] In exemplary embodiments, the binding strength of the IL-21 mutant protein to IL-21R can be described by its sensitivity. KD is the equilibrium dissociation constant between the IL-21 mutant protein and IL-21R, the ratio of koff / kon. KD is inversely correlated with KA. The KD value is related to the concentration of the mutant protein (the amount of mutant protein required for a specific experiment), therefore, the lower the KD value (the lower the required concentration), the higher the affinity of the mutant protein. In exemplary samples, the binding strength of the IL-21 mutant protein to IL-21R can be described by KD. In exemplary samples, the KD of the IL-21 mutant protein provided herein is about 10⁻¹M, about 10⁻²M, about 10⁻³M, about 10⁻⁴M, about 10⁻⁵M, about 10⁻⁶M or less. In exemplary samples, the KD of the IL-21 mutant protein provided herein is micromolar, nanomolar, picomor, or femtomor. In the illustrative samples, the KD of the IL-21 mutant protein provided herein is in the range of about 10⁻⁴ to 10⁻⁶ M, or 10⁻⁷ to 10⁻⁹ M, or 10⁻¹⁰ to 10⁻¹² M, or 10⁻¹³ to 10⁻¹⁵ M. In the illustrative samples, the IL-21 mutant protein binds to human IL-21R with a KD greater than or equal to about 0.04 nM. In the illustrative samples, the IL-21 mutant protein binds to human IL-21R with a KD of about 0.01 nM to about 20 nM, 0.02 nM to 20 nM, 0.05 nM to 20 nM, 0.05 nM to 15 nM, 0.1 nM to 15 nM, 0.1 nM to 10 nM, 1 nM to 10 nM, or 5 nM to 10 nM. In the illustrative sample, the IL-21 mutant protein bound to cynomolgus monkey IL-21R with a KD greater than or equal to about 0.055 nM. In the illustrative sample, the IL-21 mutant protein bound to cynomolgus monkey IL-21R with KD values ​​of about 0.01 nM to about 20 nM, 0.02 nM to 20 nM, 0.05 nM to 20 nM, 0.05 nM to 15 nM, 0.1 nM to 15 nM, 0.1 nM to 10 nM, 1 nM to 10 nM, or 5 nM to 10 nM.

[0131] In exemplary embodiments, the IL-21 mutant protein exhibits a reduced affinity for the IL-21R α-chain. In exemplary samples, the IL-21 mutant protein exhibits a reduced affinity for the IL-21R α-chain by approximately 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 175, 200, 225, 250, and 2... 75x, 300x, 325x, 350x, 375x, 400x, 425x, 450x, 475x, 500x, 525x, 550x, 575x, 600x, 625x, 650x, 675x, 700x, 725x, 750x, 775x, 800x, 825x, 850x, 875x, 900x, 925x, 950x, 975x, 1000x or more mutant proteins (e.g., single or dual). In the illustrative morphology, the IL-21 mutant protein is a dual mutant protein exhibiting a reduced affinity for the binding affinity to the IL-21R α-chain.

[0132] In the exemplary samples, the decreased binding affinity of the aforementioned IL-21 mutant protein (e.g., single or double mutant protein) to the IL-21R α-chain compared to the affinity of wild-type human IL-21 to the IL-21R α-chain at approximately 0.025 nM resulted in a decrease in affinity to the IL-21R α-chain. Therefore, a two-fold decrease in affinity as discussed above would result in an affinity of the IL-21 mutant protein to the IL-21R α-chain of approximately 0.05 nM. Therefore, in exemplary embodiments, the IL-21 mutant protein (e.g., single or dual) has approximately 0.05 nM, 0.125 nM, 0.25 nM, 0.375 nM, 0.5 nM, 0.625 nM, 0.75 nM, 0.875 nM, 1.0 nM, 1.125 nM, 1.25 nM, 1.375 nM, 1.5 nM, 1.625 nM, 1.75 nM, 1.875 nM, 2.0 nM, 2.125 nM, 2.25 nM, 2.375 nM, 2.5 nM, 2.625 nM, 2.75 nM, 2.875 nM, 3.0 nM, 3.125 nM, 3.25 nM, 3.375 nM, 3.5 nM, 3.625 nM, 3.75 nM, etc. nM, 4.375 nM, 5 nM, 5.625 nM, 6.25 nM, 6.875 nM, 7.5 nM, 8.125 nM, 8.75 nM, 9.375 nM, 10.0 nM, 10.625 nM, 11.25 nM, 11.875 nM, 12.5 nM, 13.125 nM, 13.75 nM, 14.375 nM, 15.0 nM, 15.625 nM, 16.25 nM, 16.875 nM, 17.5 nM, 18.125 nM, 18.75 nM, 19.375 nM, 20.0 nM, 20.625 nM, 21.25 nM, 21.875 nM, 22.5 nM, 23.125 Affinities for the IL-21R α-chain of nM, 23.75 nM, 24.375 nM, 25 nM, or more are observed. In the exemplary morphology, the IL-21 mutant protein is a double mutant protein exhibiting reduced binding affinity for the IL-21R α-chain.

[0133] In exemplary embodiments, the IL-21 mutant protein exhibited reduced activity as measured by in vitro STAT3 phosphorylation analysis. In exemplary samples, the IL-21 mutant protein, as measured by STAT3 phosphorylation analysis, showed approximately 2-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold, 105-fold, 110-fold, 115-fold, 120-fold, 125-fold, 130-fold, 135-fold, 140-fold, 145-fold, 150-fold, 175-fold, and 200-fold reductions. 225-fold, 250-fold, 275-fold, 300-fold, 325-fold, 350-fold, 375-fold, 400-fold, 425-fold, 450-fold, 475-fold, 500-fold, 525-fold, 550-fold, 575-fold, 600-fold, 625-fold, 650-fold, 675-fold, 700-fold, 725-fold, 750-fold, 775-fold, 800-fold, 825-fold, 850-fold, 875-fold, 900-fold, 925-fold, 950-fold, 975-fold, 1000-fold, or more of reduced activity mutant proteins (e.g., single or dual). In exemplary samples, as measured by STAT3 phosphorylation analysis, the IL-21 mutant protein is a dual mutant protein exhibiting reduced activity. IL-21 mutant protein conjugates

[0134] The present invention also provides conjugates comprising one or more of the IL-21 mutant proteins of the present invention linked to a heterologous portion. As used herein, the term "heterologous portion" is synonymous with the term "binding portion" and refers to any molecule (chemical or biochemical, naturally occurring or not encoded) that is different from the IL-21 mutant protein described herein. Exemplary binding portions that may be linked to any of the IL-21 mutant proteins described herein include (but are not limited to) heteropeptides or polypeptides (including, for example, immunoglobulins or portions thereof (e.g., variable regions, CDRs or Fc regions)), targets, diagnostic markers (such as radioisotopes, fluorescent or enzyme labels), polymers comprising water-soluble polymers, or other therapeutic or diagnostic agents. In some embodiments, conjugates comprising the IL-21 mutant protein of the present invention and immunoglobulins are provided. In some embodiments, the conjugate comprises one or more of the IL-21 mutant proteins described herein and one or more of the following: peptides (different from the IL-21 mutant proteins described herein), polypeptides, nucleic acid molecules, antibodies or fragments thereof, polymers, quantum dots, small molecules, toxins, diagnostic agents, carbohydrates, and amino acids.

[0135] In exemplary embodiments, the conjugates of the present invention comprise an IL-21 mutant protein as described herein and a heterologous portion, the heterologous portion being a polypeptide (e.g., a polypeptide different from any of the IL-21 mutant proteins described herein), and the conjugate is a fusion polypeptide or fusion protein or chimeric protein or chimeric polypeptide. Further description of such conjugates is provided herein under "fusion proteins".

[0136] In some embodiments, the heterologous portion is linked to the IL-21 mutant protein of the present invention via non-covalent or covalent bonds. In exemplary samples, the bond between the IL-21 mutant protein and the heterologous portion is achieved via covalent chemical bonds, such as peptide bonds, disulfide bonds, and the like, or via physical forces, such as electrostatic, hydrogen, ionic, van der Waal, or hydrophobic or hydrophilic interactions. A variety of non-covalent coupling systems can be used, including, for example, biotin-avidin, ligand / receptor, enzyme / receptor, nucleic acid / nucleic acid binding protein, lipid / lipid binding protein, cell adhesion molecule conjugates; or any conjugates or fragments thereof with affinity for each other.

[0137] In an exemplary embodiment, the IL-21 mutant protein is attached to the binding moiety via a direct covalent bond by reacting the target amino acid residues of the IL-21 mutant protein with an organic derivatizing agent capable of reacting with selected side chains or N- or C-terminal residues of such target amino acids. Reactive groups on the IL-21 mutant protein or the binding moiety include, for example, aldehydes, amino groups, esters, thiols, α-haloacetyl groups, maleic anhydride groups, or hydrazide groups. Derivatizing agents include, for example, maleic anhydride benzoylsulfosuccinimide (bound via cysteine ​​residues), N-hydroxysuccinimide (bound via lysine residues), glutaraldehyde, succinic anhydride, or other reagents known in the art. Alternatively, the binding moiety may be indirectly attached to the IL-21 mutant protein via an intermediate carrier, such as a polysaccharide or peptide carrier. Examples of polysaccharide carriers include aminoglucan. Examples of suitable peptide carriers include polylysine, polyglutamic acid, polyaspartic acid, copolymers thereof, and mixed polymers of these amino acids with other amino acids such as serine, to compare the desired solubility properties of the resulting carriers.

[0138] Cysteine ​​residues most commonly react with α-haloacetic esters (and corresponding amines), such as chloroacetic acid and chloroacetamide, to give carboxymethyl or carboxyaminomethyl derivatives. Cysteine ​​residues are also derivatized by reactions with bromotrifluoroacetone, α-bromo-β-(5-imidazolyl)propionic acid, chloroacetyl phosphate, N-alkylcis-butene diimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuric benzoate, 2-chloromercuric-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0139] The histidine acetyl residue is derived by reacting with diethyl pyrocarbonate at pH 5.5-7.0, as this reagent is relatively specific to the histidine acetyl side chain. It is also applicable to bromobenzoylmethyl bromide; the reaction is preferably carried out in 0.1 M sodium dimethylarsinate at pH 6.0.

[0140] The lysine group and amino-terminal residues react with succinic acid or other carboxylic anhydrides. Derivatization with these reagents reverses the charge of the lysine group residues. Other suitable reagents for derivatizing residues containing α-amino groups include imine esters, such as methyl pyridinium imide, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactions with glyoxylate esters.

[0141] Arginine residues are modified by reacting with one or more conventional reagents, particularly benzoxaldehyde, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. The derivatization of arginine residues requires the reaction to be carried out under basic conditions because the guanidine functional group has a high pKa. Furthermore, these reagents can react with lysine and the ε-amino group of arginine.

[0142] Specific modifications can be made to tyramine residues, with particular interest in introducing spectral labeling into tyramine residues through reaction with aromatic diazo compounds or tetranitromethane. Most commonly, N-acetylimazole and tetranitromethane are used to form O-acetylityramine compounds and 3-nitro derivatives, respectively.

[0143] The carboxyl side group (aspartic acid or glutamine) is selectively modified by reacting with carbodiimide (RN=C=N-R'), where R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholino-4-ethyl)carbodiimide or 1-ethyl-3-(4-azacation-4,4-dimethylphenyl)carbodiimide. Furthermore, the aspartic acid and glutamine residues are converted into aspartic acid and glutamine residues by reacting with ammonium ions.

[0144] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of serine or threonine residues, methylation of the α-amino groups of the side chains of lysine, arginine and histidine (TE Creighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)), deacetylation of aspartic acid or glutamic acid, acetylation of the N-terminal amine and / or acetylation or esterification of the C-terminal carboxylic acid group.

[0145] Another type of covalent modification involves chemically or enzymatically coupling glycosides to the IL-21 mutant protein. The sugar may be attached to (a) arginine and histidine; (b) a free carboxyl group; (c) a free sulfhydryl group, such as the free sulfhydryl group of cysteine; (d) a free hydroxyl group, such as the free hydroxyl group of serine, threonine, or hydroxyproline; (e) an aromatic residue, such as an aromatic residue of tyrosine or tryptophan; or (f) an amide group of glutamic acid. These methods are described in WO87 / 05330, published September 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0146] In exemplary samples, the heterologous portion is attached to the IL-21 mutant protein of the present invention via a linker. In some samples, the linker comprises a chain having a length of 1 to about 60, 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms. In some embodiments, all chain atoms are carbon atoms. In some embodiments, the chain atoms in the linker backbone are selected from the group consisting of C, O, N, and S. The chain atoms and linker may be selected according to their intended solubility (hydrophilicity) to provide a more soluble conjugate. In some embodiments, the linker provides functional groups that are readily cleaved by enzymes or other catalysts or hydrolytic conditions found in the target tissue or organ or cell. In some embodiments, the linker is long enough to reduce the possibility of steric hindrance. If the linker is a covalent or peptide bond and the conjugate is a polypeptide, the entire conjugate may be a fusion protein. Such peptide-based linkers may be of any length. Exemplary peptide linkers are about 1 to 50 amino acids long, 5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids long, and are flexible or rigid. In an exemplary sample, the linker is a peptide containing about 2 to about 20 amino acids. In an exemplary sample, the linker is a peptide containing about 2 to about 15 amino acids, about 2 to about 10 amino acids, or about 2 to about 5 amino acids. Suitable peptide linkers are known in this art. See, for example, Chen et al., Adv Drug Delivery Reviews 65(10): 1357-1369 (2013); Arai et al., Protein Eng Des Sel 14(8): 529-532 (2001); and Wriggers et al., Curr Trends in Peptide Science 80(6): 736-746 (2005). In the exemplary sample, the linker is a peptide containing the amino acid sequence GGGGS (SEQ ID NO: 262). Fusion protein

[0147] In exemplary embodiments, the IL-21 mutant protein is linked to a polypeptide different from any of the IL-21 mutant proteins described herein, and the conjugate is a fusion polypeptide, fusion protein, chimeric protein, or chimeric polypeptide. Therefore, the present invention provides a fusion polypeptide or fusion protein comprising the IL-21 mutant protein of the present invention and a heterologous polypeptide or peptide. In exemplary samples, the fusion protein of the present invention comprises the IL-21 mutant protein of the present invention linked to an antigen-binding protein. In exemplary samples, the antigen-binding protein is an antibody or immunoglobulin, or an antigen-binding antibody fragment or antibody protein product thereof.

[0148] Generally, antibodies are formed from the plasma protein family of immunoglobulins and consist of immunoglobulin domains. (Janeway et al., Immunobiology: The Immune System in Health and Disease, 4th ed., Elsevier Science Ltd. / Garland Publishing, 1999). As used herein, the term "antibody" refers to a protein having a known immunoglobulin structure, comprising heavy and light chains and containing variable and constant regions. For example, an antibody can be IgG, which has a "Y-shaped" structure with two pairs of identical polypeptide chains, each pair having a "light" chain (typically with a molecular weight of about 25 kDa) and a "heavy" chain (typically with a molecular weight of about 50-70 kDa). Antibodies have variable and constant regions. In the IgG format, the variable region is generally about 100-110 or more amino acids, containing three complementarity-determining regions (CDRs), primarily responsible for antigen recognition, and varies substantially in other antibodies binding to different antigens. The constant region allows the antibody to recruit cells and molecules of the immune system. The variable region is formed by the N-terminal regions of each light chain and heavy chain, while the constant region is formed by the C-terminal regions of each heavy chain and light chain. (Janeway et al., "Structure of the Antibody Molecule and the Immunoglobulin Genes", Immunobiology: The Immune System in Health and Disease, 4th edition, Elsevier Science / Garland Publishing, (1999)).

[0149] The general structure and properties of antibody CDRs have been described in this technique. In short, in the antibody backbone, CDRs are embedded within a framework of heavy and light chain variable regions, wherein these CDRs constitute the regions primarily responsible for antigen binding and recognition. The variable regions typically contain at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342: 877-883) within framework regions (referred to as framework regions 1-4, FR1, FR2, FR3, and FR4, Kabat et al., 1991; see also Chothia and Lesk, 1987, above).

[0150] Antibodies may contain any constant regions known in this art. Human light chains are divided into κ and λ light chains. Heavy chains are divided into μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including (but not limited to) IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including (but not limited to) IgM1 and IgM2. Embodiments of the present invention include all such classes or isotypes of antibodies. Light chain constant regions may be, for example, κ or λ type light chain constant regions, such as human κ or λ type light chain constant regions. Heavy chain constant regions may be, for example, α, δ, ε, γ, or μ type heavy chain constant regions, such as human α, δ, ε, γ, or μ type heavy chain constant regions. Therefore, in exemplary embodiments, the antibody is an antibody of the same type IgA, IgD, IgE, IgG or IgM, including any one of IgG1, IgG2, IgG3 or IgG4.

[0151] The antibody may be a monoclonal antibody or a polyclonal antibody. In some embodiments, the antibody comprises a sequence substantially similar to that of naturally occurring antibodies produced by mammals, such as mice, rabbits, goats, horses, chickens, hamsters, humans, and similar animals. In this regard, the antibody may be considered a mammalian antibody, such as mouse antibodies, rabbit antibodies, goat antibodies, horse antibodies, chicken antibodies, hamster antibodies, human antibodies, and similar animal antibodies. In some forms, the antibody is a human antibody. In some forms, the antibody is a chimeric antibody or a humanized antibody. The term "chimeric antibody" refers to an antibody containing domains from two or more different antibodies. A chimeric antibody may, for example, contain a constant domain from one species and a variable domain from a second species, or more generally, may contain extensions of amino acid sequences from at least two species. Chimeric antibodies may also contain domains from two or more different antibodies within the same species. The term "humanized," in relation to the use of the antibody, refers to an antibody having at least a CDR region from a non-human source, which is engineered to have a structure and immune function more similar to a real human antibody than the original source antibody. For example, humanization may include transplanting a CDR from a non-human antibody, such as a mouse antibody, into a human antibody. Humanization may also include selecting amino acid substitutions to make a non-human sequence more similar to a human sequence.

[0152] Antibodies can be cleaved into fragments by enzymes such as papain and pepsin. Papain cleaves the antibody to produce two Fab fragments and a single Fc fragment. Pepsin cleaves the antibody to produce an F(ab')2 fragment and a pFc' fragment. In an exemplary embodiment of the present invention, the fusion protein of the present invention comprises an antigen-binding antibody fragment. As used herein, the term "antigen-binding antibody fragment" refers to a portion of an antibody molecule capable of binding to an antibody antigen and is also called an "antigen-binding fragment" or "antigen-binding moiety". In an exemplary case, the antigen-binding antibody fragment is a Fab fragment or an F(ab')2 fragment.

[0153] Antibody construction has been used to generate an ever-growing range of alternative formats, spanning at least the molecular weight range of about 12-150 kDa and having valences (n) ranging from monomers (n = 1), dimers (n = 2), trimers (n = 3), tetramers (n = 4), and possibly higher; such alternative formats are referred to herein as "antibody protein products." Antibody protein products include products based on the complete antibody structure and products that mimic antibody fragments while retaining complete antigen-binding capacity, such as scFv, Fab, and VHH / VH (discussed below). The smallest antigen-binding antibody fragment retaining a complete antigen-binding site is the Fv fragment, which consists entirely of a variable (V) region. Soluble, flexible amino peptide linkers are used to connect the V region to the scFv (single-chain variable fragment) fragment to stabilize the molecule, or to add a constant (C) domain to the V region to generate the Fab fragment [antigen-binding fragment]. Both scFv and Fab fragments are readily generated in host cells, such as prokaryotic host cells. Other antibody protein products include disulfide-stabilized scFv (ds-scFv), single-chain Fab (scFab), and dimer and multimer antibody formats, such as bifunctional, trifunctional, and tetrafunctional antibodies, or mini-abs comprising scFvs linked to oligomeric domains. The smallest fragments are VHH / VH of camel heavy chain Abs and single-domain Abs (sdAbs). The most commonly used building blocks for generating novel antibody formats are single-chain variable (V) domain antibody fragments (scFv), which contain V domains (VH and VL domains) from the heavy and light chains linked by peptide linkers of about 15 amino acid residues. Peptides or peptide-Fc fusions are another type of antibody protein product. The structure of a peptide body consists of a bioactive peptide grafted onto the Fc domain. Peptides are well described in this technique. See, for example, Shimamoto et al., mAbs 4(5): 586-591 (2012).

[0154] Other antibody protein products include single-chain antibodies (SCA); bifunctional antibodies; trifunctional antibodies; tetrafunctional antibodies; bispecific or trispecific antibodies and their analogues. Bispecific antibodies can be divided into five categories: BsIgG, attached IgG, BsAb fragments, bispecific fusion proteins, and BsAb conjugates. See, for example, Spiess et al., Molecular Immunology 67(2) Part A: 97-106 (2015).

[0155] In the exemplary samples, the fusion protein of the present invention comprises any of these antibody protein products. In the exemplary samples, the fusion protein of the present invention comprises any of scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibody, polyantibody (e.g., bifunctional antibody, trifunctional antibody, tetrafunctional antibody), miniAb, peptide VHH / VH of camel heavy chain antibody, sdAb, bifunctional antibody; trifunctional antibody; tetrafunctional antibody; bispecific or trispecific antibody, BsIgG, additional IgG, BsAb fragment, bispecific fusion protein, and BsAb conjugate.

[0156] In exemplary cases, the fusion protein of the present invention comprises an antibody protein product in monomeric or polymeric, oligomeric or polymeric form. In some embodiments where the antibody comprises two or more different antigen-binding region fragments, the antibody is considered bispecific, trispecific or multispecific, or divalent, trivalent or multivalent, depending on the number of different antigenic determinants recognized and bound by the antibody.

[0157] In exemplary embodiments, the antibody, antigen-binding antibody fragment, or antibody protein product binds to a tumor antigen. In exemplary samples, the tumor antigen is an antigen derived from a viral protein, an antigen derived from a point mutation, or an antigen encoded by an oncogene. In exemplary samples, the tumor antigen is p53, KRAS, NRAS, MAGEA, MAGEB, MAGEC, BAGE, GAGE, LAGE / NY-ESO1, SSX, tyrosinase, gp100 / pmel17, Melan-A / MART-1, gp75 / TRP1, TRP2, CEA, RAGE-1, HER2 / NEU, or WT1. In exemplary samples, the antibody, antigen-binding antibody fragment, or antibody protein product of the fusion protein of the present invention binds to an immunotherapeutic agent or is an immunotherapeutic agent, as described herein. In exemplary embodiments, the antibody, antigen-binding antibody fragment, or antibody protein product of the fusion protein of the present invention binds to interleukins, lymphokines, growth factors, or hematopoietic factors, as described herein.

[0158] In exemplary embodiments, the fusion protein of the present invention comprises a cytokine (e.g., the IL-21 mutant protein described herein) and an antibody, antigen-binding antibody fragment, or antibody protein product thereof that binds to a protein, tumor antigen, cytokine, lymphokine, growth factor, or other hematopoietic factor (including (but not limited to) any of the substances described herein) of an immune checkpoint pathway. In exemplary embodiments, the fusion protein of the present invention comprises a cytokine (e.g., the IL-21 mutant protein described herein) and an antibody, antigen-binding antibody fragment, or antibody protein product thereof that binds to a protein of an immune checkpoint pathway selected from the group consisting of: CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, CEACAM-1, TIGIT, LAG3, CD112, CD112R, CD96, TIM3, BTLA, or a co-stimulatory receptor: ICOS, OX40, 41BB, CD27, GITR.

[0159] In other embodiments, the fusion protein of the present invention comprises a cytokine and an antibody (or an antigen-binding antibody fragment thereof) that binds to a protein in the immune checkpoint pathway. Suitable cytokines include, for example, cytokines that enhance TH-1 type responses; and cytokines that activate STAT1, STAT3, STAT4, or STAT5. In some embodiments, the cytokine is an interleukin. In other embodiments, the cytokine is an interleukin that enhances T cell activity, such as IL-2, IL-7, IL-10, IL-12, IL-15, or IL-21. Such cytokines can be modified (e.g., via mutation) to reduce their affinity for their corresponding receptors. By reducing off-target and unwanted interactions, such mutant proteins can exhibit an improved safety profile. Therefore, cytokines can be modified to produce IL-2, IL-7, IL-10, IL-12, IL-15, or IL-21 mutant proteins. In a particular embodiment, the cytokine is the IL-21 mutant protein described herein. Suitable antibodies (or antigen-binding antibody fragments thereof) that bind to proteins along the immune checkpoint pathway include, for example, antibodies that bind to: CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, TIGIT, LAG3, CD112, TIM3, BTLA, or co-stimulatory receptors: ICOS, OX40, 41BB, or GITR. In one particular embodiment, the antibody (or its antigen-binding antibody fragment) binds to PD-1 (e.g., human PD-1).

[0160] In other embodiments, the fusion protein of the present invention is a multispecific fusion protein comprising a cytokine, an antibody (or an antigen-binding antibody fragment thereof), and at least one additional targeting moiety. For example, the fusion protein of the present invention may be a trispecific fusion protein comprising a cytokine, an antibody (or an antigen-binding antibody fragment thereof), and at least one additional targeting moiety.

[0161] In exemplary embodiments, the fusion protein of the present invention comprises the IL-21 mutant protein described herein and a PD-1 binding antagonist. The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction induced by the interaction of PD-1 with one or more binding complexes, such as PD-L1 and PD-L2. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 with one or more binding complexes. In a particular state, a PD-1 binding antagonist inhibits the binding of PD-1 with PD-L1 and / or PD-L2. For example, a PD-1 binding antagonist includes an anti-PD-1 antibody, its antigen-binding antibody fragment, an immunoadhesive, a fusion protein, an oligopeptide, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction induced by the interaction of PD-1 with PD-L1 and / or PD-L2. In one embodiment, a PD-1-binding antagonist reduces negative co-stimulatory signals mediated by or via cell surface proteins expressed on T lymphocytes and mediated via PD-1-mediated signaling, resulting in less dysfunction of dysfunctional T cells (e.g., enhanced effector responses to antigen recognition). In some embodiments, the PD-1-binding antagonist is an anti-PD-1 antibody. Examples of anti-PD-1 antibodies include nivolumab (BMS-936558), pembrolizumab (MK-3475), BMS 936558, BMS-936559, TSR-042 (Tesaro), ePDR001 (Novartis), and pidilizumab (CT-011). Additional specific examples of PD-1-binding antagonists are provided below.

