Engineered IL-21 variants and methods of use thereof

Abstract

The present disclosure relates to engineered IL-21 variants and methods of use thereof. In some embodiments, the engineered IL-21 variants comprise a non-natural disulfide bond.

Description

[0001] Cross-reference to related applications

[0002] This disclosure claims priority and benefit to U.S. Provisional Patent Application Serial No. 63 / 453,399, filed March 20, 2023, which is incorporated herein by reference in its entirety.

[0003] sequence list

[0004] This application contains a sequence list of an XML file, named 52246-0012TW1_SL_ST26.xml, which has been submitted electronically. The XML file, created on March 18, 2024, is 47,998 bytes in size. All material in the XML file is hereby incorporated by reference in its entirety. Technical Field

[0005] This disclosure relates to engineered variants of IL-21 and their usage. Background Technology

[0006] Cytokines belonging to the common γ chain (γc) family are central regulators of the development, proliferation, survival and differentiation of multiple cell lineages in the innate and adaptive immune systems, and thus have received considerable attention in anticancer therapeutic applications.

[0007] IL-21 exerts potent antitumor effects due to its ability to induce and expand cytotoxic CD8+ T cells, NK cells, and NKT cells, as well as its ability to inhibit FOXP3 expression and expand regulatory T cells (Tregs). Furthermore, IL-21 has been associated with clinical antitumor activity. However, at high concentrations, IL-21 may cause dose-limiting side effects, including grade 3 / 4 granulocytopenia and hepatotoxicity. In addition, IL-21 can promote tumorigenesis by driving sustained inflammatory responses to IL-6 and IL-17, such as those occurring during chronic colitis.

[0008] Recombinant IL-21 has been tested as an antitumor agent in various clinical trials. It has shown promising antitumor activity and acceptable toxicity. However, the short half-life of IL-21 reduces its levels in vivo and requires frequent dosing, which limits its clinical application.

[0009] Therefore, there is a need to develop cancer therapies that target the IL-21 pathway with longer half-lives, enhanced anti-tumor efficacy, and limited toxicity. Summary of the Invention

[0010] This disclosure relates to engineered IL-21 variants, protein constructs (e.g., fusion proteins or protein complexes), and methods of using them. In some embodiments, the variants and protein constructs comprise non-natural disulfide bonds formed by mutating one, two, three, four, five, six, or more than six residues (e.g., a pair of residues) of wild-type IL-21 to cysteine ​​residues without interfering with the overall structure or residues essential for IL-21 receptor interaction. Therefore, the variants or protein constructs may be more stable than wild-type IL-21. In some embodiments, the protein constructs exhibit enhanced thermal stability (e.g., increased Tg) compared to wild-type IL-21. 聚集 and T 起始 And higher efficacy in alleviating immunosuppression (e.g., Treg-mediated T cell suppression). Therefore, the engineered IL-21 variants and their protein constructs described herein can be used for cancer treatment with minimal toxicity.

[0011] This article also provides methods for screening cytokines (e.g., IL-21) that have increased stability and / or antitumor efficacy.

[0012] On one hand, this disclosure relates to engineered IL-21 peptides, which in some embodiments comprise non-natural disulfide bonds. In some embodiments, the engineered IL-21 peptide comprises an amino acid sequence that is at least 80% identical to that of SEQ ID NO:2. In some embodiments, the engineered IL-21 peptide may bind to a complex formed by the human IL-21 receptor (IL-21R) and the common cytokine γ chain (γc).

[0013] In some embodiments, the engineered IL-21 polypeptide includes one or more of the following: (a) the amino acid corresponding to position 8 of SEQ ID NO:2 is cysteine ​​(C); (b) the amino acid corresponding to position 19 of SEQ ID NO:2 is C; (c) the amino acid corresponding to position 29 of SEQ ID NO:2 is C; (d) the amino acid corresponding to position 31 of SEQ ID NO:2 is C; (e) the amino acid corresponding to position 33 of SEQ ID NO:2 is C; (f) the amino acid corresponding to position 36 of SEQ ID NO:2 is C; (g) the amino acid corresponding to position 39 of SEQ ID NO:2 is C; and (h) the amino acid corresponding to position 56 of SEQ ID NO:2 is C.