[0162] In exemplary embodiments, the PD-1-binding antagonist comprises, is substantially composed of, or is composed of an antigen-binding protein bound to PD-1. In exemplary embodiments, the antigen-binding protein is an antibody bound to PD-1, an antigen-binding antibody fragment thereof, or an antibody protein product.

[0163] In exemplary samples, the fusion protein of the present invention comprises the IL-21 mutant protein as described herein and an anti-PD-1 antibody (as described herein), its antigen-binding antibody fragment, or an anti-PD-1 antibody protein product. In exemplary cases, the anti-PD-1 antibody is a monoclonal IgG. In exemplary cases, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product is monovalent or bivalent. In exemplary samples, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product binds to human PD-1, having the amino acid sequence of SEQ ID NO: 263. In exemplary samples, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product binds to cynomolgus macaque PD-1, having the amino acid sequence of SEQ ID NO: 264. In an illustrative case, an anti-PD-1 antibody, its antigen-binding antibody fragment, or an anti-PD-1 antibody protein product binds to both human PD-1 and cynomolgus monkey PD-1. In an illustrative case, the fusion protein of the present invention comprises an IL-21 mutant protein (as described herein) and an anti-PD-1 antibody (as described herein).

[0164] In exemplary embodiments, the binding strength of the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product to PD-1 can be described according to KD. In exemplary samples, the KD of the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product provided herein is about 10⁻¹M, about 10⁻²M, about 10⁻³M, about 10⁻⁴M, about 10⁻⁵M, about 10⁻⁶M, about 10⁻⁷M, about 10⁻⁸M, about 10⁻⁹M, or less. In exemplary samples, the KD of the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product provided herein is micromolar, nanomolar, picomor, or femtomor. In the exemplary samples, the KD of the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product provided herein is in the range of about 10⁻⁴ to 10⁻⁶ M, or 10⁻⁷ to 10⁻⁹ M, or 10⁻¹⁰ to 10⁻¹² M, or 10⁻¹³ to 10⁻¹⁵ M. In the exemplary samples, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product has high affinity for human PD-1, cynomolgus monkey PD-1, or both. In the exemplary samples, the KD of the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product for human PD-1 is less than 100 pM, and as applicable, is from about 1 pM to about 50 pM. In the exemplary samples, the KD of the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product for human PD-1 is in the range of about 1 pM to about 20 pM or less than about 10 pM. In the exemplary samples, the KD of the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product against cynomolgus monkey PD-1 was less than 100 pM, and as appropriate, was from about 1 pM to about 75 pM. In the exemplary samples, the KD of the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product against cynomolgus monkey PD-1 was in the range of about 1 pM to about 20 pM or less than about 10 pM.

[0165] In an exemplary embodiment, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product is an antagonist that reduces, blocks, inhibits, eliminates, or interferes with signal transduction induced by the interaction of PD-1 with one or more binding ligands, such as PD-L1 and PD-L2. In an exemplary embodiment, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product blocks PD-1 from binding to its ligands PD-L1 or PD-L2. In an exemplary embodiment, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product inhibits the binding interaction between PD-1 and PD-L1 or PD-L2 by at least 50%. In an exemplary embodiment, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product exhibits inhibition of the binding interaction between PD-1 and PD-L1 or PD-L2 by at least about 50%, at least about 60%, or at least about 70%.

[0166] In an exemplary case, an anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product inhibits PD-1-mediated IL-2 production by T cells in a mixed lymphocyte response (MLR). In an exemplary case, the IC50 of the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product in the MLR is in the range of about 0.1 nM to about 5 nM. In an exemplary case, the IC50 of the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product in the MLR is less than 2 nM or less than 1 nM. In an exemplary case, the IC50 of the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product in the MLR is about 0.5 nM to about 2 nM.

[0167] In an exemplary case, an anti-PD-1 antibody (or an antigen-binding antibody fragment thereof) comprises: (a) a heavy chain (HC) complementarity-determining region (CDR) amino acid sequence selected from the group consisting of SEQ ID NOs: 312, 322, 332, 342, 352, 362, 372 and 382 (see Table D) or a variant thereof differing from only one or two amino acids or having at least or about 70% sequence identity; (b) an HC CDR2 amino acid sequence selected from the group consisting of SEQ ID NOs: 313, 323, 333, 343, 353, 363, 373 and 383 (see Table D) or a variant thereof differing from only one or two amino acids or having at least or about 70% sequence identity; (c) a sequence selected from SEQ ID NOs: 314, 324, 334, 344, 3545, 364, 374 and 384. (See Table D) The HC CDR3 amino acid sequences of the group consisting of HC CDR3 or differing from only one or two amino acids or variant sequences having at least or about 70% sequence identity; (d) Selected from SEQ ID NO: 315, 325, 335, 345, 355, 365, 375 and 385. The light chain (LC) CDR1 amino acid sequences of the group consisting of LC CDR1 or differing from only one or two amino acids or variant sequences having at least or about 70% sequence identity; (e) Selected from SEQ ID NO: 316, 326, 336, 346, 356, 366, 376 and 386. The LC CDR2 amino acid sequences of the group consisting of LC CDR2 or differing from only one or two amino acids or variant sequences having at least or about 70% sequence identity; (f) Selected from SEQ ID NO: The LC CDRs of the group consisting of 317, 327, 337, 347, 357, 367, 377, and 387 (see Table D) are amino acid sequences that differ from only one or two amino acids or variant sequences thereof having at least or about 70% sequence identity; or combinations of any two, three, four, five, or six of (g) (a)-(f). In some embodiments, the anti-PD-1 antibody protein product comprises such CDRs. Table D 20A2 20C1 22D4 20C1.006 20C1.009 20A2.003 22D4.006 22D4.017 HC CDR1 312 322 332 342 352 362 372 382 HC CDR2 313 323 333 343 353 363 373 383 HC CDR3 314 324 334 344 354 364 374 384 LC CDR1 315 325 335 345 355 365 375 385 LC CDR2 316 326 336 346 356 366 376 386 LC CDR3 317 327 337 347 357 367 377 387 The numbers represent the relevant SEQ ID NO.

[0168] In the exemplary sample, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises at least one or two of the LC CDR1, LC CDR2, and LC CDR3 amino acid sequences described in Table D, and the HC CDR amino acid sequences described in Table D. In some embodiments, the anti-PD-1 antibody protein product comprises such CDRs.

[0169] In an exemplary embodiment, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises 3, 4, 5, or all 6 amino acid sequences represented by SEQ ID NO: in a single row of Table D. In an exemplary embodiment, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises each of the LC CDR amino acid sequences represented by SEQ ID NO: in a single row of Table D, and at least 1 or 2 of the HC CDR amino acid sequences represented by SEQ ID NO: in the same or another single row of Table D. In an exemplary embodiment, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises each of the HC CDR amino acid sequences represented by SEQ ID NO: in a single row of Table D, and at least 1 or 2 of the LC CDR amino acid sequences represented by SEQ ID NO: in the same or another single row of Table D. In an exemplary embodiment, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 312-317; (b) SEQ ID NO: 322-327; (c) SEQ ID NO: 332-337; (d) SEQ ID NO: 342-347; (e) SEQ ID NO: 352-357; (f) SEQ ID NO: 362-367; (g) SEQ ID NO: 372-377; and (h) SEQ ID NO: 382-387. In certain embodiments, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises all six CDR amino acid sequences of any one of the antibodies 20A2, 20C1, 22D4, 20C1.006, 20C1.009, 20A2.003, 22D4.006, or 22D4.017 in Table D. In some embodiments, the anti-PD-1 antibody protein product comprises such CDRs.

[0170] In an exemplary case, the amino acid sequence of Table D is separated by at least one or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) inserted amino acids. In an exemplary case, there are about 10 to about 20 amino acids between the sequences of LC CDR1 and LC CDR2, and about 25 to about 40 amino acids between the sequences of LC CDR2 and LC CDR3. In an exemplary case, there are about 14 to about 16 amino acids between the sequences of LC CDR1 and LC CDR2, and about 30 to about 35 amino acids between the sequences of LC CDR2 and LC CDR3. In an exemplary case, there are about 10 to about 20 amino acids between the sequences of HC CDR1 and HC CDR2, and about 25 to about 40 amino acids between the sequences of HC CDR2 and HC CDR3. In an exemplary case, there are approximately 14 to approximately 16 amino acids between the sequences of HC CDR1 and HC CDR2, and approximately 30 to approximately 35 amino acids between the sequences of HC CDR2 and HC CDR3.

[0171] In exemplary embodiments, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises (a) a heavy chain variable region amino acid sequence selected from the group consisting of 318, 328, 338, 348, 358, 368, 378, and 388 (see Table E) or a variant sequence differing from only one or two amino acids or having at least or about 70% sequence identity; or (b) a light chain variable region amino acid sequence selected from the group consisting of 319, 329, 339, 349, 359, 369, 379, and 389 (see Table E) or a variant sequence differing from only one or two amino acids or having at least or about 70% sequence identity; or (c) (a) and (b). In some embodiments, the anti-PD-1 antibody protein product comprises such variable regions. Table E 20A2 20C1 22D4 20C1.006 20C1.009 20A2.003 22D4.006 22D4.017 HC variable 318 328 338 348 358 368 378 388 LC variable 319 329 339 349 359 369 379 389 HC (Full Length) 320 330 340 350 360 370 380 390 LC (Full Length) 321 331 341 351 361 371 381 391 The number indicates the relevant SEQ ID NO.

[0172] In an exemplary embodiment, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 318 and 319; (b) SEQ ID NO: 328 and 329; (c) SEQ ID NO: 338 and 339; (d) SEQ ID NO: 348 and 349; (e) SEQ ID NO: 358 and 359; (f) SEQ ID NO: 368 and 369; (g) SEQ ID NO: 378 and 379; and (h) SEQ ID NO: 388 and 389.

[0173] In exemplary embodiments, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises (a) a heavy chain variable region amino acid sequence selected from the group consisting of 320, 330, 340, 350, 360, 370, 380, and 390 (see Table E) or a variant sequence differing from only one or two amino acids or having at least or about 70% sequence identity; or (b) a light chain variable region amino acid sequence selected from the group consisting of 321, 331, 341, 351, 361, 371, 381, and 391 (see Table E) or a variant sequence differing from only one or two amino acids or having at least or about 70% sequence identity; or (c) (a) and (b). In some embodiments, the anti-PD-1 antibody protein product comprises such variable regions.

[0174] In exemplary embodiments, an anti-PD-1 antibody (or an antigen-binding antibody fragment thereof) comprises a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 320 and 321; (b) SEQ ID NO: 330 and 331; (c) SEQ ID NO: 340 and 341; (d) SEQ ID NO: 350 and 351; (e) SEQ ID NO: 360 and 361; (f) SEQ ID NO: 370 and 371; (g) SEQ ID NO: 380 and 381; and (h) SEQ ID NO: 390 and 391. In some embodiments, the anti-PD-1 antibody protein product includes such regions.

[0175] In the exemplary state, the heavy chain amino acid sequence of the anti-PD-1 antibody (or its antigen-binding antibody fragment) contains a set of charge-pair mutations as described herein. In the specific state, the heavy chain amino acid sequence of the anti-PD-1 antibody (or its antigen-binding antibody fragment) contains charge-pair mutations selected from V1, V103, and V131 charge-pair mutations.

[0176] In the exemplary samples, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product contains an amino acid sequence similar to the amino acid sequences mentioned above, and the antigen-binding protein essentially retains its biological function, such as its ability to bind to PD-1, such as human PD-1, cynomolgus monkey PD-1, or to reduce, block, inhibit, eliminate, or interfere with signal transduction caused by the interaction of PD-1 with one or more binding complexes, such as PD-L1 or PD-L2.

[0177] In exemplary samples, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product contains an amino acid sequence that differs from the aforementioned amino acid sequences by only 1, 2, 3, 4, 5, 6, or more amino acids. In exemplary samples, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product contains a variant of the aforementioned sequences, which differs from the aforementioned sequences by only one or two amino acids. In exemplary samples, the antigen-binding protein contains one or more amino acid substitutions outside the CDR, for example, the one or more amino acid substitutions appearing in the framework region of the heavy or light chain. In exemplary samples, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product contains one or more amino acid substitutions, while the antigen-binding protein retains an amino acid sequence of six CDRs. In the exemplary samples, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product comprises an amino acid sequence having only one, two, three, four, five, six, or more conserved amino acid substitutions relative to the amino acid sequences mentioned above.

[0178] In the exemplary sample, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product contains an amino acid sequence having more than or about 30%, more than or about 50%, or more than or about 70% sequence identity with the amino acid sequences mentioned above. In the exemplary sample, the antigen-binding protein contains an amino acid sequence having at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or more than 90% sequence identity with the amino acid sequences mentioned above. In the exemplary sample, the antigen-binding protein contains an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, or more than 90% sequence identity along the full length of the amino acid sequences mentioned above. In the exemplary sample, the antigen-binding protein contains an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity along the full length of the amino acid sequences mentioned above.

[0179] In the exemplary samples, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product contains a variant sequence of the mentioned sequence, which has at least or about 70% sequence identity with the aforementioned sequence. In the exemplary samples, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product contains a variant sequence of the mentioned sequence, which has at least or about 80% sequence identity with the aforementioned sequence. In the exemplary samples, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product contains a variant sequence of the mentioned sequence, which has at least or about 90% sequence identity with the aforementioned sequence. In the exemplary samples, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product contains a variant sequence of the mentioned sequence, which has at least or about 95% sequence identity with the aforementioned sequence.

[0180] In an exemplary embodiment, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises one, two, three, four, or five sequences of SEQ ID NO. in a single row of Table D and at least one variant sequence having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity with any of SEQ ID NO: 312-387. In exemplary embodiments, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises one, two, three, four, or five sequences selected from one of the following groups of sequences: (a) SEQ ID NO: 312-317; (b) SEQ ID NO: 322-327; (c) SEQ ID NO: 332-337; (d) SEQ ID NO: 342-347; (e) SEQ ID NO: 352-357; (f) SEQ ID NO: 362-367; (g) SEQ ID NO: 372-377; and (h) SEQ ID NO: 382-387, wherein the antibody or its fragment further comprises at least one variant sequence having at least 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity with at least one sequence from the group of sequences. For example, in the exemplary sample, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises four sequences of SEQ ID NO: 312-317, i.e., SEQ ID NO: 312-315, wherein the antibody or its fragment comprises two variant sequences: one variant sequence has at least or about 70% (e.g., at least about 80%, at least about 90%) sequence identity with SEQ ID NO: 316, and the other variant sequence has at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity with SEQ ID NO: 317. In some embodiments, the anti-PD-1 antibody protein product includes such regions.

[0181] In an exemplary embodiment, the anti-PD-1 antibody (or its antigen-binding antibody fragment) product comprises a pair of variant sequences having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity with any one of SEQ ID NO: 318, 319, 328, 329, 338, 339, 348, 349, 358, 359, 368, 369, 378, 379, 388 and 389. In exemplary cases, the antibody or a fragment thereof comprises a pair of variant sequences having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity with one of the following: (a) SEQ ID NO: 318 and 319; (b) SEQ ID NO: 328 and 329; (c) SEQ ID NO: 338 and 339; (d) SEQ ID NO: 348 and 349; (e) SEQ ID NO: 358 and 359; (f) SEQ ID NO: 368 and 369; (g) SEQ ID NO: 378 and 379; and (h) SEQ ID NO: 388 and 389. In an exemplary embodiment, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises a pair of sequences: one sequence is a sequence listed in Table E, and the other sequence is a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity with any one of SEQ ID NO: 318, 319, 328, 329, 338, 339, 348, 349, 358, 359, 368, 369, 378, 379, 388, and 389. In an exemplary embodiment, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises a pair of sequences: one sequence is selected from (a) SEQ ID NO: 318 and 319; (b) SEQ ID NO: 328 and 329; (c) SEQ ID NO: 338 and 339; (d) SEQ ID NO: 348 and 349; (e) SEQ ID NO: 358 and 359; (f) SEQ ID NO: 368 and 369; (g) SEQ ID NO: 378 and 379; and (h) SEQ ID NO: 388 and 389, and the other sequence is a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity with the sequences (a)-(u).For example, in the exemplary sample, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises the sequence of SEQ ID NO: 318 and further comprises a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity with SEQ ID NO 319. In some embodiments, the anti-PD-1 antibody protein product comprises such regions.

[0182] In an exemplary embodiment, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises a pair of variant sequences having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity with any one of SEQ ID NO: 320, 321, 330, 331, 340, 341, 350, 351, 360, 361, 370, 371, 380, 381, 390 and 391. In an exemplary case, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises a pair of variant sequences having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity with one of the following: (a) SEQ ID NO: 320 and 321; (b) SEQ ID NO: 330 and 331; (c) SEQ ID NO: 340 and 341; (d) SEQ ID NO: 350 and 351; (e) SEQ ID NO: 360 and 361; (f) SEQ ID NO: 370 and 371; (g) SEQ ID NO: 380 and 381; and (h) SEQ ID NO: 390 and 391. In an exemplary embodiment, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises a pair of sequences: one sequence is one of the sequences in Table E, and the other sequence is a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity with any one of SEQ ID NO: 320, 321, 330, 331, 340, 341, 350, 351, 360, 361, 370, 371, 380, 381, 390, and 391. In an exemplary embodiment, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises a pair of sequences: one sequence is selected from (a) SEQ ID NO: 320 and 321; (b) SEQ ID NO: 330 and 331; (c) SEQ ID NO: 340 and 341; (d) SEQ ID NO: 350 and 351; (e) SEQ ID NO: 360 and 361; (f) SEQ ID NO: 370 and 371; (g) SEQ ID NO: 380 and 381; and (h) SEQ ID NO: 390 and 391, and the other sequence is a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity with the sequences (a)-(u).For example, in the exemplary sample, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises the sequence of SEQ ID NO: 320 and further comprises a variant sequence having at least or about 70% (e.g., at least about 80%, at least about 90%, at least about 95%) sequence identity with SEQ ID NO 321. In some embodiments, the anti-PD-1 antibody protein product comprises such regions.

[0183] In other exemplary cases, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product contains one or more amino acid modifications relative to its naturally occurring counterpart, thereby improving half-life / stability or making the antibody more suitable for expression / manufacturing (e.g., as a fusion protein with the IL-21 mutant protein). In exemplary cases, the anti-PD-1 antibody is designed to prevent or reduce the interaction between the anti-PD-1 antibody and the Fc receptor. In exemplary cases, the anti-PD-1 antibody is a stable effectorless function (SEFL) antibody containing a constant region lacking the ability to interact with the Fcγ receptor. SEFL antibodies are known in this art. See, for example, Liu et al., J Biol Chem 292: 1876-1883 (2016); and Jacobsen et al., J. Biol. Chem. 292: 1865-1875 (2017). In the exemplary samples, the SEFL antibody contains one or more of the following mutations, according to EU system numbers: L242C, A287C, R292C, N297G, V302C, L306C, and / or K334C. In the exemplary samples, the SEFL antibody contains N297G. In the exemplary samples, the SEFL antibody contains A287C, N297G, and L306C. In other exemplary samples, the SEFL antibody contains R292C, N297G, and V302C (i.e., SEFL2-2).

[0184] Anti-PD-1 antibodies, their antigen-binding antibody fragments, or anti-PD-1 antibody protein products may contain other half-life extension (HLE) modifications. In exemplary cases, the HLE modification is present in the heavy chain constant region and contains one or more of the following mutations, according to EU system numbers: M252Y, S254T, and T256E. In exemplary cases, anti-PD-1 antibodies, their antigen-binding antibody fragments, or anti-PD-1 antibody protein products contain one or two of M252Y, S254T, and T256E. In exemplary cases, anti-PD-1 antibodies, their antigen-binding antibody fragments, or anti-PD-1 antibody protein products contain all three of M252Y, S254T, and T256E. In the illustrative example, the heavy chain constant region contains an amino acid sequence of SEQ ID NO: 545, SEQ ID NO: 547, or SEQ ID NO: 549, or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with SEQ ID NO: 545, SEQ ID NO: 547, or SEQ ID NO: 549. In the illustrative case, the HLE modification is present in the heavy chain constant region and contains one or more of the following mutations, according to EU system numbers: L309D, Q311H, and N434S. In exemplary cases, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product comprises one, two, or all three of L309D, Q311H, and N434S. In the exemplary sample, the heavy chain constant region contains an amino acid sequence of SEQ ID NO: 544, SEQ ID NO: 546, or SEQ ID NO: 548, or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with SEQ ID NO: 544, SEQ ID NO: 546, or SEQ ID NO: 548.

[0185] In the exemplary sample, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product contains SEFL2-2 modification and HLE modification. In some cases, the HLE modification contains one or two or all three of M252Y, S254T, and T256E. In the exemplary sample, the heavy chain constant region contains an amino acid sequence of SEQ ID NO: 551, SEQ ID NO: 553, or SEQ ID NO: 555, or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with SEQ ID NO: 551, SEQ ID NO: 553, or SEQ ID NO: 555. In some cases, the HLE modification comprises one or two or all three of L309D, Q311H, and N434S. In the exemplary sample, the heavy chain constant region comprises an amino acid sequence of SEQ ID NO: 550, SEQ ID NO: 552, or SEQ ID NO: 554, or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with SEQ ID NO: 550, SEQ ID NO: 552, or SEQ ID NO: 554. In the exemplary sample, the heavy chain further comprises charge-pair mutations as described below.

[0186] In eukaryotic cells, two types of glycosylation reactions occur: (1) N-linked glycosylation, in which the glycan is attached to an aspartic acid with the recognition sequence Asn-X-Thr / Ser, where "X" is any amino acid other than proline; and (2) O-linked glycosylation, in which the glycan is attached to serine or threonine. N-linked glycosylation begins in the endoplasmic reticulum (ER), where a complex set of reactions leads to the attachment of a core glycan structure consisting essentially of two GlcNAc residues and three Man residues. The glycan complex formed in the ER is modified by enzymes in the Gorgite body. If the enzyme has relatively little access to the sugar, it usually retains its initial HM form. If the enzyme has access to the sugar, many Man residues cleave and further modify the sugar, producing a complex N-glycan structure. For example, mannosidase-1, located in the cis-horgite structure, can cleave or hydrolyze HM glycans, while fucosyltransferase FUT-8, located in the intermediate horgite structure, fucosylates the glycan (Hanrue Imai-Nishiya (2007), BMC Biotechnology, 7:84). In the exemplary sample, the anti-PD-1 antibody is N-glycosylated, for example, containing sugar moieties (e.g., glycans, sugars) of one or more specific amino acids covalently attached to the heavy chain. In the alternative sample, the anti-PD-1 antibody is unglycosylated or does not contain any sugar moieties (e.g., glycans, sugars) of specific amino acids covalently attached to the heavy chain.

[0187] In the exemplary sample, the anti-PD-1 antibody comprises a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 284 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with SEQ ID NO: 284. In the exemplary sample, the anti-PD-1 antibody comprises a heavy chain constant region containing the amino acid sequence of SEQ ID NO: 284 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with SEQ ID NO: 284, and further comprises a linker. In an exemplary case, the linker contains the amino acid sequence of SEQ ID NO: 262. Therefore, in some exemplary samples, the anti-PD-1 antibody comprises the heavy chain constant region amino acid sequence of SEQ ID NO: 287 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with SEQ ID NO: 287. In the exemplary sample, the anti-PD-1 antibody comprises a heavy chain constant region containing an amino acid sequence of SEQ ID NO: 284 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with SEQ ID NO: 284, wherein the C-terminal Lys is truncated or removed. In this regard, in some samples, the anti-PD-1 antibody contains a heavy chain constant region lacking a C-terminal Lys group and includes the amino acid sequence of SEQ ID NO: 285 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with SEQ ID NO: 285. Generally, during expression, the C-terminal lysine of the antibody is cleaved by a carboxypeptidase.The absence of the C-terminal Lys heavy chain constant region advantageously prevents carboxypeptidase action on the heavy chain of the anti-PD-1 antibody. In exemplary samples, the anti-PD-1 antibody comprises a heavy chain constant region lacking the C-terminal Lys and further comprises a linker. In exemplary cases, the linker comprises the amino acid sequence of SEQ ID NO: 262. Thus, in some exemplary samples, the anti-PD-1 antibody comprises the heavy chain constant region amino acid sequence of SEQ ID NO: 286 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with SEQ ID NO: 286. In the exemplary sample, the anti-PD-1 antibody comprises a heavy chain constant region containing an amino acid sequence of SEQ ID NO: 284 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with SEQ ID NO: 284, wherein the region contains one or more SEFL mutations that prevent or reduce the interaction between the anti-PD-1 antibody and the Fc receptor, including (but not limited to) L242C, A287C, R292C, N297G, V302C, L306C, and / or K334C. In the exemplary sample, the SEFL mutation is a SEFL2-2 mutation: R292C, N297G, and V302C, such that the anti-PD-1 antibody includes the heavy chain constant region amino acid sequence of SEQ ID NO: 265 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with SEQ ID NO: 265. In the exemplary sample, the anti-PD-1 antibody includes a heavy chain constant region having a SEFL2-2 mutation and with the C-terminal Lys truncated or removed. Such a heavy chain constant region may include the sequence of SEQ ID NO: 266. In the exemplary sample, the anti-PD-1 antibody comprises a heavy chain constant region having a SEFL2-2 mutation, with the C-terminal Lys truncated or removed, and having a linker. Such a heavy chain constant region may comprise the sequence of SEQ ID NO: 267. In the exemplary sample, the anti-PD-1 antibody comprises a heavy chain constant region having a SEFL2-2 mutation and a linker, but without truncating the C-terminal Lys. Such a heavy chain constant region may comprise the sequence of SEQ ID NO: 282.

[0188] In the exemplary sample, the IL-21 mutant protein is attached to the Fc of the anti-PD-1 antibody. In the exemplary sample, the IL-21 mutant protein is attached to one of the two heavy chains of the antibody. In the exemplary sample, the IL-21 mutant protein is attached to the C-terminus of one of the two heavy chains of the antibody.

[0189] In the exemplary sample, the fusion protein contains only one IL-21 mutant protein (i.e., the fusion protein contains an IL-21 mutant protein monomer). In the exemplary sample, the IL-21 mutant protein is attached to the C-terminus of one of the two heavy chains of the antibody. In the exemplary sample, when the fusion protein contains only one IL-21 mutant protein, the Fc of the antibody contains modifications designed to drive heterodimerization of the two heavy chains (one heavy chain fused with the IL-21 mutant protein and the other heavy chain lacking the IL-21 mutant protein). Such modifications include Fc mutations, such as mortar-and-mortar, DuoBodies, Azymetric, charge pairs, HA-TF, SEEDbody, and modifications with differential protein A affinity. See, for example, Spiess et al., Molecular Immunology, 67(2, Part A), 2015, pp. 95-106. The mortar-and-groove mutation includes T366W in the first heavy chain and T366S, L368A, and / or Y407V in the second heavy chain. See, for example, Ridgway et al., Protein Eng., 9 (1996), pp. 617-621; and Atwell et al., J. Mol. Biol., 270 (1997), pp. 26-35. The DuoBody mutation includes F405L in the first heavy chain and K409R in the second heavy chain. See, for example, Labrijn et al., Proc. Natl. Acad. Sci. USA, 110 (2013), pp. 5145-5150. The Azymetric mutation includes T350V, L351Y, F405A, and / or Y407V in the first heavy chain and T350V, T366L, K392L, and / or T394W in the second heavy chain. See, for example, Von Kreudenstein et al., mAbs, 5 (2013), pp. 646-654. HA-TF mutations include S364H and / or F405A in the first heavy chain and Y349T and / or T394F in the second heavy chain. See, for example, Moore et al., mAbs, 3 (2011), pp. 546-557. SEEDbody mutations include IgG / A chimeric mutations in both the first and second heavy chains. See, for example, Davis et al., Protein Eng. Des. Sel., 23 (2010), pp. 195-202. Differential protein A affinity mutations include H435R in one heavy chain and no mutation in the other. See, for example, U.S. Patent No. 8,586,713.

[0190] In one particular instance, the mutation is a charge-pair mutation. Examples of such charge-pair mutations are given below, according to EU system designations. Charge-pair mutations include K409D in the first heavy chain and D399K in the second heavy chain; K392D in the first heavy chain and E356K in the second heavy chain; or K409D and K392D in the first heavy chain and D399K and E356K in the second heavy chain (the latter is referred to herein as "V1"). See, for example, Gunasekaran et al., J Biol Chem 285: 19637-19646 (2010). In another particular instance, the charge-pair mutation includes K439D, K392D, and K409D in the first heavy chain; and E356K and D399K in the second heavy chain (referred to herein as "V103"). In yet another specific instance, charge-pair mutations include K360E, K370E, K392E, and K409D in the first heavy chain; and E357K and D399K in the second heavy chain (referred to herein as "V131"). Charge-pair mutations may also include K370D in the first heavy chain and E357K in the second heavy chain; or all three of K409D, K392D, and K370D in the first heavy chain and all three of D399K, E357K, and E356K in the second heavy chain (the latter referred to herein as "V4"). Additional charge-pair mutations also include D221E, P228E, and / or L368E in the first heavy chain and D221R, P228R, and / or K409R in the second heavy chain. See, for example, Strop et al., J. Mol. Biol., 420 (2012), pp. 204-219.

[0191] In embodiments where the fusion protein comprises only one IL-21 mutant protein (i.e., the fusion protein comprises an IL-21 mutant protein monomer) and the heavy chain contains a V1 charge pair mutation, the IL-21 mutant protein may attach to a heavy chain containing K409D and K392D mutations (e.g., the IL-21 mutant protein attaches to a heavy chain containing SEQ ID NO: 294, 296, or 298) or a heavy chain containing D399K and E356K mutations (e.g., the IL-21 mutant protein attaches to a heavy chain containing SEQ ID NO: 295, 297, or 299). In a particular embodiment, the IL-21 mutant protein attaches to a heavy chain containing D399K and E356K mutations.

[0192] In embodiments where the fusion protein comprises only one IL-21 mutant protein (i.e., the fusion protein comprises an IL-21 mutant protein monomer) and the heavy chain contains a V4 charge pair mutation, the IL-21 mutant protein may attach to a heavy chain containing K409D, K392D, and K370D mutations (e.g., the IL-21 mutant protein attaches to a heavy chain containing SEQ ID NO: 288, 290, or 292) or a heavy chain containing D399K, E357K, and E356K mutations (e.g., the IL-21 mutant protein attaches to a heavy chain containing SEQ ID NO: 289, 291, or 293). In a particular embodiment, the IL-21 mutant protein attaches to a heavy chain containing D399K, E357K, and E356K mutations.

[0193] In embodiments where the fusion protein comprises only one IL-21 mutant protein (i.e., the fusion protein comprises an IL-21 mutant protein monomer) and the heavy chain contains a V103 charge pair mutation, the IL-21 mutant protein may attach to a heavy chain containing K439D, K392D, and K409D mutations (e.g., the IL-21 mutant protein attaches to a heavy chain containing SEQ ID NO: 472, 474, or 476) or a heavy chain containing E356K and D399K mutations (e.g., the IL-21 mutant protein attaches to a heavy chain containing SEQ ID NO: 473, 475, or 477). In a particular embodiment, the IL-21 mutant protein attaches to a heavy chain containing E356K and D399K mutations.

[0194] In embodiments where the fusion protein comprises only one IL-21 mutant protein (i.e., the fusion protein comprises an IL-21 mutant protein monomer) and the heavy chain contains a V131 charge pair mutation, the IL-21 mutant protein may attach to a heavy chain containing K360E, K370E, K392E, and K409D mutations (e.g., the IL-21 mutant protein attaches to a heavy chain containing SEQ ID NO: 478, 480, or 482) or a heavy chain containing E357K and D399K mutations (e.g., the IL-21 mutant protein attaches to a heavy chain containing SEQ ID NO: 479, 481, or 483). In a particular embodiment, the IL-21 mutant protein attaches to a heavy chain containing E357K and D399K mutations.

[0195] Therefore, in the exemplary state, the anti-PD-1 antibody (or its antigen-binding antibody fragment) contains a set of charge-pair mutations as described herein. In the specific state, the anti-PD-1 antibody (or its antigen-binding antibody fragment) contains charge-pair mutations selected from V1, V103, and V131 charge-pair mutations.

[0196] In the exemplary sample, the anti-PD-1 antibody comprises a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 284 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with SEQ ID NO: 284, and having one or more charge pair mutations, such as V1, V4, V103, or V131 charge pair mutations. In the exemplary sample, the anti-PD-1 antibody comprises a heavy chain constant region containing an amino acid sequence of SEQ ID NO: 284 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with SEQ ID NO: 284, and having a V1 charge pair mutation, wherein the first heavy chain constant region contains K409 and K392D mutations and the second heavy chain constant region contains D399K and E356K mutations. Such a first heavy chain constant region may contain the sequence of SEQ ID NO: 294 and such a second heavy chain constant region may contain the sequence of SEQ ID NO: 295. Such first and second heavy chain constant regions may have their C-terminal Lys segments truncated or removed, such that the first and second heavy chain constant regions may contain SEQ ID NO: 296 and 297, respectively. Such first and second heavy chain constant regions may have their C-terminal Lys segments truncated or removed and have a linker, such that the first and second heavy chain constant regions may contain SEQ ID NO: 298 and 299, respectively. In exemplary samples, the anti-PD-1 antibody contains heavy chain constant regions containing V1 charge pair mutations and SEFL2-2 mutations. Such first heavy chain constant regions containing V1 charge pair mutations and SEFL2-2 mutations may contain the sequence of SEQ ID NO: 306, and such second heavy chain constant regions containing V1 charge pair mutations and SEFL2-2 mutations may contain the sequence of SEQ ID NO: 307. Additional variations of such first and second heavy chains include, for example, heavy chains with truncated or removed C-terminal Lys (SEQ ID NO: 308 and 309) and heavy chains with truncated or removed C-terminal Lys and having connectors (SEQ ID NO: 310 and 311).

[0197] In the exemplary sample, the anti-PD-1 antibody comprises a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 284 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with SEQ ID NO: 284, having a V4 charge pair, wherein the first heavy chain constant region comprises K409, K392D, and K370D mutations, and the second heavy chain constant region comprises D399K, E356K, and E357K mutations. Such a first heavy chain constant region may comprise the sequence of SEQ ID NO: 288, and such a second heavy chain constant region may comprise the sequence of SEQ ID NO: 289. Such first and second heavy chain constant regions may have their C-terminal Lys segments truncated or removed, such that the first and second heavy chain constant regions may contain SEQ ID NO: 290 and 291, respectively. Such first and second heavy chain constant regions may have their C-terminal Lys segments truncated or removed and have a linker, such that the first and second heavy chain constant regions may contain SEQ ID NO: 292 and 293, respectively. In the exemplary sample, the anti-PD-1 antibody contains a heavy chain constant region containing a V4 charge pair mutation and a SEFL2-2 mutation. Such a first heavy chain constant region containing a V4 charge pair mutation and a SEFL2-2 mutation may contain the sequence of SEQ ID NO: 300, and such a second heavy chain constant region containing a V4 charge pair mutation and a SEFL2-2 mutation may contain the sequence of SEQ ID NO: 301. Additional variations of such first and second heavy chains are included, for example, heavy chains with truncated C-terminal Lys (SEQ ID NO: 302 and 303) and heavy chains with truncated or removed C-terminal Lys and having connectors (SEQ ID NO: 304 and 305).

[0198] In the exemplary sample, the anti-PD-1 antibody comprises a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 284 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with SEQ ID NO: 284, having a V103 charge pair mutation, wherein the first heavy chain constant region comprises the sequence of SEQ ID NO: 484 and the second heavy chain constant region comprises the sequence of SEQ ID NO: 485. Such first and second heavy chain constant regions may be truncated or have their C-terminal Lys removed, such that the first and second heavy chain constant regions may comprise SEQ ID NO: 486 and 487, respectively. Such first and second heavy chain constant regions may have their C-terminal Lys truncated or removed and have a linker, such that the first and second heavy chain constant regions may contain SEQ ID NO: 488 and 489, respectively. In exemplary samples, the anti-PD-1 antibody contains a heavy chain constant region containing a V103 charge pair mutation and a SEFL2-2 mutation. Such a first heavy chain constant region containing a V103 charge pair mutation and a SEFL2-2 mutation may contain the sequence of SEQ ID NO: 484, and such a second heavy chain constant region containing a V103 charge pair mutation and a SEFL2-2 mutation may contain the sequence of SEQ ID NO: 485. Additional variations covering such first and second heavy chains include, for example, heavy chains with C-terminal Lys truncated (SEQ ID NO: 486 and 487) and heavy chains with C-terminal Lys truncated or removed and having a linker (SEQ ID NO: 488 and 489).

[0199] In the exemplary sample, the anti-PD-1 antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 284 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with SEQ ID NO: 284, having a V131 charge pair mutation, wherein the first heavy chain constant region comprises the sequence of SEQ ID NO: 478 and the second heavy chain constant region comprises the sequence of SEQ ID NO: 479. Such first and second heavy chain constant regions may be truncated or have their C-terminal Lys removed, such that the first and second heavy chain constant regions may comprise SEQ ID NO: 480 and 481, respectively. Such first and second heavy chain constant regions may have their C-terminal Lys truncated or removed and have a linker, such that the first and second heavy chain constant regions may contain SEQ ID NO: 482 and 483, respectively. In exemplary samples, the anti-PD-1 antibody contains a heavy chain constant region containing a V131 charge pair mutation and a SEFL2-2 mutation. Such a first heavy chain constant region containing a V131 charge pair mutation and a SEFL2-2 mutation may contain the sequence of SEQ ID NO: 490, and such a second heavy chain constant region containing a V131 charge pair mutation and a SEFL2-2 mutation may contain the sequence of SEQ ID NO: 491. Additional variations covering such first and second heavy chains include, for example, heavy chains with C-terminal Lys truncated (SEQ ID NO: 492 and 493) and heavy chains with C-terminal Lys truncated or removed and having a linker (SEQ ID NO: 494 and 495).

[0200] In the substituted state sample, the fusion protein contains more than one IL-21 mutant protein (i.e., the fusion protein contains an IL-21 mutant protein dimer or an IL-21 mutant protein multimer). In the exemplary substituted state sample, the fusion protein contains two, three, or four (or more) IL-21 mutant proteins. In the exemplary state sample, when the fusion protein contains more than one IL-21 mutant protein, each IL-21 mutant protein contains the same structure, such as the same amino acid sequence. In the exemplary case, the fusion protein contains an IL-21 homodimer or an IL-21 homomultimer. In the substituted state sample, each IL-21 mutant protein of the fusion protein contains a different structure, such as a different amino acid sequence. In the exemplary case, the fusion protein contains an IL-21 heterodimer or an IL-21 heteromultimer. In an exemplary case, the fusion protein comprises two IL-21 mutant proteins, wherein a first IL-21 mutant protein is attached to the C-terminus of a first antibody heavy chain, and a second IL-21 mutant protein is attached to the C-terminus of a second antibody heavy chain. In the exemplary sample, each IL-21 mutant protein has the same amino acid sequence (e.g., an IL-21 mutant protein homodimer). In the exemplary sample, the first IL-21 mutant protein has an amino acid sequence different from that of the second IL-21 mutant protein (e.g., an IL-21 mutant protein heterodimer).

[0201] Regarding fusion proteins comprising one or more IL-21 mutant proteins, each IL-21 mutant protein may be attached to one of the heavy chains of an antibody with or without a linker. In an exemplary embodiment, the IL-21 mutant protein is attached to the C-terminus of one of the antibody heavy chains via a linker, and the linker is a peptide. In an exemplary case, the peptide comprises the amino acid sequence GGGGS (SEQ ID NO: 262). In an alternative embodiment, the IL-21 mutant protein is attached to the C-terminus of one of the antibody heavy chains without a linker.

[0202] In the exemplary sample, the fusion protein comprises only one IL-21 mutant protein, which is directly attached to the C-terminus of one of the heavy chains of the anti-PD-1 antibody. In the exemplary sample, the IL-21 mutant protein comprises an amino acid substitution listed in Table 4 or a sequence of SEQ ID NO: listed in Table 4. In the exemplary sample, the IL-21 mutant protein comprises an amino acid substitution listed in Table 5 or a sequence of SEQ ID NO: listed in Table 5. In the exemplary sample, the IL-21 mutant protein comprises an amino acid substitution listed in Table 7 or a sequence of SEQ ID NO: listed in Table 7. In the exemplary sample, the IL-21 mutant protein comprises an amino acid substitution listed in any of Tables 6 and 8-14 or a sequence of SEQ ID NO: listed in these tables. In the exemplary sample, the IL-21 mutant protein contains the amino acid sequence of any one of SEQ ID NO: 159, 161, 238, 241, 242, or 244. In the exemplary sample, the IL-21 mutant protein is directly attached to the anti-PD-1 antibody and does not contain a peptide linker.

[0203] In the exemplary sample, the fusion protein comprises two IL-21 mutant proteins, each mutant protein being directly attached to the C-terminus of the heavy chain of the anti-PD-1 antibody, and each mutant protein having the same amino acid sequence. In the exemplary sample, the IL-21 mutant protein comprises the amino acid substitutions listed in Table 4 or the sequence of SEQ ID NO: listed in Table 4. In the exemplary sample, the IL-21 mutant protein comprises the amino acid substitutions listed in Table 5 or the sequence of SEQ ID NO: listed in Table 5. In the exemplary sample, the IL-21 mutant protein comprises the amino acid substitutions listed in Table 7 or the sequence of SEQ ID NO: listed in Table 7. In the exemplary sample, the IL-21 mutant protein comprises the amino acid substitutions listed in any of Tables 6 and 8-14 or the sequence of SEQ ID NO: listed in these tables. In the exemplary sample, the IL-21 mutant protein comprises the amino acid sequence of any one of SEQ ID NO: 159, 161, 237, 238, 241, and 244. In the exemplary sample, the IL-21 mutant protein is directly attached to the anti-PD-1 antibody and does not contain a peptide linker.

[0204] In the illustrative sample, the fusion protein comprises an amino acid sequence of the antibody constant region described herein fused to an amino acid sequence of any IL-21 mutant protein described herein. In the illustrative sample, the fusion protein comprises an unglycosylated amino acid sequence of the antibody constant region described herein fused to an amino acid sequence of any IL-21 mutant protein described herein. In an exemplary embodiment, the fusion protein comprises a constant region fused with an IL-21 mutant protein containing any one of SEQ ID NO: 3-21, 23-56, 58-112, 114-208, 210-222, 224-255, and 283, or having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity, the constant region comprising SEQ ID NO: An amino acid sequence of any one of SEQ ID NO: 265-267, 282, 284-311, 472-495, and 544-555, or an amino acid sequence that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) identical to any one of SEQ ID NO: 265-267, 282, 284-311, 472-495, and 544-555. In the exemplary samples, the fusion protein comprises an amino acid sequence of any of SEQ ID NO: 268-281 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with SEQ ID NO: 268-281.

[0205] In exemplary embodiments, the fusion protein comprises the constructs described in Figures 4A, 4B, or 4C. In exemplary embodiments, the fusion protein comprises (i) an anti-PD-1 antibody (or an antigen-binding antibody fragment thereof) as described herein; and (ii) an IL-21 mutant protein as described herein. In other exemplary embodiments, the fusion protein comprises (i) an anti-PD-1 antibody (or an antigen-binding antibody fragment thereof) as described herein; (ii) a charge-pair mutation as described herein; and (iii) an IL-21 mutant protein as described herein (see, for example, Figure 4C). In other exemplary embodiments, the fusion protein comprises (i) an anti-PD-1 antibody (or an antigen-binding antibody fragment thereof) as described herein, wherein the heavy chain sequence of the anti-PD-1 antibody (or its antigen-binding antibody fragment) does not contain a C-terminal lysine; (ii) a charge-pair mutation as described herein; and (iii) an IL-21 mutant protein as described herein.

[0206] In exemplary cases, an anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises: (a) the heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence as described in Table D, or a sequence selected from the group consisting of SEQ ID NO: 312, 322, 332, 342, 352, 362, 372, and 382, ​​or a variant sequence differing from only one or two amino acids or having at least or about 70% sequence identity; (b) the HC CDR 2 amino acid sequence as described in Table D, or a sequence selected from the group consisting of SEQ ID NO: 313, 323, 333, 343, 353, 363, 373, and 383, or a variant sequence differing from only one or two amino acids or having at least or about 70% sequence identity; (c) the HC CDR 3 amino acid sequence as described in Table D, or a sequence selected from the group consisting of SEQ ID NO: (d) Sequences of the group consisting of SEQ ID NO: 314, 324, 334, 344, 3545, 364, 374, and 384, or sequences differing from only one or two amino acids or variant sequences having at least or about 70% sequence identity; (e) Light chain (LC) CDR1 amino acid sequences described in Table D, or sequences selected from the group consisting of SEQ ID NO: 315, 325, 335, 345, 355, 365, 375, and 385, or sequences differing from only one or two amino acids or variant sequences having at least or about 70% sequence identity; (f) LC CDR2 amino acid sequences described in Table D, or sequences selected from SEQ ID NO: The sequences comprising the group of 316, 326, 336, 346, 356, 366, 376, and 386, or differing from only one or two amino acids or variant sequences having at least or about 70% sequence identity; (f) the LC CDR3 amino acid sequence described in Table D, or the sequences selected from the group of SEQ ID NO: 317, 327, 337, 347, 357, 367, 377, and 387, or differing from only one or two amino acids or variant sequences having at least or about 70% sequence identity; or (g) any combination of two or more of (a)-(f). In the exemplary samples, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises at least one or two of the LC CDR1, LC CDR2, and LC CDR3 amino acid sequences described in Table D and the HC CDR amino acid sequences described in Table D. In exemplary embodiments, the antigen-binding protein comprises 3, 4, 5, or 6 amino acid sequences represented by SEQ ID NO: in a single row of Table D.In exemplary embodiments, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product comprises six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 312-317; (b) SEQ ID NO: 322-327; (c) SEQ ID NO: 332-337; (d) SEQ ID NO: 342-347; (e) SEQ ID NO: 352-357; (f) SEQ ID NO: 362-367; (g) SEQ ID NO: 372-377; and (h) SEQ ID NO: 382-387. In some embodiments, the anti-PD-1 antibody protein product includes such regions.

[0207] In an exemplary embodiment, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises (a) the heavy chain variable region amino acid sequence described in Table E, or a sequence selected from the group consisting of 318, 328, 338, 348, 358, 368, 378 and 388, or a variant sequence differing from only one or two amino acids or having at least or about 70% sequence identity; or (b) the light chain variable region amino acid sequence described in Table E, or a sequence selected from the group consisting of 319, 329, 339, 349, 359, 369, 379 and 389, or a variant sequence differing from only one or two amino acids or having at least or about 70% sequence identity; or (c) (a) and (b). In exemplary embodiments, the anti-PD-1 antibody (or its antigen-binding antibody fragment) comprises a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 318 and 319; (b) SEQ ID NO: 328 and 329; (c) SEQ ID NO: 338 and 339; (d) SEQ ID NO: 348 and 349; (e) SEQ ID NO: 358 and 359; (f) SEQ ID NO: 368 and 369; (g) SEQ ID NO: 378 and 379; and (h) SEQ ID NO: 388 and 389. In an exemplary case, the antibody constant region comprises an amino acid sequence of any one of SEQ ID NO: 265-267, 282, and 284-311, or an amino acid sequence that is identical to any one of SEQ ID NO: 265-267, 282, and 284-311 by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%), which is identical to any one of SEQ ID NO: 3-21, 23-56, 58-112, 114-208, 210-222, 224-255, and 283, or an amino acid sequence that is identical to any one of SEQ ID NO: 265-267, 282, and 284-31 ... IL-21 mutant protein fusions having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) amino acid sequences with sequence identity.In exemplary embodiments, the antigen-binding protein comprises a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 320 and 321; (b) SEQ ID NO: 330 and 331; (c) SEQ ID NO: 340 and 341; (d) SEQ ID NO: 350 and 351; (e) SEQ ID NO: 360 and 361; (f) SEQ ID NO: 370 and 371; (g) SEQ ID NO: 380 and 381; and (h) SEQ ID NO: 390 and 391. In exemplary embodiments, the fusion protein comprises (I) a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 320 and 321; (b) SEQ ID NO: 330 and 331; (c) SEQ ID NO: 340 and 341; (d) SEQ ID NO: 350 and 351; (e) SEQ ID NO: 360 and 361; (f) SEQ ID NO: 370 and 371; (g) SEQ ID NO: 380 and 381; and (h) SEQ ID NO: 390 and 391, and (II) any one of SEQ ID NO: 3-21, 23-56, 58-112, 114-208, 210-222, 224-255 and 283 or SEQ ID NO: IL-21 mutant proteins with amino acid sequences of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity.

[0208] In an illustrative case, the fusion protein comprises a homodimer as shown in Figure 4A, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 355-357) and three heavy chain CDRs (SEQ ID NO: 352-354) of antibody 20C1.009, and each heavy chain comprises a constant region sequence containing a SEFL2-2 mutation (e.g., SEQ ID NO: 265 or 266), wherein each of the two IL-21 mutant proteins comprises amino acid substitutions R9E and R76A (i.e., each comprising the sequence of SEQ ID NO: 244). In an illustrative case, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 358 and the light chain variable region of SEQ ID NO: 359. In an illustrative case, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. In an exemplary embodiment, the fusion protein comprises a homodimer containing the amino acid sequences of SEQ ID NO: 361 and 562 or SEQ ID NO: 361 and 563. In an exemplary embodiment, the fusion protein comprises a homodimer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 361) and two antibody heavy chains (each fused to an IL-21 mutant protein, and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 562). In an exemplary embodiment, the fusion protein comprises a homodimer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 361) and two antibody heavy chains (each fused to an IL-21 mutant protein, and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 563).

[0209] In an exemplary embodiment, the fusion protein comprises a homodimer as shown in Figure 4A, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 355-357) and three heavy chain CDRs (SEQ ID NO: 352-354) of antibody 20C1.009, and each heavy chain comprises a constant region sequence containing a SEFL2-2 mutation (e.g., SEQ ID NO: 265 or 266), wherein each of the two IL-21 mutant proteins comprises amino acid substitutions R9E and R76E (i.e., each comprises the sequence of SEQ ID NO: 245). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 358 and the light chain variable region of SEQ ID NO: 359. In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. In an exemplary embodiment, the fusion protein comprises a homodimer containing the amino acid sequences of SEQ ID NO: 361 and 564 or SEQ ID NO: 361 and 565. In an exemplary embodiment, the fusion protein comprises a homodimer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 361) and two antibody heavy chains (each fused to an IL-21 mutant protein, and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 564). In an exemplary embodiment, the fusion protein comprises a homodimer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 361) and two antibody heavy chains (each fused to an IL-21 mutant protein, and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 565).

[0210] In an illustrative case, the fusion protein comprises a homodimer as shown in Figure 4B, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 355-357) and three heavy chain CDRs (SEQ ID NO: 352-354) of antibody 20C1.009, and each heavy chain comprises a constant region sequence (e.g., SEQ ID NO: 267) containing the SEFL2-2 mutation and a linker, wherein each of the two IL-21 mutant proteins comprises amino acid substitutions R9E and R76A (i.e., each comprising the sequence of SEQ ID NO: 244). In an illustrative case, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 358 and the light chain variable region of SEQ ID NO: 359. In an illustrative case, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. In an exemplary embodiment, the fusion protein comprises a homodimer containing the amino acid sequences of SEQ ID NO: 361 and 566. In an exemplary embodiment, the fusion protein comprises a homodimer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 361) and two antibody heavy chains (each fused to an IL-21 mutant protein, and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 566).