[0014] In some embodiments, the engineered IL-21 polypeptide includes one or more of the following: (a) the amino acid corresponding to position 61 of SEQ ID NO:2 is C; (b) the amino acid corresponding to position 62 of SEQ ID NO:2 is C; (c) the amino acid corresponding to position 63 of SEQ ID NO:2 is C; (d) the amino acid corresponding to position 80 of SEQ ID NO:2 is C; (e) the amino acid corresponding to position 86 of SEQ ID NO:2 is C; (f) the amino acid corresponding to position 105 of SEQ ID NO:2 is C; (g) the amino acid corresponding to position 106 of SEQ ID NO:2 is C; (h) the amino acid corresponding to position 107 of SEQ ID NO:2 is C; (i) the amino acid corresponding to position 110 of SEQ ID NO:2 is C; (j) the amino acid corresponding to position 112 of SEQ ID NO:2 is C; and (k) the amino acid corresponding to position 117 of SEQ ID NO:2 is C.In some embodiments, the engineered IL-21 polypeptide comprises one or more of the following: (a) the amino acid corresponding to position 8 of SEQ ID NO:2 is C, and the amino acid corresponding to position 86 of SEQ ID NO:2 is C; (b) the amino acid corresponding to position 19 of SEQ ID NO:2 is C, and the amino acid corresponding to position 117 of SEQ ID NO:2 is C; (c) the amino acid corresponding to position 29 of SEQ ID NO:2 is C, and the amino acid corresponding to position 110 of SEQ ID NO:2 is C; (d) the amino acid corresponding to position 31 of SEQ ID NO:2 is C, and the amino acid corresponding to position 62 of SEQ ID NO:2 is C; (e) the amino acid corresponding to position 31 of SEQ ID NO:2 is C, and the amino acid corresponding to position 63 of SEQ ID NO:2 is C; (f) the amino acid corresponding to position 33 of SEQ ID NO:2 is C, and the amino acid corresponding to position 61 of SEQ ID NO:2 is C; (g) the amino acid corresponding to position 33 of SEQ ID NO:2 is C, and the amino acid corresponding to position 86 of SEQ ID NO:2 is C, and the amino acid corresponding to position 87 of SEQ ID NO:2 is C; The amino acid corresponding to position 63 of SEQ ID NO:2 is C; (h) the amino acid corresponding to position 36 of SEQ ID NO:2 is C, and the amino acid corresponding to position 106 of SEQ ID NO:2 is C; (i) the amino acid corresponding to position 36 of SEQ ID NO:2 is C, and the amino acid corresponding to position 107 of SEQ ID NO:2 is C; (j) the amino acid corresponding to position 39 of SEQ ID NO:2 is C, and the amino acid corresponding to position 105 of SEQ ID NO:2 is C; (k) the amino acid corresponding to position 39 of SEQ ID NO:2 is C, and the amino acid corresponding to position 107 of SEQ ID NO:2 is C; (l) the amino acid corresponding to position 39 of SEQ ID NO:2 is C, and the amino acid corresponding to position 112 of SEQ ID NO:2 is C; and (m) the amino acid corresponding to position 56 of SEQ ID NO:2 is C, and the amino acid corresponding to position 80 of SEQ ID NO:2 is C.

[0015] In some embodiments, the engineered IL-21 polypeptide includes one or more of the following: (a) the amino acid corresponding to position 12 of SEQ ID NO:2 is M; (b) the amino acid corresponding to position 16 of SEQ ID NO:2 is R; (c) the amino acid corresponding to position 19 of SEQ ID NO:2 is I; (d) the amino acid corresponding to position 23 of SEQ ID NO:2 is D; (e) the amino acid corresponding to position 105 of SEQ ID NO:2 is E; (f) the amino acid corresponding to position 114 of SEQ ID NO:2 is E; (g) the amino acid corresponding to position 118 of SEQ ID NO:2 is S; (h) the amino acid corresponding to position 121 of SEQ ID NO:2 is Q; (i) the amino acid corresponding to position 122 of SEQ ID NO:2 is K; (j) the amino acid corresponding to position 124 of SEQ ID NO:2 is I; (k) the amino acid corresponding to position 125 of SEQ ID NO:2 is H; and (l) the amino acid corresponding to position 128 of SEQ ID NO:2 is L.

[0016] In some embodiments, the engineered IL-21 peptide described herein comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the engineered IL-21 peptide comprises at least 90% of the same amino acid sequence as SEQ ID NO:3. In some embodiments, the engineered IL-21 peptide comprises at least 90% of the same amino acid sequence as SEQ ID NO:5. In some embodiments, the engineered IL-21 peptide comprises at least 90% of the same amino acid sequence as SEQ ID NO:9. In some embodiments, the engineered IL-21 peptide comprises at least 90% of the same amino acid sequence as SEQ ID NO:10.

[0017] In some embodiments, the engineered IL-21 peptide can induce the proliferation of immune cells (e.g., T cells or NK cells). In some embodiments, the engineered IL-21 peptide can induce STAT-3 phosphorylation.

[0018] In one aspect, this disclosure relates to a fusion protein comprising the engineered IL-21 polypeptide described herein. In some embodiments, the fusion protein described herein further comprises human serum albumin (HSA). In some embodiments, the HSA comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:26. In some embodiments, the engineered IL-21 polypeptide is linked to the C-terminus of the HSA via a linker peptide. In some embodiments, the linker peptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:25. In some embodiments, the fusion protein further optionally includes a His tag at the N-terminus. In some embodiments, the fusion protein comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:17, 18, 19, 20, or 21.

[0019] In one aspect, this disclosure relates to a fusion protein, which optionally comprises, from its N-terminus to its C-terminus: (a) an optional His tag; (b) an HSA; (c) a adaptor peptide; and (d) an engineered IL-21 polypeptide. In some embodiments, the His tag comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:22. In some embodiments, the HSA comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:26. In some embodiments, the adaptor peptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:25. In some embodiments, the engineered IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0020] In some embodiments, the fusion protein described herein further includes an Fc region.

[0021] In some embodiments, the fusion protein can alleviate Treg-mediated T cell suppression. In some embodiments, the fusion protein can induce primary NK cell toxicity.