[0211] In an illustrative case, the fusion protein comprises a homodimer as shown in Figure 4B, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 355-357) and three heavy chain CDRs (SEQ ID NO: 352-354) of antibody 20C1.009, and each heavy chain comprises a constant region sequence (e.g., SEQ ID NO: 267) containing the SEFL2-2 mutation and a linker, wherein each of the two IL-21 mutant proteins comprises amino acid substitutions R9E and R76E (i.e., each comprising the sequence of SEQ ID NO: 245). In an illustrative case, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 358 and the light chain variable region of SEQ ID NO: 359. In an illustrative case, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. In an exemplary embodiment, the fusion protein comprises a homodimer containing the amino acid sequences of SEQ ID NO: 361 and 567. In an exemplary embodiment, the fusion protein comprises a homodimer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 361) and two antibody heavy chains (each fused to the IL-21 mutant protein and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 567).

[0212] In an exemplary case, the fusion protein comprises the IL-21 mutant protein monomer shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 355-357) of antibody 20C1.009, three heavy chain CDRs (SEQ ID NO: 352-354) of antibody 20C1.009, and a pair of heavy chains (e.g., SEQ ID NO: 306 and 307 or SEQ ID NO: 308 and 309) comprising a constant region sequence containing a SEFL2-2 mutation and a V1 charge pair mutation, wherein the IL-21 mutant protein monomer is attached to a heavy chain containing an E356K and D399K V1 charge pair mutation (e.g., the IL-21 mutant protein monomer is attached to a heavy chain comprising any of SEQ ID NO: 309), and wherein the IL-21 mutant protein comprises amino acid substitutions for R9E and R76A (i.e., the sequence comprising SEQ ID NO: 244). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 358 and the light chain variable region of SEQ ID NO: 359. In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. In an exemplary embodiment, the fusion protein comprises a monomer containing the amino acid sequences of SEQ ID NO: 361 and 568. In an exemplary embodiment, the fusion protein comprises a monomer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 361) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 568, and one not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 574).

[0213] In an exemplary case, the fusion protein comprises the IL-21 mutant protein monomer shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 355-357) of antibody 20C1.009, three heavy chain CDRs (SEQ ID NO: 352-354) of antibody 20C1.009, and a pair of heavy chains (e.g., SEQ ID NO: 484 and 485 or SEQ ID NO: 486 and 487) comprising a constant region sequence containing a SEFL2-2 mutation and a V103 charge pair mutation, wherein the IL-21 mutant protein monomer is attached to the heavy chain containing the E356K and D399K V103 charge pair mutations (e.g., the IL-21 mutant protein monomer is attached to the heavy chain containing SEQ ID NO: 487), and wherein the IL-21 mutant protein comprises amino acid substitutions R9E and R76A (i.e., the sequence containing SEQ ID NO: 244). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 358 and the light chain variable region of SEQ ID NO: 359. In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. In an exemplary embodiment, the fusion protein comprises a monomer containing the amino acid sequences of SEQ ID NO: 361 and 569. In an exemplary embodiment, the fusion protein comprises a monomer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 361) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 569, and one not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 575).

[0214] In an exemplary case, the fusion protein comprises an IL-21 mutant protein monomer as shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 355-357) of antibody 20C1.009, three heavy chain CDRs (SEQ ID NO: 352-354) of antibody 20C1.009, and a pair of heavy chains (e.g., SEQ ID NO: 490 and 491 or SEQ ID NO: 492 and 493) comprising a constant region sequence containing a SEFL2-2 mutation and a V131 charge pair mutation, wherein the IL-21 mutant protein monomer is attached to the heavy chain containing the E357K and D399K V131 charge pair mutations (e.g., the IL-21 mutant protein monomer is attached to the heavy chain containing SEQ ID NO: 493), and wherein the IL-21 mutant protein comprises amino acid substitutions R9E and R76A (i.e., the sequence containing SEQ ID NO: 244). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 358 and the light chain variable region of SEQ ID NO: 359. In an exemplary embodiment, the anti-PD-1 antibody comprises either the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. In an exemplary embodiment, the fusion protein comprises a monomer containing the amino acid sequences of SEQ ID NO: 361 and 570. In an exemplary embodiment, the fusion protein comprises a monomer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 361) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 570, and one not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 576).

[0215] In an exemplary case, the fusion protein comprises an IL-21 mutant protein monomer as shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 355-357) of antibody 20C1.009, three heavy chain CDRs (SEQ ID NO: 352-354) of antibody 20C1.009, and a pair of heavy chains (e.g., SEQ ID NO: 306 and 307 or SEQ ID NO: 308 and 309) comprising a constant region sequence containing a SEFL2-2 mutation and a V1 charge pair mutation, wherein the IL-21 mutant protein monomer is attached to the heavy chain containing E356K and D399K V1 charge pair mutations (e.g., the IL-21 mutant protein monomer is attached to the heavy chain comprising SEQ ID NO: 309), and wherein the IL-21 mutant protein comprises amino acid substitutions R9E and R76E (i.e., the sequence comprising SEQ ID NO: 245). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 358 and the light chain variable region of SEQ ID NO: 359. In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. In an exemplary embodiment, the fusion protein comprises a monomer containing the amino acid sequences of SEQ ID NO: 361 and 571. In an exemplary embodiment, the fusion protein comprises a monomer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 361) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 571, and one not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 574).

[0216] In an exemplary case, the fusion protein comprises an IL-21 mutant protein monomer as shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 355-357) of antibody 20C1.009, three heavy chain CDRs (SEQ ID NO: 352-354) of antibody 20C1.009, and a pair of heavy chains (e.g., SEQ ID NO: 484 and 485 or SEQ ID NO: 486 and 487) comprising a constant region sequence containing a SEFL2-2 mutation and a V103 charge pair mutation, wherein the IL-21 mutant protein monomer is attached to the heavy chain containing E356K and D399K V103 charge pair mutations (e.g., the IL-21 mutant protein monomer is attached to the heavy chain containing SEQ ID NO: 487), and wherein the IL-21 mutant protein comprises amino acid substitutions R9E and R76E (i.e., the sequence containing SEQ ID NO: 245). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 358 and the light chain variable region of SEQ ID NO: 359. In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. In an exemplary embodiment, the fusion protein comprises a monomer containing the amino acid sequences of SEQ ID NO: 361 and 572. In an exemplary embodiment, the fusion protein comprises a monomer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 361) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 572, and one not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 575).

[0217] In an exemplary case, the fusion protein comprises an IL-21 mutant protein monomer as shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 355-357) of antibody 20C1.009, three heavy chain CDRs (SEQ ID NO: 352-354) of antibody 20C1.009, and a pair of heavy chains (e.g., SEQ ID NO: 490 and 491 or SEQ ID NO: 492 and 493) comprising a constant region sequence containing a SEFL2-2 mutation and a V131 charge pair mutation, wherein the IL-21 mutant protein monomer is attached to a heavy chain containing an E357K and D399K V1 charge pair mutation (e.g., the IL-21 mutant protein monomer is attached to a heavy chain comprising SEQ ID NO: 493), and wherein the IL-21 mutant protein comprises amino acid substitutions R9E and R76E (i.e., the sequence comprising SEQ ID NO: 245). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 358 and the light chain variable region of SEQ ID NO: 359. In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 360 or the light chain of SEQ ID NO: 361. In an exemplary embodiment, the fusion protein comprises a monomer containing the amino acid sequences of SEQ ID NO: 361 and 573. In an exemplary embodiment, the fusion protein comprises a monomer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 573, and one not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 576).

[0218] In an illustrative case, the fusion protein comprises a homodimer as shown in Figure 4A, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 385-387) and three heavy chain CDRs (SEQ ID NO: 382-384) of antibody 22D4.017, and each heavy chain comprises a constant region sequence containing a SEFL2-2 mutation (e.g., SEQ ID NO: 265 or 266), wherein each of the two IL-21 mutant proteins comprises amino acid substitutions R9E and R76A (i.e., each comprises the sequence of SEQ ID NO: 244). In an illustrative case, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 388 and the light chain variable region of SEQ ID NO: 389. In an illustrative case, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. In exemplary cases, the fusion protein comprises a homodimer containing the amino acid sequences of SEQ ID NO: 389 and 496 or SEQ ID NO: 389 and 519. In exemplary samples, the fusion protein comprises a homodimer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each fused to the IL-21 mutant protein, and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 496). In exemplary samples, the fusion protein comprises a homodimer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each fused to the IL-21 mutant protein, and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 519).

[0219] In an exemplary embodiment, the fusion protein comprises a homodimer as shown in Figure 4A, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 385-387) and three heavy chain CDRs (SEQ ID NO: 382-384) of antibody 22D4.017, and each heavy chain comprises a constant region sequence containing a SEFL2-2 mutation (e.g., SEQ ID NO: 265 or 266), wherein each of the two IL-21 mutant proteins comprises amino acid substitutions R9E and R76E (i.e., each comprises the sequence of SEQ ID NO: 245). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 388 and the light chain variable region of SEQ ID NO: 389. In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. In exemplary cases, the fusion protein comprises a homodimer containing the amino acid sequences of SEQ ID NO: 389 and 497 or SEQ ID NO: 389 and 498. In exemplary samples, the fusion protein comprises a homodimer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each fused to an IL-21 mutant protein, and the fusion heavy chain-IL-21 mutant protein containing the amino acid sequence of SEQ ID NO: 497). In exemplary samples, the fusion protein comprises a homodimer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each fused to an IL-21 mutant protein, and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 498).

[0220] In an exemplary embodiment, the fusion protein comprises a homodimer as shown in Figure 4B, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 385-387) and three heavy chain CDRs (SEQ ID NO: 382-384) of antibody 22D4.017, and each heavy chain comprises a constant region sequence (e.g., SEQ ID NO: 267) containing the SEFL2-2 mutation and a linker, wherein each of the two IL-21 mutant proteins comprises amino acid substitutions R9E and R76A (i.e., each comprising the sequence of SEQ ID NO: 244). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 388 and the light chain variable region of SEQ ID NO: 389. In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. In an exemplary embodiment, the fusion protein comprises a homodimer containing the amino acid sequences of SEQ ID NO: 389 and 499. In an exemplary embodiment, the fusion protein comprises a homodimer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each fused to the IL-21 mutant protein and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 499).

[0221] In an exemplary case, the fusion protein comprises a homodimer as shown in Figure 4B, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 385-387) and three heavy chain CDRs (SEQ ID NO: 382-384) of antibody 22D4.017, and each heavy chain comprises a constant region sequence (e.g., SEQ ID NO: 267) containing the SEFL2-2 mutation and a linker, wherein each of the two IL-21 mutant proteins comprises amino acid substitutions R9E and R76E (i.e., each comprising the sequence of SEQ ID NO: 245). In an exemplary case, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 388 and the light chain variable region of SEQ ID NO: 389. In an exemplary case, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. In an exemplary embodiment, the fusion protein comprises a homodimer containing the amino acid sequences of SEQ ID NO: 389 and 500. In an exemplary embodiment, the fusion protein comprises a homodimer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each fused to the IL-21 mutant protein and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 500).

[0222] In an exemplary case, the fusion protein comprises an IL-21 mutant protein monomer as shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 385-387) of antibody 22D4.017, three heavy chain CDRs (SEQ ID NO: 382-384) of antibody 22D4.017, and a pair of heavy chains (e.g., SEQ ID NO: 306 and 307 or SEQ ID NO: 308 and 309) comprising a constant region sequence containing a SEFL2-2 mutation and a V1 charge pair mutation, wherein the IL-21 mutant protein monomer is attached to the heavy chain containing E356K and D399K V1 charge pair mutations (e.g., the IL-21 mutant protein monomer is attached to the heavy chain containing SEQ ID NO: 309), and wherein the IL-21 mutant protein comprises amino acid substitutions R9E and R76A (i.e., the sequence containing SEQ ID NO: 244). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 388 and the light chain variable region of SEQ ID NO: 389. In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. In an exemplary embodiment, the fusion protein comprises a monomer containing the amino acid sequences of SEQ ID NO: 389 and 501. In an exemplary embodiment, the fusion protein comprises a monomer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 501, and one not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 556).

[0223] In an exemplary case, the fusion protein comprises the IL-21 mutant protein monomer shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 385-387) of antibody 22D4.017, three heavy chain CDRs (SEQ ID NO: 382-384) of antibody 22D4.017, and a pair of heavy chains (e.g., SEQ ID NO: 484 and 485 or SEQ ID NO: 486 and 487) comprising a constant region sequence containing a SEFL2-2 mutation and a V103 charge pair mutation, wherein the IL-21 mutant protein monomer is attached to the heavy chain containing the E356K and D399K V103 charge pair mutations (e.g., the IL-21 mutant protein monomer is attached to the heavy chain containing SEQ ID NO: 487), and wherein the IL-21 mutant protein comprises amino acid substitutions R9E and R76A (i.e., the sequence containing SEQ ID NO: 244). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 388 and the light chain variable region of SEQ ID NO: 389. In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. In an exemplary embodiment, the fusion protein comprises a monomer containing the amino acid sequences of SEQ ID NO: 389 and 502. In an exemplary embodiment, the fusion protein comprises a monomer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 502, and one not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 557).

[0224] In an exemplary case, the fusion protein comprises an IL-21 mutant protein monomer as shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 385-387) of antibody 22D4.017, three heavy chain CDRs (SEQ ID NO: 382-384) of antibody 22D4.017, and a pair of heavy chains (e.g., SEQ ID NO: 490 and 491 or SEQ ID NO: 492 and 493) comprising a constant region sequence containing a SEFL2-2 mutation and a V131 charge pair mutation, wherein the IL-21 mutant protein monomer is attached to the heavy chain containing E357K and D399K V131 charge pair mutations (e.g., the IL-21 mutant protein monomer is attached to the heavy chain containing SEQ ID NO: 493), and wherein the IL-21 mutant protein comprises amino acid substitutions R9E and R76A (i.e., the sequence containing SEQ ID NO: 244). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 388 and the light chain variable region of SEQ ID NO: 389. In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. In an exemplary embodiment, the fusion protein comprises a monomer containing the amino acid sequences of SEQ ID NO: 389 and 503. In an exemplary embodiment, the fusion protein comprises a monomer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 503, and one not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 558).

[0225] In an exemplary case, the fusion protein comprises an IL-21 mutant protein monomer as shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 385-387) of antibody 22D4.017, three heavy chain CDRs (SEQ ID NO: 382-384) of antibody 22D4.017, and a pair of heavy chains (e.g., SEQ ID NO: 306 and 307 or SEQ ID NO: 308 and 309) comprising a constant region sequence containing a SEFL2-2 mutation and a V1 charge pair mutation, wherein the IL-21 mutant protein monomer is attached to the heavy chain containing E356K and D399K V1 charge pair mutations (e.g., the IL-21 mutant protein monomer is attached to the heavy chain containing SEQ ID NO: 309), and wherein the IL-21 mutant protein comprises amino acid substitutions R9E and R76E (i.e., the sequence containing SEQ ID NO: 245). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 388 and the light chain variable region of SEQ ID NO: 389. In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. In an exemplary embodiment, the fusion protein comprises a monomer containing the amino acid sequences of SEQ ID NO: 389 and 504. In an exemplary embodiment, the fusion protein comprises a monomer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 504, and one not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 556).

[0226] In an exemplary case, the fusion protein comprises an IL-21 mutant protein monomer as shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 385-387) of antibody 22D4.017, three heavy chain CDRs (SEQ ID NO: 382-384) of antibody 22D4.017, and a pair of heavy chains (e.g., SEQ ID NO: 484 and 485 or SEQ ID NO: 486 and 487) comprising a constant region sequence containing a SEFL2-2 mutation and a V103 charge pair mutation, wherein the IL-21 mutant protein monomer is attached to the heavy chain containing E356K and D399K V103 charge pair mutations (e.g., the IL-21 mutant protein monomer is attached to the heavy chain containing SEQ ID NO: 487), and wherein the IL-21 mutant protein comprises amino acid substitutions R9E and R76E (i.e., the sequence containing SEQ ID NO: 245). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 388 and the light chain variable region of SEQ ID NO: 389. In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. In an exemplary embodiment, the fusion protein comprises a monomer containing the amino acid sequences of SEQ ID NO: 389 and 505. In an exemplary embodiment, the fusion protein comprises a monomer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 505, and one not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 557).

[0227] In an exemplary case, the fusion protein comprises the IL-21 mutant protein monomer shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 385-387) of antibody 22D4.017, three heavy chain CDRs (SEQ ID NO: 382-384) of antibody 22D4.017, and a pair of heavy chains (e.g., SEQ ID NO: 490 and 491 or SEQ ID NO: 492 and 493) comprising a constant region sequence containing a SEFL2-2 mutation and a V131 charge pair mutation, wherein the IL-21 mutant protein monomer is attached to a heavy chain containing E357K and D399K V131 charge pair mutations (e.g., the IL-21 mutant protein monomer is attached to a heavy chain comprising any of SEQ ID NO: 493), and wherein the IL-21 mutant protein comprises amino acid substitutions R9E and R76E (i.e., comprising SEQ ID NO: (Sequence 245). In an illustrative case, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 388 and the light chain variable region of SEQ ID NO: 389. In an illustrative case, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 390 or the light chain of SEQ ID NO: 391. In an illustrative case, the fusion protein comprises a monomer containing the amino acid sequences of SEQ ID NO: 389 and 506. In an illustrative case, the fusion protein comprises a monomer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 506, and one not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 558).

[0228] In an illustrative case, the fusion protein comprises a homodimer as shown in Figure 4A, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 365-367) and three heavy chain CDRs (SEQ ID NO: 362-364) of antibody 20A2.003, and each heavy chain comprises a constant region sequence containing a SEFL2-2 mutation (e.g., SEQ ID NO: 265 or 266), wherein each of the two IL-21 mutant proteins comprises amino acid substitutions R9E and R76A (i.e., each comprises the sequence of SEQ ID NO: 244). In an illustrative case, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 368 and the light chain variable region of SEQ ID NO: 369. In an illustrative case, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. In an exemplary embodiment, the fusion protein comprises a homodimer containing the amino acid sequences of SEQ ID NO: 369 and 507 or SEQ ID NO: 369 and 508. In an exemplary embodiment, the fusion protein comprises a homodimer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each fused to an IL-21 mutant protein, and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 507). In an exemplary embodiment, the fusion protein comprises a homodimer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each fused to an IL-21 mutant protein, and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 508).

[0229] In an exemplary embodiment, the fusion protein comprises a homodimer as shown in Figure 4A, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 365-367) and three heavy chain CDRs (SEQ ID NO: 362-364) of antibody 20A2.003, and each heavy chain comprises a constant region sequence containing a SEFL2-2 mutation (e.g., SEQ ID NO: 265 or 266), wherein each of the two IL-21 mutant proteins comprises amino acid substitutions R9E and R76E (i.e., each comprises the sequence of SEQ ID NO: 245). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 368 and the light chain variable region of SEQ ID NO: 369. In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. In exemplary cases, the fusion protein comprises a homodimer containing the amino acid sequences of SEQ ID NO: 369 and 509 or SEQ ID NO: 369 and 510. In exemplary samples, the fusion protein comprises a homodimer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each fused to an IL-21 mutant protein, and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 509). In exemplary samples, the fusion protein comprises a homodimer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each fused to an IL-21 mutant protein, and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 510).

[0230] In an exemplary embodiment, the fusion protein comprises a homodimer as shown in Figure 4B, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 365-367) and three heavy chain CDRs (SEQ ID NO: 362-364) of antibody 20A2.003, and each heavy chain comprises a constant region sequence (e.g., SEQ ID NO: 267) containing the SEFL2-2 mutation and a linker, wherein each of the two IL-21 mutant proteins comprises amino acid substitutions R9E and R76A (i.e., each comprising the sequence of SEQ ID NO: 244). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 368 and the light chain variable region of SEQ ID NO: 369. In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. In an exemplary embodiment, the fusion protein comprises a homodimer containing the amino acid sequences of SEQ ID NO: 369 and 511. In an exemplary embodiment, the fusion protein comprises a homodimer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each fused to the IL-21 mutant protein and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 511).

[0231] In an illustrative case, the fusion protein comprises a homodimer as shown in Figure 4B, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 365-367) and three heavy chain CDRs (SEQ ID NO: 362-364) of antibody 20A2.003, and each heavy chain comprises a constant region sequence (e.g., SEQ ID NO: 267) containing the SEFL2-2 mutation and a linker, wherein each of the two IL-21 mutant proteins comprises amino acid substitutions R9E and R76E (i.e., each comprising the sequence of SEQ ID NO: 245). In an illustrative case, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 368 and the light chain variable region of SEQ ID NO: 369. In an illustrative case, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. In an exemplary embodiment, the fusion protein comprises a homodimer containing the amino acid sequences of SEQ ID NO: 369 and 512. In an exemplary embodiment, the fusion protein comprises a homodimer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each fused to the IL-21 mutant protein and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 512).

[0232] In an exemplary case, the fusion protein comprises an IL-21 mutant protein monomer as shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 365-367) of antibody 20A2.003, three heavy chain CDRs (SEQ ID NO: 362-364) of antibody 20A2.003, and a pair of heavy chains (e.g., SEQ ID NO: 306 and 307 or SEQ ID NO: 308 and 309) comprising a constant region sequence containing a SEFL2-2 mutation and a V1 charge pair mutation, wherein the IL-21 mutant protein monomer is attached to a heavy chain containing an E356K and D399K V1 charge pair mutation (e.g., the IL-21 mutant protein monomer is attached to a heavy chain comprising any of SEQ ID NO: 309), and wherein the IL-21 mutant protein comprises amino acid substitutions for R9E and R76A (i.e., the sequence comprising SEQ ID NO: 244). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 368 and the light chain variable region of SEQ ID NO: 369. In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. In an exemplary embodiment, the fusion protein comprises a monomer containing the amino acid sequences of SEQ ID NO: 369 and 513. In an exemplary embodiment, the fusion protein comprises a monomer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 513, and one not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 559).

[0233] In an exemplary case, the fusion protein comprises an IL-21 mutant protein monomer as shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 365-367) of antibody 20A2.003, three heavy chain CDRs (SEQ ID NO: 362-364) of antibody 20A2.003, and a pair of heavy chains (e.g., SEQ ID NO: 484 and 485 or SEQ ID NO: 486 and 487) comprising a constant region sequence containing a SEFL2-2 mutation and a V103 charge pair mutation, wherein the IL-21 mutant protein monomer is attached to the heavy chain containing E356K and D399K V103 charge pair mutations (e.g., the IL-21 mutant protein monomer is attached to the heavy chain containing SEQ ID NO: 487), and wherein the IL-21 mutant protein comprises amino acid substitutions R9E and R76A (i.e., the sequence containing SEQ ID NO: 244). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 368 and the light chain variable region of SEQ ID NO: 369. In an exemplary embodiment, the anti-PD-1 antibody comprises either the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. In an exemplary embodiment, the fusion protein comprises a monomer containing the amino acid sequences of SEQ ID NO: 369 and 514. In an exemplary embodiment, the fusion protein comprises a monomer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 514, and one not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 560).

[0234] In an exemplary case, the fusion protein comprises an IL-21 mutant protein monomer as shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 365-367) of antibody 20A2.003, three heavy chain CDRs (SEQ ID NO: 362-364) of antibody 20A2.003, and a pair of heavy chains (e.g., SEQ ID NO: 490 and 491 or SEQ ID NO: 492 and 493) comprising a constant region sequence containing a SEFL2-2 mutation and a V131 charge pair mutation, wherein the IL-21 mutant protein monomer is attached to the heavy chain containing the E357K and D399K V131 charge pair mutations (e.g., the IL-21 mutant protein monomer is attached to the heavy chain containing SEQ ID NO: 493), and wherein the IL-21 mutant protein comprises amino acid substitutions R9E and R76A (i.e., the sequence containing SEQ ID NO: 244). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 368 and the light chain variable region of SEQ ID NO: 369. In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. In an exemplary embodiment, the fusion protein comprises a monomer containing the amino acid sequences of SEQ ID NO: 369 and 515. In an exemplary embodiment, the fusion protein comprises a monomer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 515, and one not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 561).

[0235] In an exemplary case, the fusion protein comprises an IL-21 mutant protein monomer as shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 365-367) of antibody 20A2.003, three heavy chain CDRs (SEQ ID NO: 362-364) of antibody 20A2.003, and a pair of heavy chains (e.g., SEQ ID NO: 306 and 307 or SEQ ID NO: 308 and 309) comprising a constant region sequence containing a SEFL2-2 mutation and a V1 charge pair mutation, wherein the IL-21 mutant protein monomer is attached to the heavy chain containing E356K and D399K V1 charge pair mutations (e.g., the IL-21 mutant protein monomer is attached to the heavy chain containing SEQ ID NO: 309), and wherein the IL-21 mutant protein comprises amino acid substitutions R9E and R76E (i.e., the sequence containing SEQ ID NO: 245). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 368 and the light chain variable region of SEQ ID NO: 369. In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. In an exemplary embodiment, the fusion protein comprises a monomer containing the amino acid sequences of SEQ ID NO: 369 and 516. In an exemplary embodiment, the fusion protein comprises a monomer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 516, and one not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 559).

[0236] In an exemplary case, the fusion protein comprises an IL-21 mutant protein monomer as shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 365-367) of antibody 20A2.003, three heavy chain CDRs (SEQ ID NO: 362-364) of antibody 20A2.003, and a pair of heavy chains (e.g., SEQ ID NO: 484 and 485 or SEQ ID NO: 486 and 487) comprising a constant region sequence containing a SEFL2-2 mutation and a V103 charge pair mutation, wherein the IL-21 mutant protein monomer is attached to the heavy chain containing E356K and D399K V103 charge pair mutations (e.g., the IL-21 mutant protein monomer is attached to the heavy chain containing SEQ ID NO: 487), and wherein the IL-21 mutant protein comprises amino acid substitutions R9E and R76E (i.e., the sequence containing SEQ ID NO: 245). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 368 and the light chain variable region of SEQ ID NO: 369. In an exemplary embodiment, the anti-PD-1 antibody comprises either the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. In an exemplary embodiment, the fusion protein comprises a monomer containing the amino acid sequences of SEQ ID NO: 369 and 517. In an exemplary embodiment, the fusion protein comprises a monomer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 517, and one not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 560).

[0237] In an exemplary case, the fusion protein comprises an IL-21 mutant protein monomer as shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 365-367) of antibody 20A2.003, three heavy chain CDRs (SEQ ID NO: 362-364) of antibody 20A2.003, and a pair of heavy chains (e.g., SEQ ID NO: 490 and 491 or SEQ ID NO: 492 and 493) comprising a constant region sequence containing a SEFL2-2 mutation and a V131 charge pair mutation, wherein the IL-21 mutant protein monomer is attached to the heavy chain containing E357K and D399K V1 charge pair mutations (e.g., the IL-21 mutant protein monomer is attached to the heavy chain containing SEQ ID NO: 493), and wherein the IL-21 mutant protein comprises amino acid substitutions R9E and R76E (i.e., the sequence containing SEQ ID NO: 245). In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain variable region of SEQ ID NO: 368 and the light chain variable region of SEQ ID NO: 369. In an exemplary embodiment, the anti-PD-1 antibody comprises the heavy chain of SEQ ID NO: 370 or the light chain of SEQ ID NO: 371. In an exemplary embodiment, the fusion protein comprises a monomer containing the amino acid sequences of SEQ ID NO: 369 and 518. In an exemplary embodiment, the fusion protein comprises a monomer containing two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 518, and one not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 561).

[0238] In an exemplary case, the fusion protein comprises an IL-21 mutant protein homodimer as shown in FIG4A or 4B or an IL-21 mutant protein monomer as shown in FIG4C, and an anti-PD-1 antibody comprising three light chain CDRs (SEQ ID NO: 365-367) of antibody 20A2.003, three heavy chain CDRs (SEQ ID NO: 362-364) of antibody 20A2.003, and a heavy chain constant region sequence comprising any one of SEQ ID NO 544-555. In an exemplary case, the fusion protein comprises an IL-21 mutant protein homodimer as shown in Figure 4A or 4B, or an IL-21 mutant protein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 365-367) of antibody 20A2.003, three heavy chain CDRs (SEQ ID NO: 362-364) of antibody 20A2.003, and a heavy chain constant region sequence comprising SEQ ID NO: 525 or 527.

[0239] In an exemplary case, the fusion protein comprises an IL-21 mutant protein homodimer as shown in FIG4A or 4B or an IL-21 mutant protein monomer as shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 385-387) of antibody 22D4.017, three heavy chain CDRs (SEQ ID NO: 382-384) of antibody 22D4.017, and a heavy chain constant region sequence comprising any one of SEQ ID NO: 544-555. In an exemplary case, the fusion protein comprises an IL-21 mutant protein homodimer as shown in Figure 4A or 4B, or an IL-21 mutant protein monomer as shown in Figure 4, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 385-387) of antibody 22D4.017, three heavy chain CDRs (SEQ ID NO: 382-384) of antibody 22D4.017, and a heavy chain constant region sequence comprising SEQ ID NO 529 or 531.

[0240] In an exemplary case, the fusion protein comprises an IL-21 mutant protein homodimer as shown in FIG4A or 4B or an IL-21 mutant protein monomer as shown in FIG4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 355-357) of antibody 20C1.009, three heavy chain CDRs (SEQ ID NO: 352-354) of antibody 20C1.009, and a heavy chain constant region sequence comprising any one of SEQ ID NO: 544-555. In exemplary cases, the fusion protein comprises an IL-21 mutant protein homodimer as shown in Figure 4A or 4B, or an IL-21 mutant protein monomer as shown in Figure 4C, wherein the anti-PD-1 antibody comprises three light chain CDRs (SEQ ID NO: 355-357) of antibody 20C1.009, three heavy chain CDRs (SEQ ID NO: 352-354) of antibody 20C1.009, and a heavy chain constant region sequence comprising SEQ ID NO: 521 or 523. Antigen-binding protein

[0241] The present invention provides a PD-1 antigen-binding protein. In exemplary samples, the PD-1 antigen-binding protein is an anti-PD-1 antibody described herein, an antigen-binding antibody fragment thereof, or an anti-PD-1 antibody protein product. In exemplary cases, an anti-PD-1 antibody, its antigen-binding antibody fragment, or an anti-PD-1 antibody protein product comprises: (a) the heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence described in Table D, or a sequence selected from the group consisting of SEQ ID NO: 312, 322, 332, 342, 352, 362, 372, and 382, ​​or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) the HC CDR 2 amino acid sequence described in Table D, or a sequence selected from the group consisting of SEQ ID NO: 313, 323, 333, 343, 353, 363, 373, and 383, or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) the HC CDR 3 amino acid sequence described in Table D, or a sequence selected from the group consisting of SEQ ID NO: (d) Sequences of the light chain (LC) CDR1 amino acid sequences described in Table D, or sequences selected from the group consisting of 314, 324, 334, 345, 355, 365, 375, and 385, or sequences differing from only one or two amino acids ... The CDR3 amino acid sequence, or a sequence selected from the group consisting of 317, 327, 337, 347, 357, 367, 377, and 387, or a sequence differing by only one or two amino acids or a variant thereof having at least or about 70% sequence identity; or a combination of any two or more of (g) (a)-(f). In exemplary samples, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product comprises at least one or two of the LC CDR1, LC CDR2, and LC CDR3 amino acid sequences described in Table D, and the HC CDR amino acid sequences described in Table D. In exemplary embodiments, the antigen-binding protein comprises at least three, four, or five of the amino acid sequences represented by SEQ ID NO: in a single row of Table D.

[0242] In exemplary embodiments, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product comprises six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 312-317; (b) SEQ ID NO: 322-327; (c) SEQ ID NO: 332-337; (d) SEQ ID NO: 342-347; (e) SEQ ID NO: 352-357; (f) SEQ ID NO: 362-367; (g) SEQ ID NO: 372-377; and (h) SEQ ID NO: 382-387. In exemplary cases, the amino acid sequences in Table D are separated by at least one or more (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inserting amino acids. In exemplary embodiments, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product comprises (a) the heavy chain variable region amino acid sequence illustrated in Table E, or a sequence selected from the group consisting of 318, 328, 338, 348, 358, 368, 378, and 388, or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or (b) the light chain variable region amino acid sequence illustrated in Figure E, or a sequence selected from the group consisting of 319, 329, 339, 349, 359, 369, 379, and 389, or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or (c) (a) and (b). In exemplary embodiments, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product comprises a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 318 and 319; (b) SEQ ID NO: 328 and 329; (c) SEQ ID NO: 338 and 339; (d) SEQ ID NO: 348 and 349; (e) SEQ ID NO: 358 and 359; (f) SEQ ID NO: 368 and 369; (g) SEQ ID NO: 378 and 379; and (h) SEQ ID NO: 388 and 389.In the exemplary samples, the anti-PD-1 antibody, its antigen-binding antibody fragment, or the anti-PD-1 antibody protein product comprises: (I) a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 318 and 319; (b) SEQ ID NO: 328 and 329; (c) SEQ ID NO: 338 and 339; (d) SEQ ID NO: 348 and 349; (e) SEQ ID NO: 358 and 359; (f) SEQ ID NO: 368 and 369; (g) SEQ ID NO: 378 and 379; and (h) SEQ ID NO: 388 and 389; and (II) a constant region comprising any one of SEQ ID NO: 265-267, 282, 284-311, 472-495, and 544-555. In exemplary embodiments, the antigen-binding protein comprises a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 320 and 321; (b) SEQ ID NO: 330 and 331; (c) SEQ ID NO: 340 and 341; (d) SEQ ID NO: 350 and 351; (e) SEQ ID NO: 360 and 361; (f) SEQ ID NO: 370 and 371; (g) SEQ ID NO: 380 and 381; and (h) SEQ ID NO: 390 and 391. In exemplary embodiments, the antigen-binding protein comprises an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identity with one or more of the above SEQ ID NO:

[0243] In some embodiments, the antigen-binding protein is an anti-PD-1 antibody or an antigen-binding antibody fragment thereof.

[0244] The present invention further provides conjugates comprising the PD-1 antigen-binding protein described herein and a heterologous portion. The heterologous portion may be any molecule different from the PD-1 antigen-binding protein described herein. In exemplary samples, the heterologous portion is a heteropeptide or polypeptide, a targeting agent, a diagnostic marker, a polymer, a nucleic acid, a quantum dot, a small molecule, a toxin, a carbohydrate, an amino acid, or other therapeutic or diagnostic agent. In exemplary samples, the heterologous portion is the IL-21 mutant protein as described herein.

[0245] The present invention further provides a fusion protein comprising the PD-1 antigen-binding protein described herein and a heterologous polypeptide or peptide. In the exemplary samples, the heterologous polypeptide is the IL-21 mutant protein as described herein. Antibody preparation method

[0246] Suitable methods for preparing antibodies, antigen-binding antibody fragments, and antibody protein products are known in this art. For example, standard hybridoma methods for producing antibodies are described, for instance, in Harlow and Lane (eds.), *Antibodies: A Laboratory Manual*, CSH Press (1988), and CA. Janeway et al. (eds.), *Immunobiology*, 5th edition, Garland Publishing, New York, NY (2001)). An exemplary method for preparing the anti-PD-1 monoclonal antibody of the present invention is provided in the examples herein.

[0247] Depending on the host species, a variety of adjuvants can be used to enhance the immune response, causing the host to produce a larger amount of antibodies. Such adjuvants include (but are not limited to) Freund's adjuvants, mineral gels (such as aluminum hydroxide) and surfactants (such as lysophosphatidylcholine), pluronic polyols, polyanionic peptides, oil emulsions, keyhole hemocyanin, and dinitrophenol. BCG (Bacillus Calmette-Guérin) and Corynebacterium parvum are potentially suitable human adjuvants.

[0248] Other methods for generating antibodies are summarized in Table F. Table F technology Illustrative references EBV-hybridoma method and phage vector expression system Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984); Roder et al., Methods Enzymol., 121, 140-67 (1986); and Huse et al., Science, 246, 1275-81 (1989) Methods for generating antibodies in non-human animals U.S. Patents 5,545,806, 5,569,825, and 5,714,352, and U.S. Patent Application Publication No. 2002 / 0197266 Inducing in vivo production in a lymphocyte population, or by screening a recombinant immunoglobulin library or a group of highly specific binding reagents. Orlandi et al. (Proc Natl Acad Sci 86: 3833-3837; 1989), and Winter G and Milstein C (Nature 349: 293-299, 1991) Methods for producing recombinant proteins Protein production and purification” Nat Methods 5(2): 135-146 (2008) bacteriophage presentation Janeway et al., above; Huse et al., above; and U.S. Patent 6,265,150. Related methods are also described in U.S. Patent Nos. 5,403,484; 5,571,698; 5,837,500; and 5,702,892. The technology described below is in: U.S. Patent Nos. 5,780,279; 5,821,047; 5,824,520; 5,855,885; 5,858,657; 5,871,907; 5,969,108; 6,057,098; and 6,225,447. Antibodies can be produced by transgenic mice. U.S. Patents 5,545,806 and 5,569,825, and Janeway et al., above.

[0249] Methods for testing the ability of an antibody to bind to PD-1 regardless of how the antibody is produced are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), ELISA, Western ink dot assay, immunoprecipitation, SPR, and competitive inhibition assay (see, for example, Janeway et al., hereinafter, and U.S. Patent Application Publication No. 2002 / 0197266, and the section above concerning competitive assays). Other binding assays, such as competitive binding assays or competitive assays, for testing the ability of an antibody to compete with a second antibody for binding to an antigen or its antigenic determinant, are known in the art and can be used to test the ability of an antibody to bind to PD-1. See, for example, U.S. Patent Application Publication No. US20140178905; Chand et al., Biologicals 46: 168-171 (2017); Liu et al., Anal Biochem 525: 89-91 (2017); and Goolia et al., J Vet Diagn Invest 29(2): 250-253 (2017). Other methods for comparing two antibodies are also known in this art, including, for example, surface plasma resonance (SPR). SPR can be used to determine the binding constants of an antibody and a second antibody and to compare the two binding constants. Heterogeneous components: polymers, carbohydrates, lipids, and therapeutic agents.

[0250] In exemplary embodiments, the conjugate of the present invention comprises an IL-21 mutant protein linked to a polymer. In some embodiments, the polymer is selected from the group consisting of: polyamides, polycarbonates, polyalkylene compounds and their derivatives, including polymers of polyalkylene glycols, polyoxyethylenes, polyalkylene terephthalates, acrylates and methacrylates, including poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(laurate methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate) and poly(octadecyl acrylate); polyethylene polymers, including Polyvinyl alcohol, polyvinyl ether, polyvinyl ester, polyhalogenated vinyl, poly(vinyl acetate), and polyvinylpyrrolidone; polyglycolic acid, polysiloxane, polyurethane, and copolymers thereof; cellulose, including alkyl cellulose, hydroxyalkyl cellulose, cellulose ether, cellulose ester, nitrocellulose, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxyethyl cellulose, cellulose triacetate, and sodium cellulose sulfate; polypropylene, polyethylene, including poly(ethylene glycol), poly(ethylene oxide), and poly(ethylene terephthalate), and polystyrene. In certain embodiments, the polymer is a polyalkylene glycol, including, for example, polyethylene glycol (PEG).

[0251] In an exemplary embodiment, the conjugate of the present invention comprises an IL-21 mutant protein linked to a carbohydrate. In some embodiments, the carbohydrate is a monosaccharide (e.g., glucose, galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose), an oligosaccharide (e.g., raffinose, stachyose), or a polysaccharide (e.g., starch, amylase, amylopectin, cellulose, chitin, callose, laminarin, xylan, mannan, fucoidan, or galactomannan).

[0252] In some embodiments, the heterologous portion is a lipid. In some embodiments, the lipid is a fatty acid, eicosanoic acid, prostaglandin, leukotrienes, thrombin, N-acetylglucosamine, glycerides (e.g., monosubstituted, disubstituted, trisubstituted glycerol), glycerophospholipids (e.g., phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine), sphingolipids (e.g., sphingosine, ceramide), sterol lipids (e.g., steroids, cholesterol), isopentenyl alcohol lipids, glycolipids or polyketides, oils, waxes, cholesterol, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, or phospholipids.

[0253] In an exemplary embodiment, the conjugate of the present invention comprises an IL-21 mutant protein linked to a therapeutic agent. The therapeutic agent may be any of those known in the art. In an exemplary embodiment, the therapeutic agent is an immunotherapeutic agent, provided that the therapeutic agent stimulates an immune response. In an exemplary embodiment, the immunotherapeutic agent is a cancer vaccine. In an exemplary embodiment, the immunotherapeutic agent is a monoclonal antibody. In an exemplary embodiment, the immunotherapeutic agent is an immune checkpoint inhibitor, such as an inhibitor of CTLA4, PD-1, or PD-L1. In an exemplary case, the monoclonal antibody is specific to a protein in the immune checkpoint pathway. Proteins in the immune checkpoint pathway may be, for example, CTLA4, PD-1, PD-L1, B7-H3, B7H4, or TIM3. For example, the antigen-binding protein of the present invention can bind to atezolizumab, avelumab, ipilimumab, tremelimumab, BMS-936558, MK3475, CT-011, AM-224, MDX-1105, IMP321, and MGA271.

[0254] In exemplary cases, the therapeutic agent is a cytokine, lymphokine, growth factor, or hematopoietic factor that effectively inhibits tumor metastasis and / or has an antiproliferative effect on at least one cell population. Such cytokines, lymphokines, growth factors, or other hematopoietic factors include (but are not limited to): M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNFα, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and erythropoietin.Other growth factors used in this article include angiopoietin, bone morphogenetic protein-1, bone morphogenetic protein-2, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein-5, bone morphogenetic protein-6, bone morphogenetic protein-7, bone morphogenetic protein-8, bone morphogenetic protein-9, bone morphogenetic protein-10, bone morphogenetic protein-11, bone morphogenetic protein-12, and bone morphogenetic protein-13. -14. Bone morphogenesis proteins -15. Bone morphogenesis protein receptor IA, Bone morphogenesis protein receptor IB, Brain-derived neurotrophic factor, Ciliary nerve growth factor, Ciliary nerve growth factor receptor α, Cytokine-induced neutrophil chemokine 1, Cytokine-induced neutrophil chemokine 2α, Cytokine-induced neutrophil chemokine 2β, β-endothelial growth factor, Endothelin 1, Epithelial-derived neutrophil inducer, Glial cell line-derived neurotrophic factor receptor α1, Glial cell line-derived... Neurotrophic factor receptor α2, growth-related proteins, growth-related protein α, growth-related protein β, growth-related protein γ, heparin-binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor α, nerve growth factor, nerve growth factor receptor, neurotrophin-3, neurotrophin-4, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor α, transforming growth factor β, transforming growth factor β1, transforming growth factor β1.2, transforming growth factor β2, transforming growth factor β3, transforming growth factor β5, potential transforming growth factor β1, transforming growth factor β-binding protein I, transforming growth factor β-binding protein II, transforming growth factor β-binding protein III, type I tumor necrosis factor receptor, type II tumor necrosis factor receptor, urokinase-type plasminogen activator receptor and chimeric proteins and their biological or immune-active fragments. In exemplary embodiments, the therapeutic agent comprises an antibody specific to any of the aforementioned cytokines, lymphokines, growth factors, or other hematopoietic factors. Nucleic acid.

[0255] The present invention further provides a nucleic acid comprising a nucleotide sequence encoding the IL-21 mutant protein of the present invention, a conjugate comprising the IL-21 mutant protein, or a fusion protein comprising the IL-21 mutant protein. For example, the nucleic acid may comprise a nucleotide sequence encoding a heavy chain of an anti-PD-1 antibody, followed by a nucleotide sequence encoding the IL-21 mutant protein of the present invention. The nucleotide sequences encoding the heavy chain and the nucleotide sequences encoding the IL-21 mutant protein may be side-linked with nucleotide sequences encoding a peptide linker comprising the amino acid sequence GGGGS (SEQ ID NO: 262). In an alternative state, the nucleic acid does not comprise a nucleotide sequence encoding a peptide linker, and the nucleotide sequence encoding the heavy chain of the anti-PD-1 antibody is tandemly linked with the nucleotide sequence encoding the IL-21 mutant protein of the present invention.

[0256] In the exemplary sample, the nucleic acid comprises a nucleotide sequence encoding an IL-21 mutant protein comprising an amino acid sequence of SEQ ID NO: 3-21, 23-56, 58-112, 114-208, 210-222, 224-255 and 283 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%) sequence identity with the amino acid sequence of SEQ ID NO: 3-21, 23-56, 58-112, 114-208, 210-222, 224-255 and 283.

[0257] In the exemplary sample, the nucleic acid comprises a nucleotide sequence of a peptide linker encoding an amino acid sequence that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) identical to the sequence of SEQ ID NO: 262.

[0258] In the exemplary sample, the nucleic acid contains a nucleotide sequence encoding a fusion protein that contains an amino acid sequence of the antibody constant region described herein fused with an amino acid sequence of any IL-21 mutant protein described herein. In an exemplary case, the nucleic acid comprises a nucleotide sequence encoding a fusion protein comprising an amino acid sequence fused with any one of SEQ ID NO: 3-21, 23-56, 58-112, 114-208, 210-222, 224-255, and 283, or an amino acid sequence fused with any one of SEQ ID NO: 3-21, 23-56, 58-112, 114-208, 210-222, 224-255, and 283 having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) of the sequence identical to the sequence of SEQ ID NO: 265-267, and 282. Any one of SEQ ID NO: 265-267 and 282 has an amino acid sequence that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identical to the sequence of SEQ ID NO: 268-281. In the exemplary sample, the nucleic acid comprises a nucleotide sequence encoding a fusion protein comprising an amino acid sequence of any one of SEQ ID NO: 268-281 or an amino acid sequence that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or has more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) sequence identical to the sequence of SEQ ID NO: 268-281.

[0259] In the exemplary sample, the nucleic acid comprises a nucleotide sequence encoding an anti-PD-1 antibody comprising a heavy chain constant region amino acid sequence of any one of SEQ ID NO: 265-267, 282, 284-311, 472-495 and 544-555 or an amino acid sequence having at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or having more than about 90% (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99%) sequence identity with any one of SEQ ID NO: 265-267, 282, 284-311, 472-495 and 544-555.

[0260] The present invention further provides a nucleic acid comprising a nucleotide sequence encoding the PD-1 antigen-binding protein of the present invention. In an exemplary embodiment, the nucleotide sequence comprises a sequence encoding a heavy chain CDR or light chain CDR, a heavy chain variable region or light chain variable region, or a heavy chain sequence or a light chain sequence. See Table G below. In an exemplary case, the nucleotide sequence comprises any of SEQ ID NO: 392-471. The present invention further provides paired nucleotide sequences comprising: (a) SEQ ID NO: 398 and 399; (b) SEQ ID NO: 408 and 409; (c) SEQ ID NO: 418 and 419; (d) SEQ ID NO: 428 and 429; (e) SEQ ID NO: 438 and 439; (f) SEQ ID NO: 448 and 449; (g) SEQ ID NO: 458 and 459, or (h) SEQ ID NO: 468 and 469. The present invention further provides paired nucleotide sequences comprising the following: (a) SEQ ID NO: 400 and 401; (b) SEQ ID NO: 410 and 411; (c) SEQ ID NO: 420 and 421; (d) SEQ ID NO: 430 and 431; (e) SEQ ID NO: 440 and 441; (f) SEQ ID NO: 450 and 451; (g) SEQ ID NO: 460 and 461; or (h) SEQ ID NO: 470 and 471. Table G 20A2 20C1 22D4 20C1.006 20C1.009 20A2.003 22D4.006 22D4.017 HC CDR1 392 402 412 422 432 442 452 462 HC CDR2 393 403 413 423 433 443 453 463 HC CDR3 394 404 414 424 434 444 454 464 LC CDR1 395 405 415 425 435 445 455 465 LC CDR2 396 406 416 426 436 446 456 466 LC CDR3 397 407 417 427 437 447 457 467 HC variable 398 408 418 428 438 448 458 468 LC variable 399 409 419 429 439 449 459 469 HC full length 400 410 420 430 440 450 460 470 LC full length 401 411 421 431 441 451 461 471

[0261] In the exemplary samples, the nucleic acid molecule comprises a nucleotide sequence encoding the conjugate or fusion protein of the present invention. As used herein, "nucleic acid" includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule," and generally refers to a polymer or modified form of DNA or RNA, which may be single-stranded or double-stranded, synthetic or derived from natural sources (e.g., isolated and / or purified), may contain naturally occurring, non-natural, or modified nucleotides, and may contain natural, non-natural, or modified internucleotide bonds, such as aminophosphate bonds or thiophosphate bonds, instead of the phosphodiester bonds found between the nucleotides of unmodified oligonucleotides. The nucleic acid may comprise any nucleotide sequence encoding any of the antigen-binding proteins or polypeptides of the present invention. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions, and / or substitutions. In other embodiments, the nucleic acid comprises one or more insertions, deletions, inversions, and / or substitutions.

[0262] In some embodiments, the nucleic acids of the present invention are recombinant. As used herein, the term "recombinant" means (i) a molecule constructed outside of living cells by conjugating a natural or synthetic nucleic acid segment with a nucleic acid molecule capable of replicating in living cells; or (ii) a molecule produced by replication of the molecule described in (i) above. For purposes herein, replication may be in vitro or in vivo.

[0263] In some cases, nucleic acids are constructed using procedures known in this art based on chemical synthesis and / or enzymatic ligation reactions. See, for example, Sambrook et al., above; and Ausubel et al., above. For instance, nucleic acids can be chemically synthesized using naturally occurring nucleotides or nucleotides with various modifications, which are designed to increase the biological stability of the molecule or the physical stability of the double strand formed after hybridization (e.g., phosphate thioester derivatives and acridine-substituted nucleotides). Examples of modified nucleotides that can be used to generate nucleic acids include (but are not limited to) 5-Fluorouracil, 5-Bromouracil, 5-Chlorouracil, 5-Iodouracil, Hypoxanthine, Xanthine, 4-Ethylcytosine, 5-(Carboxyhydroxymethyl)uracil, 5-Carboxymethylaminomethyl-2-thiouridine, 5-Carboxymethylaminomethyluracil, Dihydrouracil, β-D-galactosylpiperidine, Inosine, N6-Isopentenyladenine, 1-Methylguanine, 1-Methylinosine, 2,2-Dimethylguanine, 2-Methyladenine, 2-Methylguanine, 3-Methylcytosine, 5-Methylcytosine, N-substituted adenine The nucleic acids are 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylpiperidine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, piperidine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl uracil-5-oxyacetic acid, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic acids of this invention may be purchased from companies such as Macromolecular Resources (Fort Collins, CO) and Synthegen (Houston, TX). Vector

[0264] In some embodiments, the nucleic acid of the present invention is incorporated into the vector. In this regard, the present invention provides vectors comprising any of the nucleic acids of the present invention. In exemplary embodiments, the vector is a recombinant expression vector. For purposes herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, allows the cell to express the mRNA, protein, polypeptide, or peptide when the vector is in contact with a host cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed intracellularly. The vectors of the present invention are generally not naturally occurring. However, a portion of the vector may be naturally occurring. The vectors of the present invention may contain any class of nucleotides, including (but not limited to) DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially derived from natural sources, and may contain natural, non-natural, or modified nucleotides. The vector may contain naturally occurring or non-natural nucleotide inter-bonds or both. In some embodiments, modified nucleotides or non-natural nucleotide inter-bonds do not impede transcription or replication of the vector.

[0265] The vector of the present invention can be any suitable vector and can be used for transformation or transfection of any suitable host. Suitable vectors include vectors designed for propagation and amplification or for expression, or both, such as plastids and viruses. Vectors can be selected from the group consisting of: pUC series (Fermentas Life Sciences), pBluescript series (Stratagene, LaJoIIa, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, CA). Phage vectors such as λGTIO, λGTl 1, λZapII (Stratagene), λEMBL4, and λNMl149 can also be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). In some cases, the vector is a viral vector, such as a retroviral vector.