[0022] In one aspect, this disclosure relates to a protein complex comprising: (a) a first polypeptide comprising, from its N-terminus to its C-terminus: an optional first hinge region, a first Fc region, an optional linker peptide, and an engineered IL-21 polypeptide as described herein; and (b) a second polypeptide comprising, from its N-terminus to its C-terminus, an optional second hinge region and a second Fc region. In some embodiments, the first hinge region, the first Fc region, the second hinge region, and / or the second Fc region are derived from human IgG4. In some embodiments, the first Fc region and / or the second Fc region comprises one or more kilometre (KIH) mutations. In some embodiments, the first Fc region comprises at least 80%, 90%, 95%, or 100% identical sequences to SEQ ID NO:33, and the second Fc region comprises at least 80%, 90%, 95%, or 100% identical sequences to SEQ ID NO:32. In some embodiments, the first hinge region and / or the second hinge region comprises at least 80%, 90%, 95%, or 100% identical sequences to SEQ ID NO:31. In some embodiments, the adapter peptide comprises at least 80%, 90%, 95%, or 100% of the same sequence as SEQ ID NO:34. In some embodiments, the protein complex described herein comprises one of the following: (1) the first polypeptide comprises at least 80%, 90%, 95%, or 100% of the sequence identical to SEQ ID NO:35, and the first polypeptide comprises at least 80%, 90%, 95%, or 100% of the sequence identical to SEQ ID NO:29; (2) the first polypeptide comprises at least 80%, 90%, 95%, or 100% of the sequence identical to SEQ ID NO:36, and the first polypeptide comprises at least 80%, 90%, 95%, or 100% of the sequence identical to SEQ ID NO:29; (3) the first polypeptide comprises at least 80%, 90%, 95%, or 100% of the sequence identical to SEQ ID NO:37, and the first polypeptide comprises at least 80%, 90%, 95%, or 100% of the sequence identical to SEQ ID NO:29; (4) the first polypeptide comprises at least 80%, 90%, 95%, or 100% of the sequence identical to SEQ ID NO:38, and the first polypeptide comprises at least 80%, 90%, 95%, or 100% of the sequence identical to SEQ ID NO:35. NO:29 is at least 80%, 90%, 95% or 100% identical to the sequence; or (5) the first polypeptide comprises at least 80%, 90%, 95% or 100% identical to the sequence of SEQ ID NO:39, and the first polypeptide comprises at least 80%, 90%, 95% or 100% identical to the sequence of SEQ ID NO:29.

[0023] In some embodiments, the protein complex can induce STAT-3 phosphorylation and / or alleviate Treg-mediated T cell suppression. In some embodiments, the protein complex can induce the proliferation of immune cells (e.g., T cells or NK cells) and / or induce primary NK cell toxicity.

[0024] In one aspect, this disclosure relates to a pharmaceutical composition comprising an engineered IL-21 peptide, fusion protein, or protein complex as described herein, and a pharmaceutically acceptable carrier.

[0025] On one hand, this disclosure relates to a nucleic acid encoding an engineered IL-21 polypeptide, fusion protein, or protein complex as described herein. On the other hand, this disclosure relates to a vector comprising the nucleic acid described herein.

[0026] On one hand, this disclosure relates to a cell that includes the nucleic acids or vectors described herein. In some embodiments, the cell is an Expi293 cell or a CHO-S cell.

[0027] On one hand, this disclosure relates to a method for producing an engineered IL-21 peptide or a fusion protein comprising said engineered IL-21 peptide, said method comprising (a) culturing said cells under conditions sufficient to cause said engineered IL-21 peptide or said fusion protein to be produced by said cells; and (b) collecting said engineered IL-21 peptide, said fusion protein or said protein complex produced by said cells.

[0028] In one aspect, this disclosure relates to a method of treating a subject with cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an engineered IL-21 peptide, fusion protein, or protein complex as described herein. In some embodiments, the subject has a solid tumor or hematologic malignancy. In some embodiments, the cancer is melanoma, renal cell carcinoma (RCC), lymphoma, esophageal adenocarcinoma, lung cancer, hepatocellular carcinoma, bile duct carcinoma, gallbladder cancer, gastric cancer, pancreatic cancer, colorectal cancer, endometrial cancer, ovarian cancer, bladder cancer, or prostate cancer.

[0029] On one hand, this disclosure relates to a method for reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of a composition comprising an engineered IL-21 peptide, fusion protein, or protein complex as described herein.

[0030] On one hand, this disclosure relates to a method for killing tumor cells, the method comprising contacting tumor cells with an effective amount of a composition comprising an engineered IL-21 peptide, fusion protein, or protein complex as described herein.

[0031] On one hand, this disclosure relates to a method for improving the stability of a protein (e.g., a cytokine), the method comprising (a) providing a 3D structure of the protein (e.g., a cytokine); and (b) measuring the C-value of one or more amino acid residues in the 3D structure. α (c) the distance between atoms; and (d) selecting two amino acid residues from the one or more amino acid residues, wherein in some embodiments, the C0 of the two selected amino acid residues is... α The atoms are in the range of 3-7 angstroms (e.g., 4.5-6.5 angstroms). In some embodiments, the method described herein further includes expressing a protein variant (e.g., a cytokine variant), which in some embodiments comprises a non-natural disulfide bond formed by mutating the two selected amino acid residues to cysteine. In some embodiments, mutating the two selected amino acid residues to cysteine ​​does not substantially alter the 3D structure of the protein (e.g., a cytokine).

[0032] On one hand, this disclosure relates to a method for screening cytokine variants having improved antitumor efficacy, the method comprising (a) providing a 3D structure of the cytokine; and (b) measuring the C-value of one or more amino acid residues in the 3D structure. α (c) the distance between atoms; and (d) selecting two amino acid residues from the one or more amino acid residues, wherein in some embodiments, the C0 of the two selected amino acid residues is... α The atoms are in the range of 3-7 angstroms (e.g., 4.5-6.5 angstroms). In some embodiments, the method described herein further includes: (d) expressing a cytokine variant, in some embodiments, the variant comprising a non-natural disulfide bond formed by mutating the two selected amino acid residues to cysteine; (e) administering the cytokine variant to a tumor-bearing animal; and (f) determining tumor growth in the tumor-bearing animal (e.g., by measuring tumor volume).

[0033] In some embodiments, the protein or cytokine described herein is IL-21 (e.g., human IL-21).

[0034] In some embodiments, the protein or cytokine has no more than 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues.

[0035] As used herein, the term "non-natural disulfide bond" refers to a disulfide bond that is not naturally present in wild-type proteins. In some embodiments, a non-natural disulfide bond is formed by two cysteine ​​residues, at least one of which is mutated. In some embodiments, both of the two cysteine ​​residues are mutated. In some embodiments, at least one or two cysteine ​​residues are introduced by insertion. In some embodiments, deletion alters the distance between two existing cysteine ​​residues, which then forms a disulfide bond that is not present in wild-type proteins.

[0036] As used herein, the term "engineered IL-21 peptide" refers to a peptide derived from or a portion thereof of the wild-type IL-21 peptide, optionally having one or more mutations (e.g., insertions, deletions, or substitutions). In some embodiments, the engineered IL-21 peptide comprises or consists of an amino acid sequence corresponding to amino acids 25-162 of human IL-21 (SEQ ID NO:1). In some embodiments, the engineered IL-21 peptide has one or more mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) (e.g., amino acid substitution with cysteine).

[0037] As used herein, the term "protein construct" refers to a complex having one or more polypeptides. In some embodiments, a protein construct is a fusion protein, such as a fusion protein comprising an HSA and an engineered IL-21 polypeptide (e.g., any engineered IL-21 polypeptide described herein). In some embodiments, a protein construct has two or more polypeptides, wherein the polypeptides may associate with each other to form a dimer or a multimer (e.g., a trimer). In some embodiments, a protein construct is a heterodimeric Fc-fused IL-21 (e.g., any heterodimeric Fc-fused IL-21 or a variant thereof described herein).

[0038] As used herein, the term "cancer" refers to cells with an uncontrolled capacity for autonomous growth. Examples of such cells include cells exhibiting an abnormal state or condition characterized by rapid proliferation of cell growth. The term is intended to encompass cancerous growth, such as tumors; carcinogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. It also includes malignant tumors of various organ systems, such as those of the respiratory, cardiovascular, renal, reproductive, hematologic, nervous, hepatic, gastrointestinal, and endocrine systems; and adenocarcinomas, which include malignant tumors such as most colon cancers, renal cell carcinomas, prostate and / or testicular tumors, non-small cell lung cancer, and small bowel cancer. "Spontaneously occurring" cancer includes any cancer not experimentally induced by transplanting cancer cells into a subject, and includes, for example, spontaneously occurring cancer, cancer resulting from patient exposure to carcinogens, cancer resulting from the insertion of transgenic oncogenes or the knockout of tumor suppressor genes, and cancer resulting from infection, such as viral infection. The term "carcinoma" is recognized in the art and refers to a malignant tumor of epithelial or endocrine tissue. The term also includes carcinosarcoma, which comprises malignant tumors composed of cancerous and sarcomatous tissue. "Adenocarcinoma" refers to cancer originating from glandular tissue or cancer in which tumor cells form identifiable glandular structures. The term "sarcoma" is widely accepted in the field and refers to a mesenchymal-derived malignant tumor. The term "hematopoietic neoplasia" includes diseases involving proliferative / tumorous cells of hematopoietic origin. Hematopoietic neoplasia may arise from bone marrow, lymphatic, or erythroid cells or their precursor cells. Blood cancers are cancers that begin in hematopoietic tissues such as bone marrow or immune system cells. Examples of blood cancers include, for example, leukemia, lymphoma, and multiple myeloma.

[0039] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe animals, humans, or non-humans provided with treatment according to the method of the invention. Veterinary and non-veterinary applications are contemplated in this disclosure. Human patients can be adults or adolescents (e.g., persons under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. This includes, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), rabbits, swine animals (e.g., pigs, miniature pigs), horses, dogs, cats, cattle, and other livestock, farm animals, and zoo animals.

[0040] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers of any length of amino acids, consisting of at least two amino acids.

[0041] As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably to refer to a polymer of nucleotides of any length of at least two nucleotides, and include, but are not limited to, DNA, RNA, DNA / RNA hybrids, and modifications thereof.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. This document describes the methods and materials used in this invention; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All disclosures, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail.

[0043] Other features and advantages of the invention will become apparent from the following detailed description, drawings, and claims. Attached Figure Description

[0044] Figure 1 This is a table summarizing the purification status of IL-21 variants in Expi293 and CHO-S cells. "H8" represents the 8XHis tag.

[0045] Figure 2A The efficacy of the IL-21 variant in inducing STAT-3 phosphorylation was demonstrated.

[0046] Figure 2B The efficacy of IL-21 variants in inducing T cell proliferation was demonstrated.

[0047] Figure 3A The SDS-PAGE results for the selected HSA fusion of the IL-21 variant are shown. NR: Non-reduced. R: Reduced.

[0048] Figure 3B This is a table summarizing the HPLC-SEC analysis results of the HSA-fused IL-21 variant.

[0049] Figure 3C This is a table summarizing the DLS / SLS analysis results of the IL-21 variants fused with HSA.