[0266] The vector of the present invention can be prepared using, for example, the standard recombinant DNA techniques described above by Sambrook et al. and Ausubel et al. The constructs of the expression vector, which are circular or linear, can be prepared to contain a replication system that functions in prokaryotic or eukaryotic host cells. The replication system can be derived from, for example, CoIEl, 2μ plasmid, λ, SV40, bovine papillomavirus and analogues.

[0267] In some forms, the vector contains regulatory sequences, such as transcription and translation start and stop codons, which are specific to the type of host (e.g., bacteria, fungi, plants or animals) to which the vector will be introduced, depending on whether the vector is based on DNA or RNA.

[0268] The vector may include one or more marker genes that allow selection of the host for transformation or transfection. Marker genes include biocidal resistance (e.g., resistance to antibiotics, heavy metals, etc.), supplementation of the original nutrient in auxotrophic hosts, and analogues thereof. Marker genes suitable for the expression vector of the present invention include, for example, neomycin / G418 resistance genes, hygromycin resistance genes, histamine resistance genes, tetracycline resistance genes, and ampicillin resistance genes.

[0269] The vector may contain a natural or standard promoter operatively linked to a nucleotide sequence encoding a polypeptide (including its functional portion and functional variants) or a nucleotide sequence complementary to or hybridized to a nucleotide sequence encoding IL-21, its conjugate, or a fusion protein. For example, the selection of strong, weak, inducible, tissue-specific, and developmentally specific promoters is within the scope of the ordinary skill of a person skilled in the art. Similarly, the combination of nucleotide sequences and promoters is also within the scope of the skill of a person skilled in the art. The promoter may be a non-viral promoter or a viral promoter, such as the cytomegalovirus (CMV) promoter, the SV40 promoter, the RSV promoter, and promoters found in long terminal repeat sequences of murine stem cell viruses. Host cell

[0270] This document provides host cells comprising the nucleic acids or vectors of the present invention. As used herein, the term "host cell" refers to any type of cell that may contain the vectors of the present invention and is capable of producing expression products (e.g., mRNA, proteins) encoded by nucleic acids. In some embodiments, the host cell is an adhesive cell or a suspension cell, i.e., a cell that grows in suspension. In exemplary embodiments, the host cell is a cultured cell or a primary cell, i.e., directly isolated from an organism, such as a human. The host cell may belong to any cell type, may be derived from any type of tissue, and may be at any developmental stage.

[0271] In the exemplary samples, the cells are eukaryotic cells, including (but not limited to) yeast cells, filamentous fungal cells, protozoan cells, algal cells, insect cells, or mammalian cells. Such host cells are described in this technique. See, for example, Frenzel et al., Front Immunol 4: 217 (2013). In the exemplary samples, the eukaryotic cells are mammalian cells. In the exemplary samples, the eukaryotic cells are non-human mammalian cells. In some samples, the cells included Chinese ovarian (CHO) cells and their derivatives (e.g., CHO-K1, CHO pro-3, CS9), mouse myeloma cells (e.g., NS0, GS-NS0, Sp2 / 0), engineered cells lacking dihydrofolate reductase (DHFR) activity (e.g., DUKX-X11, DG44), human embryonic kidney 293 (HEK293) cells or their derivatives (e.g., HEK293T, HEK293-EBNA), African green monkey kidney cells (e.g., COS cells, VERO cells), human cervical cancer cells (e.g., HeLa), human osteosarcoma epithelial cells U2-OS, adenocarcinoma human alveolar basal epithelial cells A549, human fibrosarcoma cells HT1080, mouse brain tumor cells CAD, embryonic carcinoma cells P19, and mouse embryonic fibroblasts NIH. 3T3, mouse fibroblasts L929, mouse neuroblastoma cells N2a, human breast cancer cells MCF-7, retinoblastoma cells Y79, human retinoblastoma cells SO-Rb50, human liver cancer cells Hep G2, mouse B myeloma cells J558L, or young hamster kidney (BHK) cells (Gaillet et al. 2007; Khan, Adv Pharm Bull 3(2): 257-263 (2013)). In one particular embodiment, the host cell was CS9 (CHO cell line).

[0272] is an amplification or replication vector, and in some cases the host cell is a prokaryotic cell, such as a bacterial cell.

[0273] The present invention also provides a cell population comprising at least one of the host cells described herein. In some embodiments, the cell population is a heterogeneous population comprising host cells containing said vector, in addition to at least one other cell type that does not contain any vector. Alternatively, in some embodiments, the cell population is a substantially homogeneous population, wherein the population primarily comprises (e.g., substantially consists of) host cells containing the vector. In some embodiments, the population is a pure-line cell population, wherein all cells in the population are pure lines of a single host cell containing the vector, such that all cells in the population contain the vector. In exemplary embodiments of the present invention, the cell population is a pure-line population comprising host cells containing the vector as described herein. Pharmaceutical Composition

[0274] This document provides compositions comprising IL-21 mutant protein, conjugates comprising IL-21 mutant protein, fusion proteins comprising IL-21 mutant protein and polypeptides, PD-1 antigen-binding proteins (e.g., anti-PD-1 antibodies), conjugates comprising PD-1 antigen-binding proteins (e.g., anti-PD-1 antibodies), fusion proteins comprising PD-1 antigen-binding proteins (e.g., anti-PD-1 antibodies), nucleic acids, vectors, or host cells or combinations thereof. In some embodiments, the compositions comprise, in isolated and / or purified forms, the IL-21 mutant protein, PD-1 antigen-binding protein (e.g., anti-PD-1 antibody), conjugates, fusion proteins, nucleic acids, vectors, or host cells or combinations thereof. In some forms, the composition comprises a single type (e.g., structure) of the present invention’s IL-21 mutant protein, PD-1 antigen-binding protein (e.g., anti-PD-1 antibody), conjugate, fusion protein, nucleic acid, vector, or host cell, or a combination comprising two or more different types of the present invention’s IL-21 mutant protein, PD-1 antigen-binding protein, conjugate, fusion protein, nucleic acid, vector, or host cell.

[0275] In exemplary samples, the composition comprises an agent that enhances the chemophysical characteristics of, for example, IL-21 mutant proteins or fusion proteins, such as by stabilizing them at certain temperatures (e.g., room temperature), increasing their shelf life, reducing degradation (e.g., oxidative protease-mediated degradation), increasing their half-life, etc., of IL-21 mutant proteins, PD-1 antigen-binding proteins (e.g., anti-PD-1 antibodies), conjugates, fusion proteins, nucleic acids, vectors, or host cells. In some samples, the composition comprises any of the agents disclosed herein as a heterologous portion or conjugate portion, which may be mixed with the IL-21 mutant protein, conjugate, fusion protein, nucleic acid, vector, or host cell of the present invention, as appropriate.

[0276] In exemplary embodiments of the present invention, the composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the IL-21 mutant protein, PD-1 antigen-binding protein (e.g., anti-PD-1 antibody), conjugate, fusion protein, nucleic acid, vector, or host cell (hereinafter referred to as the "active agent") disclosed herein are formulated into a pharmaceutical composition comprising the active agent and a pharmaceutically acceptable carrier, diluent, or excipient. In this regard, the present invention further provides a pharmaceutical composition comprising the active agent (i.e., any one of the IL-21 mutant protein, PD-1 antigen-binding protein (e.g., anti-PD-1 antibody), conjugate, fusion protein, nucleic acid, vector, or host cell) intended for administration to an individual, such as a mammal.

[0277] In some embodiments, the active agent is present in the pharmaceutical composition at a purity level suitable for administration to a patient. In some embodiments, the active agent has a purity level of at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, and a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments, the composition contains the active agent at a concentration of about 0.001 to about 30.0 mg / ml.

[0278] In the exemplary samples, the pharmaceutical composition comprises a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, emulsions (such as oil / water or water / oil emulsions), and various types of wetting agents. The term also covers any preparation approved by a regulatory agency of the United States federal government or listed in the United States Pharmacopeia for use in animals, including humans.

[0279] Pharmaceutical compositions may contain any pharmaceutically acceptable ingredients, including, for example, acidifiers, additives, adsorbents, aerosol propellants, degassing agents, alkalizing agents, anti-caking agents, anticoagulants, antimicrobial preservatives, antioxidants, bactericides, matrices, binders, buffers, chelating agents, coating agents, colorants, desiccants, detergents, diluents, disinfectants, disintegrants, dispersants, solubilizers, dyes, softeners, emulsifiers, emulsion stabilizers, fillers, film-forming agents, etc. Flavor enhancers, flavoring agents, flow enhancers, gelling agents, granulating agents, wetting agents, lubricants, mucosal adhesives, ointment bases, ointments, oily mediators, organic bases, soft lozenge bases, pigments, plasticizers, polishing agents, preservatives, masking agents, skin penetrants, solubilizers, solvents, stabilizers, suppository bases, surfactants, surfactants, suspending agents, sweeteners, therapeutic agents, thickeners, tensioning agents, toxic agents, viscous agents, water-miscible cosolvents, water softeners, or wetting agents. See, for example, the Handbook of Pharmaceutical Excipients, 3rd edition, AH Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated in its entirety by reference. Remington's Pharmaceutical Sciences, 16th edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated in its entirety by reference.

[0280] In exemplary embodiments, the pharmaceutical composition contains a formulation substance that is non-toxic to the recipient at the dosage and concentration used. In specific embodiments, the pharmaceutical composition comprises an active agent and one or more pharmaceutically acceptable salts; polyols; surfactants; osmotic balancers; toning agents; antioxidants; antibiotics; antifungal agents; extenders; lyophilization protectants; defoamers; chelating agents; preservatives; colorants; analgesics; or other agents. In exemplary embodiments, the pharmaceutical composition may, as appropriate, include (but is not limited to) one or more excipients such as pharmaceutically acceptable salts, osmotic balancers (toning agents), antioxidants, antibiotics, antifungal agents, extenders, lyophilization protectants, defoamers, chelating agents, preservatives, colorants, and analgesics, as well as one or more polyols and / or one or more surfactants.

[0281] In some embodiments, the pharmaceutical composition may contain formulation substances for altering, maintaining or preserving, for example, the pH, permeability, viscosity, transparency, color, isotonicity, odor, sterility, stability, decomposition or release rate, surface absorption or permeation of the composition. In such embodiments, suitable formulations include (but are not limited to) amino acids (such as glycine, glutamine, aspartic acid, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); extenders (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); and low molecular weight... Peptides; salt-forming ions (such as sodium); preservatives (such as algaecides, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (such as glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronic acid, PEG, dehydrated sorbitol esters, polysorbates (such as polysorbate 20), polysorbate, triton, thiazolinone, lecithin cholesterol, tyloxapal); stability enhancers (such as sucrose or sorbitol); tension enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery mediators; diluents; excipients and / or pharmaceutical adjuvants. See REMINGTON'S PHARMACEUTICAL SCIENCES, 18th edition, (edited by AR Genrmo), 1990, Mack Publishing.

[0282] The pharmaceutical composition may be formulated to achieve a physiologically compatible pH value. In some embodiments, the pH value of the pharmaceutical composition may be, for example, between about 4 or about 5 and about 8.0, or between about 4.5 and about 7.5, or between about 5.0 and about 7.5. In exemplary embodiments, the pH value of the pharmaceutical composition is between 5.5 and 7.5. Dosage route

[0283] For the present invention, the active agent or pharmaceutical composition containing the active agent may be administered to an individual via any suitable route of administration. For example, the active agent may be administered to an individual via non-enteral, nasal, oral, pulmonary, topical, vaginal, or rectal administration. The following discussion of routes of administration is provided for illustrative purposes only and should not be construed as limiting the scope in any way.

[0284] Suitable for non-enteral administration of formulations include aqueous and non-aqueous isotonic sterile injectable solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The term "non-enteral" means not via the digestive tract, but via other routes, such as subcutaneous, intramuscular, intraspinal, or intravenous. The active agent of this invention can be administered with physiologically acceptable diluents in pharmaceutical carriers (such as sterile liquids or liquid mixtures, including water, physiological saline, dextran aqueous solutions and related sugar solutions, alcohols (such as ethanol or cetyl alcohol), glycols (such as propylene glycol or polyethylene glycol), dimethyl sulfoxide, glycerol, ketals (such as 2,2-dimethyl-153-dioxolane-4-methanol), ethers, poly(ethylene glycol) 400, oils, fatty acids, fatty acid esters or glycerides or acetylated fatty acid glycerides), with or without the addition of pharmaceutically acceptable surfactants, such as fatty acid salts or detergents, suspending agents (such as pectin, carbomer, methylcellulose, hydroxypropyl methylcellulose or carboxymethylcellulose), or emulsifiers and other pharmaceutical adjuvants.

[0285] Oils that can be used in non-enteric formulations include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, paraffin oil, and mineral oil. Suitable fatty acids for non-enteric formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0286] Suitable fatty acid salts for use in non-enteric formulations include aliphatic alkali metal salts, ammonium salts and triethanolamine salts, and suitable detergents include (a) cationic detergents, such as halodimethyl dialkylammonium and haloalkylpyridinium; (b) anionic detergents, such as alkyl, aryl and olefin sulfonates, alkyl, olefin, ether and monoglyceride sulfates and sulfosuccinates; (c) nonionic detergents, such as fatty amine oxides, fatty acid alkanolamides and polyoxyethylene ethyl polypropylene copolymers; (d) amphoteric detergents, such as alkyl-β-aminopropionates and 2-alkyl-imidazoline quaternary ammonium salts; and (e) mixtures thereof.

[0287] In some embodiments, the non-enteric formulation contains about 0.5% to about 25% by weight of the active agent of the present invention in solution. Preservatives and buffers may be used. To minimize or eliminate irritation to the injection site, such compositions may contain one or more nonionic surfactants with a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations is typically in the range of about 5% to about 15% by weight. Suitable surfactants include polyethylene glycol sorbitan fatty acid esters, such as sorbitan monooleate, and high molecular weight adducts of ethylene oxide with a hydrophobic matrix formed by the condensation of propylene oxide and propylene glycol. In some forms, the non-enteric formulation is presented in single-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored under lyophilized (freeze-dried) conditions, requiring only the addition of a sterile liquid excipient, such as water for injection, just before use. In some cases, sterile powders, granules, and tablets of the previously described types are used to prepare injectable solutions and suspensions for immediate use.

[0288] The injectable formulation is based on the present invention. The requirements for effective pharmaceutical carriers used in injectable compositions are well known to those skilled in the art (see, for example, *Pharmaceutics and Pharmacy Practice*, JB Lippincott Company, Philadelphia, PA, edited by Banker and Chalmers, pp. 238-250 (1982), and *ASHP Handbook on Injectable Drugs*, Toissel, 4th edition, pp. 622-630 (1986)). Dosage

[0289] It is believed that the active agent of the present invention can be used in methods for inhibiting PD-1 signaling while providing IL-21 signaling as described herein, and therefore it is believed that it can be used in methods for treating or preventing one or more diseases, such as cancer. For the purposes of the present invention, the amount or dose of the active agent administered shall be sufficient to achieve, for example, a therapeutic or preventive effect in an individual or animal within a reasonable time frame. For example, the dose of the active agent of the present invention shall be sufficient to treat the cancer described herein for about 1 to 4 minutes, 1 to 4 hours, or 1 to 4 weeks or longer, such as 5 to 20 weeks or more, from the time of administration. In some embodiments, the time frame may be even longer. The dose will be determined by the efficacy of the particular active agent and the condition of the animal (e.g., a human) to be treated and the weight of the animal (e.g., a human) to be treated.

[0290] Numerous analyses for determining dosage are known in the art. For purposes herein, analyses including comparing the degree of cancer treatment following administration of a predetermined dose of the active agent of the present invention to one of a group of mammals may be used to determine the initial dose administered to the mammals, each group of mammals receiving different doses of the active agent. The degree of cancer treatment following administration of a given dose may be represented by, for example, the cytotoxicity of the active agent in a mouse xenograft model or the degree of tumor regression achieved with the active agent. Methods for measuring the cytotoxicity of the fusion protein and for analyzing tumor regression are known in the art.

[0291] The dosage of the active agent of the present invention is also determined by the presence, nature, and extent of any adverse side effects that may accompany the administration of the specific active agent of the present invention. Typically, the attending physician will consider various factors such as age, weight, overall health, diet, sex, the active agent of the present invention to be administered, the route of administration, and the severity of the condition being treated to determine the dosage of the active agent of the present invention for treating individual patients. By way of example and without intention to limit the present invention, the dosage of the active agent of the present invention may be approximately 0.0001 to approximately 1 g, approximately 0.0001 to approximately 0.001 g, or approximately 0.01 mg to approximately 1 g per kilogram of the individual body weight treated per day. Controlled release formulation

[0292] In some embodiments, the active agent described herein may be modified into an accumulation form such that the release of the active agent of the present invention into the body is controlled in terms of in vivo time and location (see, for example, U.S. Patent No. 4,450,150). The accumulation form of the active agent of the present invention may be, for example, an implantable composition comprising the active agent and a porous or non-porous material, such as a polymer, wherein the active agent is encapsulated or diffused within the material and / or the non-porous material degrades. The accumulation is then implanted into the desired location within the individual, and the active agent is released from the implant at a predetermined rate.

[0293] In some formulations, the pharmaceutical composition containing the active agent is modified to have any type of in vivo release profile. In some formulations, the pharmaceutical composition is an immediate-release, controlled-release, sustained-release, extended-release, delayed-release, or biphasic-release formulation. Methods for formulating peptides for controlled-release are known in the art. See, for example, Qian et al., J Pharm 374: 46-52 (2009) and International Patent Application Publications Nos. WO 2008 / 130158, WO2004 / 033036, WO2000 / 032218 and WO 1999 / 040942.

[0294] The compositions of the present invention may further comprise, for example, micelles or liposomes or some other encapsulation form, or may be administered in a prolonged release form to provide long-term storage and / or delivery.

[0295] In some embodiments, the fusion protein or antigen-binding protein described herein (e.g., an anti-PD-1 antibody, its antigen-binding antibody fragment, or an anti-PD-1 antibody protein product) is administered alone, and in alternative embodiments, it is administered in combination with another therapeutic agent, such as another active agent of the present invention of a different type (e.g., structure). In some embodiments, the other therapeutic agent is intended to treat or prevent cancer. In some embodiments, the other therapeutic agent is a chemotherapeutic agent. In some embodiments, the other therapeutic agent is an agent used in radiotherapy for the treatment of cancer. Therefore, in some embodiments, the fusion protein or antigen-binding protein described herein (e.g., an anti-PD-1 antibody, its antigen-binding antibody fragment, or an anti-PD-1 antibody protein product) is administered in combination with one or more of platinum coordination compounds, topoisomerase inhibitors, antibiotics, antimitotic alkaloids, and difluoronucleosides. In the exemplary samples, the IL-21 fusion protein described herein (e.g., an anti-PD-1 antibody fused to a mutant IL-21 protein) is combined with an antigen-binding protein (e.g., an anti-PD-1 antibody, its antigen-binding antibody fragment, or an anti-PD-1 antibody protein product).

[0296] In a specific embodiment, any one of antibodies 20A2, 20C1, 22D4, 20C1.006, 20C1.009, 20A2.003, 22D4.006, and 22D4.017 is administered in combination with the IL-21 fusion protein described herein, including, for example, fusion proteins comprising homodimers or monomers selected from: homodimers comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each fused to an IL-21 mutant protein and each heavy chain-IL-21 mutant protein fusion comprising the amino acid sequence of SEQ ID NO: 496); homodimers comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each fused to an IL-21 mutant protein and the fused heavy chain-IL-21 mutant protein comprising the amino acid sequence of SEQ ID NO: 391); and homodimers comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two antibody heavy chains (each fused to an IL-21 mutant protein and the fused heavy chain-IL-21 mutant protein comprising the amino acid sequence of SEQ ID NO: 391). Homodimers comprising: SEQ ID NO: 497 (amino acid sequence); SEQ ID NO: 391 (amino acid sequence) and SEQ ID NO: 498 (amino acid sequence) of two antibody light chains; SEQ ID NO: 391 (amino acid sequence) and SEQ ID NO: 499 (amino acid sequence) of two antibody heavy chains; SEQ ID NO: 500 (amino acid sequence) of two antibody light chains; SEQ ID NO: 391 (amino acid sequence) and SEQ ID NO: 498 (amino acid sequence) of two antibody heavy chains; SEQ ID NO: 499 (amino acid sequence) of two antibody light chains; SEQ ID NO: 500 (amino acid sequence) of two antibody heavy chains; SEQ ID NO: 499 (amino acid sequence) of two antibody light chains; SEQ ID NO: 391 (amino acid sequence) of two antibody heavy chains; SEQ ID NO: 500 (amino acid sequence) of two antibody light chains; SEQ ID NO: 391 (amino acid sequence) of two antibody heavy chains; SEQ ID NO: 49 ...9 (amino acid sequence) of two antibody heavy chains; SEQ ID NO: 499 (amino acid sequence) of two antibody heavy chains; SEQ ID NO: 500 (amino acid sequence) of two antibody heavy chains; SEQ ID NO: 499 (amino acid sequence) of two antibody heavy chains; SEQ ID NO: 499 (amino acid sequence) of two antibody heavy chains; SEQ ID A monomer comprising: an amino acid sequence of SEQ ID NO: 391 and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 501, and the other not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 555); and a monomer comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 502, and the other not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 556).A monomer comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 503, and the other not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 557); a monomer comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 504, and the other not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 555); a monomer comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 505, and the other not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 505). The following are considered homodimers: a monomer comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 391) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 506, and the other not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 557); a homodimer comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each fused to the IL-21 mutant protein, and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 507); and a homodimer comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each fused to the IL-21 mutant protein, and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 508). A homodimer comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each fused to an IL-21 mutant protein and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 509); A homodimer comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each fused to an IL-21 mutant protein and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 510);Homodimers comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each fused to the IL-21 mutant protein and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 511); Homodimers comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two antibody heavy chains (each fused to the IL-21 mutant protein and each heavy chain-IL-21 mutant protein fusion containing the amino acid sequence of SEQ ID NO: 512); Monomers comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 513 and one not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 558); Monomers comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 558); A monomer comprising: an amino acid sequence of SEQ ID NO: 371 and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 514, and the other not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 559); a monomer comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 515, and the other not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 560); a monomer comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 516, and the other not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 558); A monomer comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 517, and the other not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 559); or a monomer comprising two antibody light chains (each containing the amino acid sequence of SEQ ID NO: 371) and two different antibody heavy chains (one fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 518, and the other not fused to the IL-21 mutant protein and containing the amino acid sequence of SEQ ID NO: 560). Kit;

[0297] The present invention further provides a kit comprising the IL-21 mutant protein of the present invention, a PD-1 antigen-binding protein (e.g., an anti-PD-1 antibody), a conjugate, a fusion protein, a nucleic acid, a vector, or a host cell or a combination thereof. In an exemplary embodiment, the kit contains at least one IL-21 mutant protein of the present invention, a PD-1 antigen-binding protein (e.g., an anti-PD-1 antibody), a conjugate, a fusion protein, a nucleic acid, a vector, or a host cell or a combination thereof in a container. In an exemplary embodiment, at least one IL-21 mutant protein of the present invention, a PD-1 antigen-binding protein (e.g., an anti-PD-1 antibody), a conjugate, a fusion protein, a nucleic acid, a vector, or a host cell is provided in the kit in a unit dose. For purposes herein, "unit dose" refers to a discrete amount dispersed in a suitable carrier. In an exemplary embodiment, the unit dose is an amount sufficient to provide the desired effect to an individual, such as for treating cancer. In an exemplary embodiment, the kit contains several unit doses, such as a weekly or monthly supply of unit doses, each individually packaged or otherwise separated from other unit doses, as appropriate. In some embodiments, the kit / unit dose components are packaged together with instructions for patient administration. In some embodiments, the kit includes one or more devices for patient administration, such as needles and syringes and the like. In some embodiments, at least one of the IL-21 mutant proteins of the present invention, PD-1 antigen-binding proteins (e.g., anti-PD-1 antibodies), conjugates, fusion proteins, nucleic acids, vectors, or host cells, or combinations thereof, are pre-packaged in spare forms, such as syringes, intravenous bags, etc. In exemplary embodiments, the spare forms are for single use. In exemplary embodiments, the kit includes multiple single-use spare forms of at least one of the IL-21 mutant proteins of the present invention, PD-1 antigen-binding proteins (e.g., anti-PD-1 antibodies), conjugates, fusion proteins, nucleic acids, vectors, or host cells. In some embodiments, the kit further includes other therapeutic or diagnostic agents or pharmaceutically acceptable carriers (e.g., solvents, buffers, diluents, etc.), including any of those described herein. Manufacturing Method

[0298] The IL-21 mutant protein of the present invention can be obtained by methods known in this art. Suitable methods for resynthesizing peptides are described, for example, in: Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2005; Peptide and Protein Drug Analysis, edited by Reid, R., Marcel Dekker, 2000; Epitope Mapping, edited by Westwood et al., Oxford University Press, Oxford, United Kingdom, 2000; and U.S. Patent No. 5,449,752. Other exemplary methods for producing the peptides of the present invention are set forth herein.

[0299] In some embodiments, the IL-21 mutant protein described herein is commercially synthesized by companies such as Synpep (Dublin, CA), Peptide Technologies (Gaithersburg, MD), Multiple Peptide Systems (San Diego, CA), Peptide 2.0 (Chantilly, VA), and American Peptide (Sunnyvale, CA). In this respect, the IL-21 mutant protein may be synthetic, recombinant, isolated, and / or purified.

[0300] In addition, in some variants, the IL-21 mutant protein is generated using nucleic acids encoding the amino acid sequence of the peptide, recombined using standard recombination methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994.

[0301] This document provides a method for manufacturing IL-21 mutant protein. In an exemplary embodiment, the method includes culturing the host cells of the present invention to express the IL-21 mutant protein and harvesting the expressed IL-21 mutant protein.

[0302] This document also provides a method for manufacturing a fusion protein comprising an IL-21 mutant protein. In an exemplary embodiment, the method includes culturing the host cells of the present invention to express the fusion protein and harvesting the expressed fusion protein.