[0050] Figure 4A The efficacy of the HSA-fused IL-21 variant in inducing STAT-3 phosphorylation was demonstrated.

[0051] Figure 4B The efficacy of the HSA-fused IL-21 variant in inducing T cell proliferation was demonstrated.

[0052] Figure 5 The efficacy of the HSA-fused IL-21 variant in inducing NK-92 cell proliferation was demonstrated.

[0053] Figure 6 The efficacy of the HSA-fused IL-21 variant in alleviating Treg-mediated T cell suppression was demonstrated.

[0054] Figure 7 The efficacy of the HSA-fused IL-21 variant in inducing primary NK cell cytotoxicity was demonstrated.

[0055] Figure 8A This is a table showing the treatment plan and dosing schedule for HSA-fused IL-21 and its variants in a BLAB / c mouse model carrying CT26.

[0056] Figure 8B The mean tumor growth curves of BALB / c mice carrying CT26 treated with HSA-fused IL-21 and its variants are shown. Mice were injected with HSA-fused IL-21 and its variants on days 4, 7, 11, 14, and 18 (indicated by arrows) after grouping (day 0).

[0057] Figure 8C-8F The following are examples of using media ( Figure 8C ), HSA-IL21-WT Figure 8D ), HSA-IL21-C01 ( Figure 8E ) and HSA-IL21-C08 ( Figure 8F Individual tumor growth curves of BALB / c mice carrying CT26 after treatment.

[0058] Figure 8G Individual tumor volumes of each BALB / c mouse carrying CT26 are shown on day 21 (or day 25 post-vaccination) treated with HSA-fused IL-21 and its variants.

[0059] Figure 8H The mean weight changes in BALB / c mice carrying CT26 treated with HSA-fused IL-21 and its variants are shown.

[0060] Figure 8I This table shows the tumor volume (TV), mean percentage of tumor growth inhibition (TGI%), and corresponding p-values ​​in BALB / c mice carrying CT26 treated with HSA-fused IL-21 and its variants on day 21 (or day 25 post-vaccination). P-values ​​were determined using univariate ANONA analysis.

[0061] Figure 9AThe efficacy of the HSA-fused IL-21 variant in alleviating Treg-mediated T cell suppression was demonstrated. hSIRPα-Fc-mt10 (SEQ ID NO:42) was used as a negative control.

[0062] Figure 9B The activated CD8 pretreated with 30 nM HSA-IL21-WT (“WT”), HSA-IL21-C07 (“C07”), or HSA-IL21-C08 (“C08”) is shown. + Live CD8 cells after incubation of T cells (Teff) and Treg cells + CFSE 低 Percentage of cells.

[0063] Figure 10 The efficacy of the HSA-fused IL-21 variant in inducing primary NK cell cytotoxicity was demonstrated.

[0064] Figure 11A The treatment plan and dosing schedule for HSA-fused IL-21 and its variants in a BLAB / c mouse model carrying CT26 are shown.

[0065] Figure 11B The mean tumor growth curves of BALB / c mice carrying CT26 treated with HSA-fused IL-21 and its variants are shown. Mice were injected with HSA-fused IL-21 and its variants on days 4, 8, 11, 14, 18, and 21 (indicated by arrows) after grouping (day 0).

[0066] Figure 11C-11F The following are examples of using media ( Figure 11C ), HSA-IL21-WT Figure 11D ), HSA-IL21-C01 ( Figure 11E ) and HSA-IL21-C08 ( Figure 11F Individual tumor growth curves of BALB / c mice carrying CT26 after treatment.

[0067] Figure 11G The mean weight changes in BALB / c mice carrying CT26 treated with HSA-fused IL-21 and its variants are shown.

[0068] Figure 11H Survival curves of CT26-carrying BALB / c mice treated with HSA-fused IL-21 and its variants are shown.

[0069] Figure 11I-11KIndividual tumor growth curves of BALB / c mice carrying CT26 cells after re-excitation by inoculation with CT26 cells are shown. Six mice injected with the cytokine were used as controls for tumor re-excitation. Figure 11I Ten tumors smaller than 50 mm that had previously been inoculated with CT26 and then treated with HSA-IL21-C01 were selected. 3 (n=10) mice were subjected to tumor re-challenge ( Figure 11J Eleven tumors smaller than 50 mm that had previously been inoculated with CT26 and then treated with HSA-IL21-C08 were selected. 3 (n=11) mice were subjected to tumor re-challenge ( Figure 11K ).

[0070] Figure 12 The efficacy of the heterodimeric Fc-fused IL-21 variant in inducing STAT-3 phosphorylation was demonstrated. Human recombinant IFN-γ protein (BioLegend, catalog number 570208) was used as a negative control.

[0071] Figure 13A The efficacy of the heterodimer Fc-fused IL-21 variant in inducing T cell proliferation was demonstrated. hSIRPα-Fc-mt10 (SEQ ID NO:42) was used as a negative control.

[0072] Figure 13B The efficacy of the heterodimer Fc-fused IL-21 variant in inducing NK-92 cell proliferation was demonstrated. hSIRPα-Fc-mt10 (SEQ ID NO:42) was used as a negative control.

[0073] Figure 14 The efficacy of heterodimer Fc-fused IL-21 variants in inducing primary NK cell cytotoxicity was demonstrated.

[0074] Figure 15A This is a table showing the treatment plan and dosing schedule for heterodimeric Fc-fused IL-21 and its variants in a BLAB / c mouse model carrying CT26.

[0075] Figure 15B-15G The following are examples of using media ( Figure 15B ), heterodimer Fc-IL21-WT ( Figure 15C ), heterodimer Fc-IL21-C01 ( Figure 15D ), heterodimer Fc-IL21-CO3 ( Figure 15E ), heterodimer Fc-IL21-C07 ( Figure 15F ) and heterodimer Fc-IL21-C08 ( Figure 15GIndividual tumor growth curves of BALB / c mice carrying CT26 after treatment. Mice were injected with heterodimer Fc-fused IL-21 and its variants on days 4, 7, 10, 13, 17 and 20 (indicated by arrows) after grouping (day 0).

[0076] Figure 15H The mean weight changes in BALB / c mice carrying CT26 treated with IL-21 fused with heterodimer Fc and its variants are shown.

[0077] Figure 16 The amino acid sequences discussed in this disclosure are listed. Detailed Implementation

[0078] Interleukin-21 (also known as IL-21, IL21, Za11, or CVID11) is a pleiotropic cytokine composed of four α-helical bundles and is primarily produced by natural killer T (NKT) cells and T follicle helper cells (T cells). FH IL-21 is produced by TH17 cells, with many other lymphohematopoietic cell populations producing it at lower levels. IL-21 signals via a heterodimer of the IL-21 receptor (IL-21R) and the common cytokine receptor γ chain γc (encoded by IL2RG). IL-21 signals through its receptor complex, which consists of a private chain IL-21Rα and a common chain γC, the latter shared by five other cytokines: IL-2, IL-4, IL-7, IL-9, and IL-15. These cytokines collectively constitute the so-called γC family of cytokines. Despite fairly limited sequence homology (average 15% sequence identity), these γC cytokines share a highly conserved overall four-helix bundle topology.

[0079] Functional IL-21R is widely expressed in lymphohematopoietic populations, including myeloid cells. Accordingly, IL-21 acts on multiple cell types. Given the breadth of its immunomodulatory targets and the pleiotropic effects of IL-21, IL-21 and IL-21R are attractive targets for therapeutic manipulation; in fact, antibodies against IL-21 and IL-21R, as well as IL-21 antagonists, have been developed.

[0080] IL-21 signals via the Janus kinase (JAK) signaling transducer and activator of transcription (STAT) signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, and the phosphoinositol 3-kinase (PI3K) AKT signaling pathway. Like other γc family cytokines, IL-21 activates JAK1 and JAK3. Interestingly, in T cells, IL-21 activates STAT3 in a more potent and persistent manner than STAT1, STAT5A, and STAT5B8. In studies of the regulation of mouse B lymphocyte-induced maturation protein 1 (BLIMP1; a transcription factor encoded by the Prdm1 gene), IL-21 response elements were shown to consist of a bipartite element that binds both interferon regulator factor 4 (IRF4) and STAT3. Surprisingly, when analyzed using chromatin immunoprecipitation associated with next-generation sequencing (ChIP-seq), these bipartite response elements were found in a genome-wide manner and are involved globally in the regulation of many IL-21 response genes.

[0081] Detailed descriptions of IL-21 and its functions can be found in, for example, Spolski, R. et al., “Interleukin-21: a double-edged sword with therapeutic potential,” *Nature Reviews Drug Discovery* 13.5 (2014):379-395; Kang, L. et al., “Rational design of interleukin-21 antagonist through selective elimination of the γC-binding epitope,” *Journal of Biological Chemistry* 285.16 (2010):12223-12231; Croce, M. et al., “IL-21: a pleiotropic cytokine with potential applications in oncology.” "Interleukin-21 in cancer immunotherapy: Friend or foe?", Journal of Immunology Research, 2015; and Stolfi, C. et al., "Interleukin-21 in cancer immunotherapy: Friend or foe?", Oncoimmunology 1.3 (2012):351-354; each of the above references is incorporated in its entirety by citation.

[0082] This disclosure provides engineered IL-21 variants having at least one non-natural disulfide bond. In some embodiments, two amino acid residues in wild-type human IL-21 (e.g., SEQ ID NO:2) are selectively mutated to cysteine ​​residues, which can form a non-natural disulfide bond. In some embodiments, the mutation substantially does not alter the overall structure of IL-21, e.g., the relative positions of the four α-helices of IL-21. In some embodiments, the non-natural disulfide bond formed by the cysteine ​​mutation can stabilize IL-21. In some embodiments, the engineered IL-21 variants described herein can bind to the IL-21R / γc complex and induce downstream signaling pathways (e.g., STAT3 phosphorylation) and / or immune cell (e.g., T cells or NK cells) proliferation. In some embodiments, the engineered IL-21 variants comprise or consist of any engineered IL-21 polypeptide described herein.

[0083] This document also provides protein constructs (e.g., fusion proteins) further comprising human serum albumin (HSA) fused with the engineered IL-21 variant described herein. In some embodiments, the HSA may stabilize the engineered IL-21 variant. In some embodiments, the protein construct may promote immune responses (e.g., alleviate Treg-mediated T cell suppression and / or induce primary NK cell toxicity).

[0084] This document also provides protein constructs (e.g., protein complexes) in which an engineered IL-21 variant (e.g., any engineered IL-21 variant described herein) is linked to the C-terminus of human IgG4 Fc to form a heterodimer (e.g., any heterodimer Fc-fused IL-21 variant described herein). In some embodiments, the protein complex may promote an immune response (e.g., induce IL-21-STAT3 signaling, induce immune cell proliferation, and / or induce primary NK cell toxicity).