[0303] In an exemplary embodiment, the method includes culturing host cells containing nucleic acids encoding the IL-21 mutant protein or fusion protein as described herein to express the IL-21 mutant protein or fusion protein. The host cell may be any of the host cells described herein. In an exemplary embodiment, the host cell line is selected from the group consisting of: CHO cells, NSO cells, COS cells, VERO cells, and BHK cells. In an exemplary embodiment, the step of culturing the host cells includes culturing the host cells in a growth medium to support the growth and expansion of the host cells. In an exemplary embodiment, the growth medium increases cell density, culture viability, and productivity in a timely manner. In an exemplary embodiment, the growth medium contains amino acids, vitamins, inorganic salts, glucose, and serum as sources of growth factors, hormones, and adhesion factors. In an exemplary embodiment, the growth medium is a chemically defined medium consisting of amino acids, vitamins, trace elements, inorganic salts, lipids, and insulin or insulin-like growth factor. In addition to nutrients, growth media also help maintain pH and osmotic pressure. Several growth media are commercially available and described in this technique. See, for example, Arora, "Cell Culture Media: A Review" MATER METHODS 3:175 (2013).

[0304] In the exemplary embodiment, the method for producing the IL-21 mutant protein or fusion protein of the present invention includes culturing host cells in a feed medium. In the exemplary embodiment, the method includes culturing in a feed medium in a batch feed mode. Methods for producing recombinant proteins are known in the art. See, for example, Li et al., "Cell culture processes for monoclonal antibody production" MAbs 2(5): 466-477 (2010).

[0305] Methods for producing IL-21 mutant or fusion proteins may include one or more steps for purifying the mutant protein or protein from cell cultures or their supernatants, and preferably recovering the purified protein. In exemplary samples, the method includes one or more chromatographic steps, such as affinity chromatography (e.g., protein A affinity chromatography), ion exchange chromatography, and hydrophobic interaction chromatography. In exemplary samples, the method includes purifying the protein using a protein A affinity chromatography resin.

[0306] In an exemplary embodiment, the method further includes a step of formulating the purified protein, etc., to obtain a formulation containing the purified protein. Such steps are described in *Formulation and Process Development Strategies for Manufacturing*, edited by Jameel and Hershenson, John Wiley & Sons (Hoboken, NJ), 2010. Method of Use

[0307] The present invention further provides a treatment method. In an exemplary embodiment, the method is a method of treating an individual in need, comprising administering to the individual in need an amount of the pharmaceutical composition of the present invention that is effective in treating the individual.

[0308] The pharmaceutical compositions of the present invention can be used to inhibit PD-1 signaling and / or activate IL-21 signaling. Without limitation to a particular theory, [1] the PD-1 inhibitory activity of the compositions provided herein allows such entities to be used in methods of enhancing T cell activity and immune responses, particularly against tumors or cancer; and / or [2] the IL-21 activating activity of the compositions provided herein allows such entities to enhance T cell survival and effector function, limit terminal differentiation and loss of replication potential, promote T cell lifespan by altering activated effector cells to a more natural T cell phenotype (e.g., by enhancing CCR7 expression), and enhance cytotoxicity against target (e.g., cancer) cells (e.g., by increasing IFNγ and granzyme B production).

[0309] Therefore, this document provides methods for enhancing T cell activity, improving T cell survival and effector function, limiting terminal differentiation and loss of replication potential, promoting T cell lifespan, and enhancing cytotoxicity against target (e.g., cancer) cells. In exemplary embodiments, these methods include administering an effective amount of the pharmaceutical composition of the present invention to an individual. In exemplary embodiments, the T cell activity or immune response is directed against cancer cells or cancer tissue or tumor cells or tumors. In exemplary embodiments, the immune response is a humoral immune response. In exemplary embodiments, the immune response is an innate immune response. In exemplary embodiments, the enhanced immune response is a T cell-mediated immune response.

[0310] As used herein, the term "enhancement" and words derived therefrom may not mean 100% or complete enhancement or increase. Rather, it refers to varying degrees of enhancement that are considered by those skilled in the art to have potential benefit or therapeutic effect. In this regard, the pharmaceutical compositions of the present invention can enhance, for example, T-cell activity or enhance immune responses to any amount or level. In exemplary embodiments, the enhancement provided by the method of the present invention is at least or about 10% enhancement (e.g., at least or about 20% enhancement, at least or about 30% enhancement, at least or about 40% enhancement, at least or about 50% enhancement, at least or about 60% enhancement, at least or about 70% enhancement, at least or about 80% enhancement, at least or about 90% enhancement, at least or about 95% enhancement, at least or about 98% enhancement).

[0311] Methods for measuring T cell activity and immune response are known in this art. T cell activity can be measured by, for example, cytotoxicity assays, such as those described in Fu et al., PLoS ONE 5(7): e11867 (2010). Other T cell activity assays are described in Bercovici et al., Clin Diagn Lab Immunol. 7(6): 859-864 (2000). Methods for measuring immune response are described, for example, in Macatangay et al., Clin Vaccine Immunol 17(9): 1452-1459 (2010) and Clay et al., Clin Cancer Res. 7(5): 1127-35 (2001).

[0312] This document also provides methods for treating an individual suffering from cancer and methods for treating an individual with a solid tumor. In an exemplary embodiment, the method includes administering to the individual an amount of the pharmaceutical composition of the present invention that is effective in treating the individual's cancer or solid tumor. The cancer that can be treated by the methods disclosed herein can be any cancer, such as any malignant growth or tumor caused by abnormal and uncontrolled cell division that can spread to other parts of the body via the lymphatic system or bloodstream. In some cases, cancer is selected from the following groups: acute lymphoblastic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal or anorectal region, eye cancer, intrahepatic bile duct cancer, joint cancer, cancer of the neck, gallbladder or pleura, cancer of the nose, nasal cavity or middle ear, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, Hodgkin's lymphoma, tongue cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal carcinoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), small bowel cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer and bladder cancer. In a specific embodiment, the cancer is selected from the group consisting of: head and neck cancer, ovarian cancer, cervical cancer, bladder and esophageal cancer, pancreatic cancer, gastrointestinal cancer, gastric cancer, breast cancer, endometrial cancer and colorectal cancer, hepatocellular carcinoma, glioblastoma, bladder cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), and bronchioloalveolar carcinoma. In a specific embodiment, the tumor is non-small cell lung cancer (NSCLC), head and neck cancer, kidney cancer, triple-negative breast cancer, and gastric cancer. In an illustrative embodiment, an individual has a tumor (e.g., a solid tumor, a hematological malignancy, or a lymphatic malignancy) and is given a pharmaceutical composition in an amount effective in treating the individual's tumor. In other exemplary cases, the tumor is non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, kidney cancer, breast cancer, melanoma, ovarian cancer, liver cancer, pancreatic cancer, colon cancer, prostate cancer, stomach cancer, lymphoma, or leukemia, and the individual is given a dose of pharmaceutical composition effective in treating the individual's tumor.

[0313] As used herein, the term "treatment" and related terms do not necessarily imply 100% or complete cure. Rather, it refers to treatment of varying degrees that are considered potentially beneficial or therapeutic by those skilled in the art. In this respect, the cancer treatment methods of the present invention can provide any amount or level of treatment. Furthermore, treatment provided by the methods of the present invention may include treating one or more symptoms or signs of the cancer being treated. Treatment provided by the methods of the present invention may also cover slowing the progression of cancer. For example, these methods may treat cancer by enhancing T-cell activity or the immune response against cancer, reducing tumor or cancer growth, reducing tumor cell metastasis, increasing cell death of tumor or cancer cells, and similar aspects. In exemplary embodiments, the methods are performed by delaying the onset or recurrence of cancer for 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, two months, 4 months, 6 months, 1 year, 2 years, 4 years, or longer. In exemplary embodiments, these methods are performed by increasing the survival of the individual.

[0314] In some embodiments of the present invention, the individual is a mammal, including (but not limited to) rodent mammals such as mice and hamsters; and lagomorph mammals such as rabbits; mammals from the order Carnivora, including felines (cats) and canines (dogs); mammals from the order Artiodactyla, including bovids (cows) and suidae (pigs); or perissodactyls, including equines (horses). In some embodiments, the mammal belongs to the order Primates, Ceboid, or Simoid (monkeys) or Anthropoids (humans and apes). In some embodiments, the mammal is a human. Exemplary Embodiments

[0315] In an exemplary embodiment, the present invention provides an IL-21 mutant protein comprising the amino acid sequence of SEQ ID NO: 2, QGQDX HMXXM XXXXX XVDXL KNXVN DLVPE FLPAP EDVET NCEWS AFSCF QKAQL KSANT GNNEX XIXXX XXXLX XXXXX TNAGR RQKHR LTCPS CDSYE KKPPK EFLXX FXXLL XXMXX QHXSS RTHGS EDS (SEQ ID NO: 2), wherein "X" represents any amino acid, and wherein the amino acid sequence of the IL-21 mutant protein differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by at least one amino acid.

[0316] In the exemplary sample, the IL-21 mutant protein contains an amino acid sequence that differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by up to 7 amino acids. In the exemplary sample, the IL-21 mutant protein contains an amino acid sequence that differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 3, 4, 5, or 6 amino acids. In the exemplary case, the IL-21 mutant protein contains an amino acid sequence that differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 1 or 2 amino acids. In the exemplary samples, the difference between the amino acid sequence of the IL-21 mutant protein and the amino acid sequence of SEQ ID NO: 1 lies in amino acids 10-15 (including amino acids 10 and 15) or amino acids 105-123 (including amino acids 105 and 123) of SEQ ID NO: 2, wherein, as appropriate, the difference occurs at amino acids 11, 14, 15, 109, 110, 112, 113, 116, 119, 120 and / or 123 of SEQ ID NO: 2. In the exemplary samples, the amino acid sequence difference between the IL-21 mutant protein and the amino acid sequence of SEQ ID NO: 1 is within amino acids 5-25 (including amino acids 5 and 25) or amino acids 65-80 (including amino acids 65 and 80) of SEQ ID NO: 2, whereby the difference occurs at amino acids 5, 8, 9, 12, 13, 16, 19, 23, 65, 66, 69, 70, 72, 73, 75, 76, 77, 78, 79 and / or 80 of SEQ ID NO: 2.

[0317] In some forms, the IL-21 mutant protein comprises an amino acid sequence having an amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1). In some cases, the amino acid substitution occurs at positions 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120, or 123 of SEQ ID NO: 1. In the illustrative sample, amino acid substitutions occur at positions 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120, or 123 of SEQ ID NO: 1. In exemplary cases, the IL-21 mutant protein comprises amino acid substitutions at the following positions: a. Positions 5, 8, 9, 12, 14, 15, 65, 66, 69, 70, 72, 73, 75, 76, 77, 80, 116, or 119 of SEQ ID NO: 1, wherein the substituted amino acid is an aliphatic amino acid; b. Positions 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 69, 70, 72, 73, 75, 76, 77, 78, 79, 110, 112, 116, 117, 119, 120, or 123 of SEQ ID NO: 1, wherein the substituted amino acid is an acidic amino acid; c. SEQ ID NO: d. SEQ ID NO: 1 at positions 5, 9, 73, 76, 109, 113, or 116, wherein the substituted amino acid is a basic amino acid; e. SEQ ID NO: 1 at positions 5, 8, 9, 70, or 76, wherein the substituted amino acid is an aromatic amino acid; f. SEQ ID NO: 1 at positions 5, 8, 9, 12, 15, 73, 76, 116, or 119, wherein the substituted amino acid is an amino acid containing a amide side chain; g. SEQ ID NO: h. Position 65, 66, 69, 70, 72, 73, 75, 76, 77 or 80 of SEQ ID NO: 1, wherein the substituted amino acid is an imine; or i. Position 5, 9, 15, 76, 116 or 119 of SEQ ID NO: 1, wherein the substituted amino acid is an amino acid containing a sulfur-containing side chain; or i. or a combination thereof.

[0318] In the exemplary samples, the substituted amino acids are naturally occurring amino acids. In some cases, the IL-21 mutant protein contains amino acid substitutions at the positions specified in Table A. Table A is shown below. Table A amino acid position of SEQ ID NO: 1 Amino acids (by single-letter code) amino acid position of SEQ ID NO: 1 Amino acids (by single-letter code) 5 A, D, E, G, H, I, K, L, M, N, Q, S, T, V or Y 72 D, G, or P 8 A, D, E, N, S, T, V, or Y 73 A, D, E, G, H, I, N, P, Q, S, or V 9 A, D, E, G, H, I, K, L, M, N, Q, S, T, V or Y 75 D, G, or P 11 D or S 76 A, D, E, G, H, I, K, L, M, N, P, Q, S, T, V or Y 12 A, D, E, N, S, T, or V 77 D, G, or P 13 D 78 D 14 A, D, or S 79 D 15 A, E, I, M, N, Q, S, T or V 80 G or P 16 D or E 109 K 19 D 110 D twenty three D 112 D 65 D, G, or P 113 K 66 D, G, or P 116 A, D, E, I, K, L, M, N, S, T or V 68 Q 117 D 69 D, G, or P 119 A, D, E, M, N, Q, S, or T 70 E, G, P or Y 120 D 71 L 123 D

[0319] In an exemplary embodiment, the present invention provides an IL-21 mutant protein comprising an amino acid sequence comprising any one of SEQ ID NO: 3-21, 23-56, 58-112, 114-208, 210-222, 224-255 and 283.

[0320] In the exemplary sample, the IL-21 mutant protein comprises an amino acid sequence having two amino acid substitutions relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1). In the exemplary sample, the amino acid substitutions occur at positions 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120, or 123 of SEQ ID NO: 1. In the exemplary case, the amino acid substitutions occur at positions 5, 9, 15, 70, 71, 72, 73, and 76 of SEQ ID NO: 1. Depending on the situation, amino acid substitutions may occur at positions 5, 9, 73, and 76 of SEQ ID NO: 1. In some samples, one of these substitutions may occur at position 76 of SEQ ID NO: 1. In an exemplary case, the substituted amino acid at position 76 of SEQ ID NO: 1 is an aliphatic amino acid or an acidic amino acid. In an exemplary sample, the IL-21 mutant protein contains an amino acid substitution at position 5, 9, or 73 of SEQ ID NO: 1, and the substituted amino acid is an aliphatic amino acid or an acidic amino acid. In some samples, the IL-21 mutant protein contains an amino acid substitution at position 1 of SEQ ID NO: 1, and the substituted amino acid is an amino acid with a side-chain amide. In some cases, the aliphatic amino acid is alanine, the acidic amino acid is glutamic acid, or the amino acid with a side-chain amide is glutamic acid. In exemplary embodiments, the present invention provides IL-21 mutant proteins comprising amino acid sequences selected from the group consisting of: SEQ ID NO: 208, 210 to 222, 224 to 248 and 255.

[0321] Regarding any of the above-described forms, the IL-21 mutant protein can bind to the IL-21 receptor with a reduced affinity relative to wild-type IL-21. In some forms, the IL-21 receptor has the amino acid sequence of SEQ ID NO: 256 or 261. In some cases, the IL-21 mutant protein binds to the IL-21 receptor γ chain having the amino acid sequence of SEQ ID NO: 257. In an exemplary case, the IL-21 mutant protein of the present invention binds to the human IL-21 receptor at a Kd greater than or equal to about 0.04 nM.

[0322] A conjugate is further provided. In an exemplary embodiment, the conjugate comprises either the IL-21 mutant protein at the beginning and a heterologous portion. In an exemplary case, IL-21 is directly attached to the heterologous portion. In an alternative case, IL-21 is attached to the heterologous portion via a linker. In some embodiments, the linker comprises, for example, a peptide comprising the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 262). In an exemplary embodiment, the heterologous portion is a polypeptide, wherein the polypeptide is, where appropriate, an antigen-binding protein. In some cases, the heterologous polypeptide is an antibody or an antigen-binding antibody fragment thereof. In an exemplary embodiment, the antibody is an anti-PD-1 antibody. In some embodiments, the IL-21 mutant protein is directly attached to the Fc of the antibody. In an exemplary embodiment, the IL-21 mutant protein is attached to the Fc of the antibody via a linker. In some embodiments, the conjugate comprises a single IL-21 mutant protein, wherein the single IL-21 mutant protein is attached to the C-terminus of one of the two antibody heavy chains. In an exemplary embodiment, the conjugate comprises two IL-21 mutant proteins, wherein a first IL-21 mutant protein is attached to the C-terminus of a first antibody heavy chain, and a second IL-21 mutant protein is attached to the C-terminus of a second antibody heavy chain. Optionally, the first IL-21 has the same amino acid sequence as the second IL-21 mutant protein. Alternatively, the first IL-21 has a different amino acid sequence than the second IL-21 mutant protein. In exemplary embodiments, the antibody heavy chain contains a charge pair mutation (e.g., V1, V4, V103, or V131 mutation). In exemplary embodiments of the conjugate of any of the preceding segments, the IL-21 mutant protein comprises amino acid substitutions at positions 5, 9, 73, and 76 of SEQ ID NO: 1. In the exemplary sample, the conjugate comprises an IL-21 mutant protein containing the amino acid sequence of SEQ ID NO: 1, the difference being that the IL-21 mutant protein contains an amino acid substitution at any two of positions 5, 9, 73 and 76 of SEQ ID NO: 1; and an anti-PD-1 antibody, wherein the IL-21 mutant protein is linked to the C-terminus of the anti-PD-1 antibody.In the illustrative sample, the conjugate comprises an anti-PD-1 antibody comprising: (a) the heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence described in Table D, or a sequence selected from the group consisting of SEQ ID NO: 312, 322, 332, 342, 352, 362, 372, and 382, ​​or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (b) the HC CDR 2 amino acid sequence described in Table D, or a sequence selected from the group consisting of SEQ ID NO: 313, 323, 333, 343, 353, 363, 373, and 383, or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; (c) the HC CDR 3 amino acid sequence described in Table D, or a sequence selected from the group consisting of SEQ ID NO: (d) Sequences of the light chain (LC) CDR1 amino acid sequences described in Table D, or sequences selected from the group consisting of 314, 324, 334, 345, 355, 365, 375, and 385, or sequences differing from only one or two amino acids ... The CDR3 amino acid sequence, or a sequence selected from the group consisting of 317, 327, 337, 347, 357, 367, 377 and 387, or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or a combination of any two or more of (g) (a)-(f). In an exemplary case, the anti-PD-1 antibody comprises six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 312-317; (b) SEQ ID NO: 322-327; (c) SEQ ID NO: 332-337; (d) SEQ ID NO: 342-347; (e) SEQ ID NO: 352-357; (f) SEQ ID NO: 362-367; (g) SEQ ID NO: 372-377; and (h) SEQ ID NO: 382-387.In some samples, the anti-PD-1 antibody comprises a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 318 and 319; (b) SEQ ID NO: 328 and 329; (c) SEQ ID NO: 338 and 339; (d) SEQ ID NO: 348 and 349; (e) SEQ ID NO: 358 and 359; (f) SEQ ID NO: 368 and 369; (g) SEQ ID NO: 378 and 379; and (h) SEQ ID NO: 388 and 389. In exemplary samples, the anti-PD-1 antibody comprises a constant region containing an amino acid sequence of any one of SEQ ID NO: 265-267, 282, or 284-311. In some cases, anti-PD-1 antibodies comprise a pair of amino acid sequences selected from the following groups: (a) SEQ ID NO: 320 and 321; (b) SEQ ID NO: 330 and 331; (c) SEQ ID NO: 340 and 341; (d) SEQ ID NO: 350 and 351; (e) SEQ ID NO: 360 and 361; (f) SEQ ID NO: 370 and 371; (g) SEQ ID NO: 380 and 381; and (h) SEQ ID NO: 390 and 391.

[0323] In exemplary embodiments, the present invention provides a fusion polypeptide or fusion protein comprising the IL-21 mutant protein described herein and a heterologous polypeptide or peptide. In some embodiments, the fusion polypeptide or fusion protein comprises an immunoglobulin or an antigen-binding antibody fragment thereof. In exemplary embodiments, the present invention provides a nucleic acid comprising a nucleotide sequence encoding the IL-21 mutant protein described herein. In exemplary embodiments, the present invention provides a vector comprising the nucleic acid described herein. In exemplary embodiments, the present invention provides a host cell comprising the nucleic acid or vector described herein. In exemplary embodiments, the present invention provides a kit comprising the IL-21 mutant protein as described herein, a nucleic acid, a vector, a host cell, a conjugate, a fusion protein, or a combination thereof and a container.

[0324] Further, a pharmaceutical composition is provided, comprising the IL-21 mutant protein of the present invention, nucleic acid, vector, host cell, conjugate, fusion protein or combination thereof, and a pharmaceutically acceptable carrier, excipient or diluent.

[0325] A method for manufacturing IL-21 mutant protein is also provided, comprising culturing the host cells of the present invention to express the IL-21 mutant protein and harvesting the expressed IL-21 mutant protein. Further, a method for treating an individual in need is provided. This method comprises administering to the individual in need an amount of the pharmaceutical composition of the present invention that is effective in treating the individual. In an exemplary embodiment, the individual has a solid tumor and the pharmaceutical composition is administered to the individual in an amount effective in treating the solid tumor of the individual. Where appropriate, the solid tumor is selected from the group consisting of: head and neck cancer, ovarian cancer, cervical cancer, bladder and esophageal cancer, pancreatic cancer, gastrointestinal cancer, gastric cancer, breast cancer, endometrial cancer and colorectal cancer, hepatocellular carcinoma, glioblastoma, bladder cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), and bronchioloalveolar carcinoma.

[0326] In an exemplary embodiment, the present invention provides a PD-1 antigen-binding protein comprising: (a) a heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence as described in Table D, or a sequence selected from the group consisting of SEQ ID NO: 312, 322, 332, 342, 352, 362, 372, and 382, ​​or a variant sequence differing from only one or two amino acids or having at least or about 70% sequence identity; (b) an HC CDR 2 amino acid sequence as described in Table D, or a sequence selected from the group consisting of SEQ ID NO: 313, 323, 333, 343, 353, 363, 373, and 383, or a variant sequence differing from only one or two amino acids or having at least or about 70% sequence identity; (c) an HC CDR 3 amino acid sequence as described in Table D, or a sequence selected from the group consisting of SEQ ID NO: (d) Sequences of the light chain (LC) CDR1 amino acid sequences described in Table D, or sequences selected from the group consisting of 314, 324, 334, 345, 355, 365, 375, and 385, or sequences differing from only one or two amino acids ... The CDR3 amino acid sequence, or a sequence selected from the group consisting of 317, 327, 337, 347, 357, 367, 377 and 387, or a variant sequence differing by only one or two amino acids or having at least or about 70% sequence identity; or a combination of any two or more of (g) (a)-(f). In an exemplary case, the PD-1 antigen-binding protein comprises six CDR amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 312-317; (b) SEQ ID NO: 322-327; (c) SEQ ID NO: 332-337; (d) SEQ ID NO: 342-347; (e) SEQ ID NO: 352-357; (f) SEQ ID NO: 362-367; (g) SEQ ID NO: 372-377; and (h) SEQ ID NO: 382-387.In certain embodiments, the PD-1 antigen-binding protein comprises a pair of amino acid sequences selected from the group consisting of: (a) SEQ ID NO: 318 and 319; (b) SEQ ID NO: 328 and 329; (c) SEQ ID NO: 338 and 339; (d) SEQ ID NO: 348 and 349; (e) SEQ ID NO: 358 and 359; (f) SEQ ID NO: 368 and 369; (g) SEQ ID NO: 378 and 379; and (h) SEQ ID NO: 388 and 389. In exemplary cases, the PD-1 antigen-binding protein comprises a constant region containing an amino acid sequence of any one of SEQ ID NO: 265-267, 282, or 284-311. In some forms, the PD-1 antigen-binding protein comprises a pair of amino acid sequences selected from the following groups: (a) SEQ ID NO: 320 and 321; (b) SEQ ID NO: 330 and 331; (c) SEQ ID NO: 340 and 341; (d) SEQ ID NO: 350 and 351; (e) SEQ ID NO: 360 and 361; (f) SEQ ID NO: 370 and 371; (g) SEQ ID NO: 380 and 381; and (h) SEQ ID NO: 390 and 391.

[0327] In exemplary embodiments, the present invention provides a conjugate comprising the PD-1 antigen-binding protein as described in the preceding paragraph and a heterologous portion. In exemplary embodiments, the present invention provides a fusion polypeptide or fusion protein comprising the PD-1 antigen-binding protein as described in the preceding paragraph and a heterologous polypeptide or peptide. In exemplary embodiments, the present invention provides a nucleic acid comprising a nucleotide sequence encoding the PD-1 antigen-binding protein, conjugate, or fusion polypeptide or fusion protein as described in the preceding paragraph. In exemplary cases, the nucleic acid comprises the sequence of any of SEQ ID NO: 392-471. In exemplary embodiments, the present invention provides a vector comprising the nucleic acid as described above and a host cell comprising the nucleic acid or vector as described above. A kit is also provided comprising the PD-1 antigen-binding protein or conjugate, fusion polypeptide, fusion protein, nucleic acid, vector, or host cell or a combination thereof and a container as described in the preceding paragraph. This invention provides a pharmaceutical composition, in an exemplary embodiment, comprising a PD-1 antigen-binding protein or conjugate, a fusion polypeptide, a fusion protein, a nucleic acid, a carrier or host cell or a combination thereof as described in the preceding paragraph, and a pharmaceutically acceptable carrier, excipient, or diluent. This invention further provides a method for manufacturing a PD-1 antigen-binding protein, wherein, in an exemplary embodiment, the method includes culturing host cells as described in the preceding paragraph to express the PD-1 antigen-binding protein and harvesting the expressed PD-1 antigen-binding protein. This invention further provides a method for treating an individual in need, wherein, in an exemplary embodiment, the method includes administering to the individual in need an amount of the pharmaceutical composition described in the preceding paragraph that is effective in treating the individual. In the exemplary embodiment, the individual has a solid tumor and the pharmaceutical composition is administered to the individual in an amount effective in treating the individual's solid tumor. Depending on the circumstances, solid tumors are selected from the following groups: head and neck cancer, ovarian cancer, cervical cancer, bladder and esophageal cancer, pancreatic cancer, gastrointestinal cancer, stomach cancer, breast cancer, endometrial cancer and colorectal cancer, hepatocellular carcinoma, glioblastoma, bladder cancer, lung cancer (e.g., non-small cell lung cancer (NSCLC)), and bronchioloalveolar carcinoma.

[0328] The following examples are given only to illustrate the present invention and are not intended to limit its scope in any way. Example 1

[0329] This case demonstrates that combination therapy containing PD-1 blocking antibody and recombinant IL-21 is superior to the corresponding monotherapy.

[0330] In preclinical studies, the effects of combination therapy with monoclonal PD-1 blocking antibody and recombinant murine IL-21 (rmIL-21) were compared with the effects of monotherapy with PD-1 blocking antibody or rmIL-21.

[0331] On day 1, CT26 / 3E5 colon cancer cells were implanted into BALB / c mice to initiate tumor growth. On day 12, tumors were measured, and mice were randomized to four groups (n=10 per group): Group 1 received an intraperitoneal (IP) injection of 300 μg of isotype control antibody (mIgG1), Group 2 received an IP injection of 300 μg of PD-1 blocking antibody, Group 3 received 50 μg of rmIL-21, and Group 4 received both PD-1 blocking antibody (300 μg) and rmIL-21 (50 μg). Groups 1, 2, and 4 received antibodies every 3 days, while Groups 3 and 4 received rmIL-21 three times a week for 3 weeks. Administration ended on day 33.

[0332] Tumor volume was monitored throughout the study. As shown in Figures 1A-1D, the tumor size increased to the greatest extent in Group 1 and to the least extent in Group 4.

[0333] Survival, as measured by Kaplan-Meier log-rank mantel-cox analysis, was the primary endpoint of this study. As shown in Figure 2 and Table 1, Group 4 had the highest percentage survival and median survival. Notably, two individuals in Group 4 were tumor-free (Table 1). Table 1 Group Median survival time (days) Individuals without tumors 1 25 0 2 29 0 3 27 0 4 37.5 2

[0334] These results demonstrate that the combination of monoclonal PD-1 blocking antibody and recombinant mIL-21 provides a survival advantage over either component administered alone as a monotherapy. Example 2

[0335] This example illustrates the design and construction of a multi-platform system designed to provide a combination of PD-1 suppression and IL-21 signal transduction.

[0336] The results obtained in Example 1 demonstrate that the combination of IL-21 signaling and PD-1 inhibition has individual advantages for tumor treatment. However, because IL-21 can enhance CD8 T cell responses and inhibit antigen presentation and T cell activation, the delivery method of IL-21 should be carefully considered. In addition, IL-21R is widely expressed in human tissues (e.g., via antigen-presenting cells (APCs), NK cells, B cells, and T cells), so careful consideration is needed to avoid off-target effects (e.g., IL-21 activity outside the tumor environment) and IL-21 clearance when IL-21 binds to its receptor in different tissues.

[0337] A two-pronged approach was devised to illustrate the above considerations. First, an IL-21 mutant protein with reduced activity due to decreased binding of the IL-21 mutant protein to IL-21R was generated. It is conceivable that such an IL-21 mutant protein would exhibit reduced activity when it spreads throughout the body, but this activity would be "rescued" if the IL-21 mutant protein could be concentrated in target (e.g., cancer) T cells. That is, once the IL-21 mutant protein is present and concentrated in target cells, it will exhibit therapeutic IL-21 activity overall. Second, the IL-21 mutant protein was fused with a target arm such as a monoclonal antibody to target relevant cells (e.g., cancer cells). To deliver the IL-21 mutant protein to target cancer cells, it was fused with an anti-PD-1 mAb. The use of the anti-PD-1 mAb has the additional benefit of preventing PD-1 / PD-L1 signaling (thus acting as a checkpoint inhibitor).

[0338] Without being bound by any specific theory, it is assumed that a fusion protein containing an anti-PD-1 antibody and a mutant IL-21 protein will provide a more durable response against cells intended for destruction. For example, [1] CD8+ T cells expressing PD-1 will bind to the anti-PD-1 mAb of the fusion protein, and [2] the bind...

Claims

1. A conjugate comprising an IL-21 mutant protein and an anti-PD-1 antibody, wherein the IL-21 mutant protein comprises the amino acid sequence of SEQ ID NO: 2, QGQDX HMXXM XXXXX XVDXL KNXVN DLVPE FLPAP EDVET NCEWS AFSCF QKAQL KSANT GNNEX XIXXX XXXLX XXXXX TNAGR RQKHR LTCPS CDSYE KKPPK EFLXX FXXLL XXMXX QHXSS RTHGS EDS (SEQ ID NO: 2), wherein "X" represents any amino acid, and wherein the amino acid sequence of the IL-21 mutant protein differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by at least one amino acid.

2. A conjugate of claim 1, wherein the IL-21 mutant protein comprises an amino acid sequence that differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by no more than 7 amino acids.

3. A combination of claim 1, wherein the IL-21 mutant protein comprises an amino acid sequence that differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by 3, 4, 5 or 6 amino acids.

4. A combination of claim 1, wherein the IL-21 mutant protein comprises an amino acid sequence that differs from the amino acid sequence of human IL-21 (SEQ ID NO: 1) by one or two amino acids.

5. A combination of any one of claims 1 to 4, wherein the difference between the amino acid sequence of the IL-21 mutant protein and the amino acid sequence of SEQ ID NO: 1 occurs in (A) amino acids 10-15 of SEQ ID NO: 2, including amino acids 10 and 15, or amino acids 105-123, including amino acids 105 and 123, or (B) amino acids 5-25 of SEQ ID NO: 2, including amino acids 5 and 25, or amino acids 65-80, including amino acids 65 and 80.

6. The combination of claim 5, wherein the difference or such difference occurs at amino acids 11, 14, 15, 109, 110, 112, 113, 116, 119, 120 and / or 123 of SEQ ID NO: 2, or wherein the difference or such difference occurs at amino acids 5, 8, 9, 12, 13, 16, 19, 23, 65, 66, 69, 70, 72, 73, 75, 76, 77, 78, 79 and / or 80 of SEQ ID NO:

2.

7. A combination of any one of claims 1 to 4, wherein the IL-21 mutant protein comprises an amino acid sequence having an amino acid substitution relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1).

8. The combination of claim 7, wherein the amino acid substitution occurs at position 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120 or 123 of SEQ ID NO:

1.

9. A combination of any one of claims 1 to 4, wherein the IL-21 mutant protein comprises the following amino acid substitutions: a. at positions 5, 8, 9, 12, 14, 15, 65, 66, 69, 70, 72, 73, 75, 76, 77, 80, 116, or 119 of SEQ ID NO: 1, wherein the substituted amino acid is an aliphatic amino acid; b. at positions 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 69, 70, 72, 73, 75, 76, 77, 78, 79, 110, 112, 116, 117, 119, 120, or 123 of SEQ ID NO: 1, wherein the substituted amino acid is an acidic amino acid; c. at positions 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 69, 70, 72, 73, 75, 76, 77, 78, 79, 110, 112, 116, 117, 119, 120, or 123 of SEQ ID NO: 1 d. At position 5, 9, 73, 76, 109, 113, or 116 of SEQ ID NO: 1, wherein the substituted amino acid is a basic amino acid; e. At position 5, 8, 9, 70, or 76 of SEQ ID NO: 1, wherein the substituted amino acid is an aromatic amino acid; f. At position 5, 8, 9, 12, 15, 73, 76, 116, or 119 of SEQ ID NO: 1, wherein the substituted amino acid is an amino acid containing a amide side chain; g. At position 5, 8, 9, 11, 12, 14, 15, 73, 76, 116, or 119 of SEQ ID NO: 1, wherein the substituted amino acid is a non-aromatic amino acid containing a hydroxyl side chain; h. at position 65, 66, 69, 70, 72, 73, 75, 76, 77 or 80 of SEQ ID NO: 1, wherein the substituted amino acid is an imine; or i. at position 5, 9, 15, 76, 116 or 119 of SEQ ID NO: 1, wherein the substituted amino acid is an amino acid containing a sulfur-containing side chain; or i. a combination thereof.

10. A combination of any of claims 1 to 4, wherein the IL-21 mutant protein comprises amino acid substitutions at positions specified in Table A: Table A amino acid position of SEQ ID NO: 1 Amino acids (by single-letter code) amino acid position of SEQ ID NO: 1 Amino acids (by single-letter code) 5 A, D, E, G, H, I, K, L, M, N, Q, S, T, V or Y 72 D, G, or P 8 A, D, E, N, S, T, V, or Y 73 A, D, E, G, H, I, N, P, Q, S, or V 9 A, D, E, G, H, I, K, L, M, N, Q, S, T, V or Y 75 D, G, or P 11 D or S 76 A, D, E, G, H, I, K, L, M, N, P, Q, S, T, V or Y 12 A, D, E, N, S, T, or V 77 D, G, or P 13 D 78 D 14 A, D, or S 79 D 15 A, E, I, M, N, Q, S, T or V 80 G or P 16 D or E 109 K 19 D 110 D 23 D 112 D 65 D, G, or P 113 K 66 D, G, or P 116 A, D, E, I, K, L, M, N, S, T or V 68 Q 117 D 69 D, G, or P 119 A, D, E, M, N, Q, S, or T 70 E, G, P or Y 120 D 71 L 123 D 。 11. A combination of any one of claims 1 to 4, wherein the IL-21 mutant protein comprises: (a) an amino acid sequence of any one of SEQ ID NO: 3-21, 23-56, 58-112, 114-198, 249-254 and 283; or (b) an amino acid sequence of any one of SEQ ID NO: 199 to 208, 210 to 222, 224 to 248 and 255.

12. A combination of any one of claims 1 to 4, wherein the IL-21 mutant protein comprises an amino acid sequence having two amino acid substitutions relative to the amino acid sequence of human IL-21 (SEQ ID NO: 1).

13. The combination of claim 12, wherein the amino acid substitution occurs at any two of positions 5, 8, 9, 11, 12, 13, 14, 15, 16, 19, 23, 65, 66, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 109, 110, 112, 113, 116, 117, 119, 120 or 123 of SEQ ID NO:

1.

14. The combination of claim 12, wherein the amino acid substitutions occur at positions 5, 9, 73 and 76 of SEQ ID NO:

1.

15. A combination of any one of claims 1 to 4, wherein the IL-21 mutant protein comprises any two of the following: (i) the amino acid at position 5 of SEQ ID NO: 1, wherein the amino acid is selected from the group consisting of: A, D, E, G, H, I, K, L, M, N, Q, S, T, V, or Y; (ii) the amino acid at position 9 of SEQ ID NO: 1, wherein the amino acid is selected from the group consisting of: A, D, E, G, H, I, K, L, M, N, Q, S, T, V, or Y; (iii) the amino acid at position 73 of SEQ ID NO: 1, wherein the amino acid is selected from the group consisting of: A, D, E, G, H, I, N, P, Q, S, or V; and (iv) SEQ ID NO: The amino acid at position 76 of 1, wherein the amino acid is selected from the group consisting of: A, D, E, G, H, I, K, L, M, N, Q, S, T, V or Y.

16. A combination of any one of claims 1 to 4, wherein the IL-21 mutant protein comprises an amino acid substitution at position 5, 9, or 73 of SEQ ID NO: 1, wherein the substituted amino acid is selected from: amino acid position of SEQ ID NO: 1 Amino acids (single-letter codes) 5 A, D, E, G, H, I, K, L, M, N, Q, S, T, V or Y 9 A, D, E, G, H, I, K, L, M, N, Q, S, T, V or Y 73 A, D, E, G, H, I, N, P, Q, S, or V 。 17. A combination of any one of claims 1 to 4, wherein the IL-21 mutant protein comprises an amino acid substitution at position 76 of SEQ ID NO: 1, and the substituted amino acid at position 76 is an aliphatic amino acid or an acidic amino acid.

18. A combination of claim 17, wherein the aliphatic amino acid is alanine and wherein the acidic amino acid is glutamic acid.

19. A conjugate of claim 1, wherein the IL-21 mutant protein is directly attached to the Fc of the antibody.

20. The conjugate of claim 1, wherein the IL-21 mutant protein is attached to the Fc of the antibody via a linker.

21. A conjugate of claim 19 or 20, wherein the conjugate comprises a single IL-21 mutant protein, wherein the single IL-21 mutant protein is linked to the C-terminus of one of the two antibody heavy chains.

22. A conjugate of claim 19 or 20, wherein the conjugate comprises two IL-21 mutant proteins, wherein the first IL-21 mutant protein is attached to the C-terminus of the first antibody heavy chain and the second IL-21 mutant protein is attached to the C-terminus of the second antibody heavy chain.

23. A combination of claim 22, wherein the first IL-21 mutant protein has the same amino acid sequence as the second IL-21 mutant protein.

24. A combination of claim 22, wherein the first IL-21 mutant protein and the second IL-21 mutant protein have the same amino acid sequence.

25. A combination of claim 22, wherein the first IL-21 mutant protein and the second IL-21 mutant protein have different amino acid sequences.

26. A combination of any one of claims 1 to 4, wherein the IL-21 mutant protein comprises the amino acid sequence of any one of SEQ ID NO: 233 to 245.

27. A combination of claim 26, wherein the IL-21 mutant protein comprises the amino acid sequence of SEQ ID NO:

244.

28. A combination of claim 26, wherein the IL-21 mutant protein comprises the amino acid sequence of SEQ ID NO:

245.

29. A combination of any one of claims 1 to 4, wherein the combination is a fusion protein comprising an IL-21 mutant protein and an anti-PD-1 antibody.

30. A conjugate of claim 29, wherein the fusion protein comprises: (i) two light chains, each comprising the light chain of SEQ ID NO: 391, and two heavy chains-IL-21 mutant protein fusions, each comprising a heavy chain fused to the IL-21 mutant protein of any one of SEQ ID NO: 498 to 500 or 519; or (ii) two light chains, each comprising the light chain of SEQ ID NO: 391, and one heavy chain fused to the IL-21 mutant protein, comprising a heavy chain fused to the IL-21 mutant protein of any one of SEQ ID NO: 501 to 506; and one heavy chain comprising a heavy chain of any one of SEQ ID NO: 556 to 558; or (iii) two light chains, each comprising the light chain of SEQ ID NO: 371, and two heavy chains-IL-21 mutant protein fusions, each comprising a heavy chain fused to the IL-21 mutant protein of any one of SEQ ID NO: 391 to 500 or 519; (iv) A heavy chain fused to the IL-21 mutant protein, any one of SEQ ID NO: 371; or (iv) two light chains, each comprising the light chain of SEQ ID NO: 371, and a heavy chain fused to the IL-21 mutant protein, comprising the amino acid sequence of any one of SEQ ID NO: 513 to 518; and a heavy chain comprising the amino acid sequence of any one of SEQ ID NO: 559 to 561; or (v) two light chains, each comprising the variable region of the light chain of SEQ ID NO: 389, and two heavy chain-IL-21 mutant protein fusions, each comprising the heavy chain fused to the IL-21 mutant protein, any one of SEQ ID NO: 498 to 500 or 519; or (vi) two light chains, each comprising the variable region of the light chain of SEQ ID NO: 389, and a heavy chain fused to the IL-21 mutant protein, comprising the amino acid sequence of any one of SEQ ID NO: 513 to 518; and a heavy chain comprising the amino acid sequence of any one of SEQ ID NO: 513 to 518; or (vi) two light chains, each comprising the variable region of the light chain of SEQ ID NO: 389, and a heavy chain fused to the IL-21 mutant protein, comprising the amino acid sequence of any one of SEQ ID NO: 513 to 518; or (vi) two light chains, each comprising the variable region of the light chain of SEQ ID NO: 389, and a heavy chain fused to the IL-21 mutant protein, comprising the amino acid sequence of any one of SEQ ID NO: 513 to 518; or (iv ... The heavy chain fused to the IL-21 mutant protein of any one of SEQ ID NO: 501 to 506; and a heavy chain comprising the heavy chain of any one of SEQ ID NO: 556 to 558; or (vii) two light chains, each comprising the light chain variable region of SEQ ID NO: 369, and two heavy chain-IL-21 mutant protein fusions, each comprising the heavy chain fused to the IL-21 mutant protein of any one of SEQ ID NO: 508 to 512; or (viii) two light chains, each comprising the light chain variable region of SEQ ID NO: 369, and a heavy chain fused to the IL-21 mutant protein comprising the heavy chain fused to the IL-21 mutant protein of any one of SEQ ID NO: 513 to 518; and a heavy chain comprising the heavy chain of any one of SEQ ID NO: 559 to 561.Or (ix) two light chains, each containing the variable region of the light chain of SEQ ID NO: 389 and the constant region of the light chain of SEQ ID NO: 391, and two heavy chains-IL-21 mutant protein fusions, each containing a heavy chain fused to the IL-21 mutant protein of any one of SEQ ID NO: 498 to 500 or 519; or (x) two light chains, each containing the variable region of the light chain of SEQ ID NO: 389 and the constant region of the light chain of SEQ ID NO: 391, one heavy chain fused to the IL-21 mutant protein, which contains a heavy chain fused to the IL-21 mutant protein of any one of SEQ ID NO: 501 to 506; and one heavy chain containing a heavy chain of any one of SEQ ID NO: 556 to 558; or (xi) two light chains, each containing the variable region of the light chain of SEQ ID NO: 369 and the constant region of the light chain of SEQ ID NO: 371, Two heavy-chain IL-21 mutant protein fusions, each comprising a heavy chain fused to the IL-21 mutant protein of any one of SEQ ID NO: 508 to 512; or (xii) two light chains, each comprising a light chain variable region of SEQ ID NO: 369 and a light chain constant region of SEQ ID NO: 371, one heavy chain fused to the IL-21 mutant protein comprising an amino acid sequence of any one of SEQ ID NO: 513 to 518; and one heavy chain comprising a heavy chain of any one of SEQ ID NO: 559 to 561.

31. The conjugate of claim 1, wherein the conjugate is a fusion protein comprising an IL-21 mutant protein and an anti-PD-1 antibody, wherein the fusion protein comprises: (i) two light chains, each comprising LC CDR1, LC CDR2 and LC CDR3, each comprising the amino acid sequences of SEQ ID NO:385, 386 and 387, respectively; (ii) two heavy chains, each comprising HC CDR1, HC CDR2 and HC CDR3, each comprising the amino acid sequences of SEQ ID NO:382, 383 and 384, respectively; and (iii) an IL-21 mutant protein comprising the amino acid sequence of SEQ ID NO:244, wherein the IL-21 mutant protein is fused to one of the antibody heavy chains.

32. The combination of claim 31, wherein each light chain includes the light chain variable region of SEQ ID NO:389, and wherein each heavy chain includes the heavy chain variable region of SEQ ID NO:

388.

33. The conjugate of claim 1, wherein the conjugate is a fusion protein comprising an IL-21 mutant protein and an anti-PD-1 antibody, wherein the fusion protein comprises: (i) two light chains, each comprising the light chain of SEQ ID NO:391; (ii) a heavy chain comprising the heavy chain of SEQ ID NO:556; and (iii) a heavy chain fused to the IL-21 mutant protein comprising the heavy chain fused to the IL-21 mutant protein of SEQ ID NO:

501.

34. A conjugate of claim 1, wherein the conjugate is a fusion protein comprising an IL-21 mutant protein and an anti-PD-1 antibody, wherein: (a) The fusion protein comprises: (i) two light chains, each comprising the light chain variable region of SEQ ID NO:389; (ii) a heavy chain comprising the heavy chain variable region of SEQ ID NO:556; and (iii) a heavy chain fused to the IL-21 mutant protein, comprising the heavy chain fused to the IL-21 mutant protein of SEQ ID NO:501; or (b) the fusion protein comprises: (i) two light chains, each comprising the light chain variable region of SEQ ID NO:389 and the light chain constant region of SEQ ID NO:391; (ii) a heavy chain comprising the heavy chain of SEQ ID NO:556; and (iii) a heavy chain fused to the IL-21 mutant protein, comprising the heavy chain fused to the IL-21 mutant protein of SEQ ID NO:

501.

35. A conjugate of claim 1, wherein the conjugate is a fusion protein comprising an IL-21 mutant protein and an anti-PD-1 antibody, wherein the fusion protein comprises: (i) two light chains, each comprising the variable region of the light chain of SEQ ID NO:391, and two heavy chain-IL-21 mutant protein fusions, each comprising the heavy chain fused to the IL-21 mutant protein of SEQ ID NO:499; or (ii) two light chains, each comprising the variable region of the light chain of SEQ ID NO:389, and two heavy chain-IL-21 mutant protein fusions, each comprising the heavy chain fused to the IL-21 mutant protein of SEQ ID NO:499; or (iii) two light chains, each comprising the variable region of the light chain of SEQ ID NO:389 and the constant region of the light chain of SEQ ID NO:391, and two heavy chain-IL-21 mutant protein fusions, each comprising the heavy chain fused to the IL-21 mutant protein of SEQ ID NO:499; or (iv) Two light chains, each containing the light chain of SEQ ID NO:391, and two heavy chain-IL-21 mutant protein fusions, each containing the heavy chain of SEQ ID NO:519 fused to the IL-21 mutant protein; or (v) two light chains, each containing the variable region of the light chain of SEQ ID NO:389, and two heavy chain-IL-21 mutant protein fusions, each containing the heavy chain of SEQ ID NO:519 fused to the IL-21 mutant protein; or (vi) two light chains, each containing the variable region of the light chain of SEQ ID NO:389 and the constant region of the light chain of SEQ ID NO:391, and two heavy chain-IL-21 mutant protein fusions, each containing the heavy chain of SEQ ID NO:519 fused to the IL-21 mutant protein.

36. The conjugate of claim 1, wherein the conjugate is a fusion protein comprising an IL-21 mutant protein and an anti-PD-1 antibody, wherein the fusion protein comprises: (i) two light chains, each comprising LC CDR1, LC CDR2 and LC CDR3, each comprising the amino acid sequences of SEQ ID NO:365, 366 and 367, respectively; (ii) two heavy chains, each comprising HC CDR1, HC CDR2 and HC CDR3, each comprising the amino acid sequences of SEQ ID NO:362, 363 and 364, respectively; and (iii) an IL-21 mutant protein comprising the amino acid sequence of SEQ ID NO:244, wherein the IL-21 mutant protein is fused to one of the antibody heavy chains.

37. The combination of claim 36, wherein each light chain contains the light chain variable region of SEQ ID NO:369, and each heavy chain contains the heavy chain variable region of SEQ ID NO:

368.

38. A conjugate of claim 1, wherein the conjugate is a fusion protein comprising an IL-21 mutant protein and an anti-PD-1 antibody, wherein: (a) The fusion protein comprises: (i) two light chains, each comprising the light chain of SEQ ID NO:371; (ii) a heavy chain comprising the heavy chain of SEQ ID NO:559; and (iii) a heavy chain fused to the IL-21 mutant protein, comprising the heavy chain fused to the IL-21 mutant protein of SEQ ID NO:513; or (b) the fusion protein comprises: (i) two light chains, each comprising the variable region of the light chain of SEQ ID NO:369; (ii) a heavy chain comprising the heavy chain of SEQ ID NO:559; and (iii) a heavy chain fused to the IL-21 mutant protein, comprising the heavy chain fused to the IL-21 mutant protein of SEQ ID NO:513; or (c) the fusion protein comprises: (i) two light chains, each comprising the variable region of the light chain of SEQ ID NO:369 and the constant region of the light chain of SEQ ID NO:371; (ii) a heavy chain comprising the variable region of the light chain of SEQ ID NO:369 and the constant region of the light chain of SEQ ID NO:371; (iii) a heavy chain fused to the IL-21 mutant protein, comprising the heavy chain fused to the IL-21 mutant protein of SEQ ID NO:

513.

39. A conjugate of claim 1, wherein the conjugate is a fusion protein comprising an IL-21 mutant protein and an anti-PD-1 antibody, wherein the fusion protein comprises: (i) two light chains, each comprising the light chain of SEQ ID NO:371, and two heavy chains-IL-21 mutant protein fusions comprising the heavy chain of SEQ ID NO:508 fused to the IL-21 mutant protein; or (ii) two light chains, each comprising the variable region of the light chain of SEQ ID NO:369, and two heavy chains-IL-21 mutant protein fusions comprising the heavy chain of SEQ ID NO:508 fused to the IL-21 mutant protein; or (iii) two light chains, each comprising the variable region of the light chain of SEQ ID NO:369 and the constant region of the light chain of SEQ ID NO:371, and two heavy chains-IL-21 mutant protein fusions comprising the heavy chain of SEQ ID NO:508 fused to the IL-21 mutant protein; or (iv) two light chains, each comprising the light chain of SEQ ID NO:369, and the variable region of the light chain of SEQ ID NO:371, and two heavy chains-IL-21 mutant protein fusions comprising the heavy chain of SEQ ID NO:508 fused to the IL-21 mutant protein; or (iv) two light chains, each comprising the light chain of SEQ ID NO:369, and the variable region of the light chain of SEQ ID NO:369, and the constant ... (v) Two light chains, each containing the variable region of the light chain of SEQ ID NO:369, and two heavy chains-IL-21 mutant protein fusions, comprising the heavy chain of SEQ ID NO:511 fused to the IL-21 mutant protein; or (vi) Two light chains, each containing the variable region of the light chain of SEQ ID NO:369 and two heavy chains-IL-21 mutant protein fusions, comprising the heavy chain of SEQ ID NO:511 fused to the IL-21 mutant protein; or (vi) Two light chains, each containing the variable region of the light chain of SEQ ID NO:369 and the constant region of the light chain of SEQ ID NO:371, and two heavy chains-IL-21 mutant protein fusions, comprising the heavy chain of SEQ ID NO:511 fused to the IL-21 mutant protein.

40. A kit comprising a combination and a container as claimed in any one of claims 1 to 39.

41. A pharmaceutical composition comprising a combination of any one of claims 1 to 39 and a pharmaceutically acceptable carrier, excipient or diluent.

42. Use of a pharmaceutical composition as claimed in claim 41, for the preparation of a medicine for treating an individual in need.

43. As used in claim 42, wherein the individual has a solid tumor.

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