[0085] This disclosure also provides methods for screening cytokine (e.g., IL-21) variants that have higher stability and / or improved antitumor efficacy.

[0086] Engineered IL-21 variant

[0087] IL-21 is expressed in activated human CD4+ T cells, but not in most other tissues. Furthermore, IL-21 expression is upregulated in the Th2 and Th17 subsets of T helper cells and in T follicular cells. In fact, IL-21 has been shown to be useful for identifying peripheral T follicular helper cells. In addition, IL-21 is expressed in NK T cells, which regulate the function of these cells.

[0088] Human IL-21 comprises a signal peptide and a soluble chain from the N-terminus to the C-terminus. According to the UniProt database (UniProt ID: Q9HBE4), the signal peptide of human IL-21 corresponds to amino acids 1-24 of SEQ ID NO:1, and the soluble chain of human IL-21 corresponds to amino acids 25-162 of SEQ ID NO:1. The soluble chain of human IL-21 is also shown in SEQ ID NO:2. Although the average sequence homology among the γc cytokines IL-21, IL-2, and IL-4 is only about 17%, the topological structure of the four helical bundles forming the core structure of these γc cytokines is highly conserved. Specifically, the first α-helix corresponds to M12 to N30 of SEQ ID NO:2, the second α-helix corresponds to E48 to A58 of SEQ ID NO:2, the third α-helix corresponds to N67 to K78 of SEQ ID NO:2, and the fourth α-helix corresponds to P109 to L128 of SEQ ID NO:2. Based on the model of the IL-21 / IL-21R / γc complex, the following residues of IL-21 were identified as potentially key positions for binding to IL-21R or γc: amino acids corresponding to positions 12, 16, 19, 23, 105, 114, 118, 121, 122, 124, 125, and 128 in SEQ ID NO:2. Specifically, these residues are M12, R16, I19, D23, E105, E114, S118, Q121, K122, I124, H125, and L128 in SEQ ID NO:2. Further details can be found, for example, in Kang, L. et al., “Rational design of interleukin-21 antagonists by selective elimination of γC binding epitopes,” *Journal of Biochemistry* 285.16(2010):12223-12231, which is incorporated herein by reference in its entirety.

[0089] Based on the 3D structure of human IL-21, the C-values ​​of one or more amino acid residues (e.g., closely spaced amino acid residues) in the 3D structure can be determined. α The distance between atoms. C αTwo residues within the 3–7 Å (e.g., 4.5–6.5 Å) region of the atom can be selectively mutated to cysteine ​​without interfering with the overall structure of IL-21 (e.g., the core structure formed by four helical bundles). It is conceivable that the newly introduced cysteine ​​could form a non-natural disulfide bond to wild-type IL-21, which could stabilize IL-21 and / or enhance its functional potency.

[0090] Therefore, on the one hand, engineered IL-21 variants (e.g., engineered IL-21 peptides) contain a first cysteine ​​mutation and a second cysteine ​​mutation, such that these two cysteine ​​residues can form a non-natural disulfide bond. In some embodiments, the first cysteine ​​mutation occurs at amino acid residues corresponding to positions 8, 19, 29, 31, 33, 36, 39, or 56 of SEQ ID NO:2. In some embodiments, the second cysteine ​​mutation occurs at amino acid residues corresponding to positions 61, 62, 63, 80, 86, 105, 106, 107, 110, 112, or 117 of SEQ ID NO:2. In some embodiments, the C of the two selected amino acid residues... α Atoms in or In some embodiments, when the C of the two selected amino acid residues... α When the atomic distance is within the aforementioned range, any first cysteine ​​mutation in the first cysteine ​​mutation may pair with any second cysteine ​​mutation in the second cysteine ​​mutation described herein. In some embodiments, non-natural disulfide bonds may further stabilize the overall structure of IL-21, for example, by maintaining the relative positions and angles of the four helical bundles in IL-21.

[0091] In some embodiments, this document provides an engineered IL-21 peptide IL21-C01 containing a first cysteine ​​residue at position 8 (e.g., Q8) of SEQ ID NO:2 and a second cysteine ​​residue at position 86 (T86) of SEQ ID NO:2. The sequence of IL21-C01 is shown in SEQ ID NO:3.

[0092] In some embodiments, this document provides an engineered IL-21 peptide IL21-CO2 containing a first cysteine ​​residue at position 19 (e.g., I19) of SEQ ID NO:2 and a second cysteine ​​residue at position 117 (K117) of SEQ ID NO:2. The sequence of IL21-CO2 is shown in SEQ ID NO:4.

[0093] In some embodiments, this document provides an engineered IL-21 peptide IL21-C03 containing a first cysteine ​​residue at position 19 (e.g., V29) of SEQ ID NO:2 and a second cysteine ​​residue at position 110 (K110) of SEQ ID NO:2. The sequence of IL21-C03 is shown in SEQ ID NO:5.

[0094] In some embodiments, this document provides an engineered IL-21 peptide IL21-C04 containing a first cysteine ​​residue at position 31 (e.g., D31) of SEQ ID NO:2 and a second cysteine ​​residue at position 62 (S62) of SEQ ID NO:2. The sequence of IL21-C04 is shown in SEQ ID NO:6.

[0095] In some embodiments, this document provides an engineered IL-21 peptide IL21-C05 containing a first cysteine ​​residue at position 31 (e.g., D31) of SEQ ID NO:2 and a second cysteine ​​residue at position 63 (A63) of SEQ ID NO:2. The sequence of IL21-C05 is shown in SEQ ID NO:7.

[0096] In some embodiments, this document provides an engineered IL-21 peptide IL21-C06 containing a first cysteine ​​residue at position 33 (e.g., V33) of SEQ ID NO:2 and a second cysteine ​​residue at position 61 (K61) of SEQ ID NO:2. The sequence of IL21-C06 is shown in SEQ ID NO:8.

[0097] In some embodiments, this document provides an engineered IL-21 peptide IL21-C07 containing a first cysteine ​​residue at position 33 (e.g., V33) of SEQ ID NO:2 and a second cysteine ​​residue at position 63 (A63) of SEQ ID NO:2. The sequence of IL21-C07 is shown in SEQ ID NO:9.

[0098] In some embodiments, this document provides an engineered IL-21 peptide IL21-C08 containing a first cysteine ​​residue at position 36 (e.g., F36) of SEQ ID NO:2 and a second cysteine ​​residue at position 106 (K106) of SEQ ID NO:2. The sequence of IL21-C08 is shown in SEQ ID NO:10.

[0099] In some embodiments, this document provides an engineered IL-21 peptide IL21-C09 containing a first cysteine ​​residue at position 36 (e.g., F36) of SEQ ID NO:2 and a second cysteine ​​residue at position 107 (K107) of SEQ ID NO:2. The sequence of IL21-C09 is shown in SEQ ID NO:11.

[0100] In some embodiments, this document provides an engineered IL-21 peptide IL21-C10 containing a first cysteine ​​residue at position 39 (e.g., A39) of SEQ ID NO:2 and a second cysteine ​​residue at position 105 (E105) of SEQ ID NO:2. The sequence of IL21-C10 is shown in SEQ ID NO:12.

[0101] In some embodiments, this document provides an engineered IL-21 peptide IL21-C11 containing a first cysteine ​​residue at position 39 (e.g., A39) of SEQ ID NO:2 and a second cysteine ​​residue at position 107 (K107) of SEQ ID NO:2. The sequence of IL21-C11 is shown in SEQ ID NO:13.

[0102] In some embodiments, this document provides an engineered IL-21 peptide IL21-C12 containing a first cysteine ​​residue at position 39 (e.g., A39) of SEQ ID NO:2 and a second cysteine ​​residue at position 112 (F112) of SEQ ID NO:2. The sequence of IL21-C12 is shown in SEQ ID NO:14.

[0103] In some embodiments, this document provides an engineered IL-21 peptide IL21-C13 containing a first cysteine ​​residue at position 56 (e.g., Q56) of SEQ ID NO:2 and a second cysteine ​​residue at position 80 (K80) of SEQ ID NO:2. The sequence of IL21-C13 is shown in SEQ ID NO:15.

[0104] In some embodiments, this document provides an engineered IL-21 peptide comprising one or more of the following: (a) an amino acid corresponding to Q8 of SEQ ID NO:2 that is C, and an amino acid corresponding to T86 of SEQ ID NO:2 that is C; (b) an amino acid corresponding to I19 of SEQ ID NO:2 that is C, and an amino acid corresponding to K117 of SEQ ID NO:2 that is C; (c) an amino acid corresponding to V29 of SEQ ID NO:2 that is C, and an amino acid corresponding to K110 of SEQ ID NO:2 that is C; (d) an amino acid corresponding to D31 of SEQ ID NO:2 that is C, and an amino acid corresponding to S62 of SEQ ID NO:2 that is C; (e) an amino acid corresponding to D31 of SEQ ID NO:2 that is C, and an amino acid corresponding to A63 of SEQ ID NO:2 that is C; (f) an amino acid corresponding to V33 of SEQ ID NO:2 that is C, and an amino acid corresponding to K61 of SEQ ID NO:2 that is C; (g) an amino acid corresponding to Q8 of SEQ ID NO:2 that is C; (d) an amino acid corresponding to D31 of SEQ ID NO:2 that is C; (e) an amino acid corresponding to D31 of SEQ ID NO:2 that is C, and an amino acid corresponding to A63 of SEQ ID NO:2 that is C; (f) an amino acid corresponding to V33 of SEQ ID NO:2 that is C, and an amino acid corresponding to K61 of SEQ ID NO:2 that is C; (g) an amino acid corresponding to Q8 of SEQ ID NO:2 that is C; (g) an amino acid corresponding to Q8 of SEQ ID NO:2 that is C; (c) an amino acid corresponding to Q8 of SEQ ID NO:2 that is C; (d) an amino acid corresponding to D31 of SEQ ID NO:2 that is C; (e) an amino acid corresponding to D31 of SEQ ID NO:2 that is C; The amino acid corresponding to V33 of SEQ ID NO:2 is C, and the amino acid corresponding to A63 of SEQ ID NO:2 is C; (h) the amino acid corresponding to F36 of SEQ ID NO:2 is C, and the amino acid corresponding to K106 of SEQ ID NO:2 is C; (i) the amino acid corresponding to F36 of SEQ ID NO:2 is C, and the amino acid corresponding to K107 of SEQ ID NO:2 is C; (j) the amino acid corresponding to A39 of SEQ ID NO:2 is C, and the amino acid corresponding to E105 of SEQ ID NO:2 is C; (k) the amino acid corresponding to A39 of SEQ ID NO:2 is C, and the amino acid corresponding to K107 of SEQ ID NO:2 is C; (l) the amino acid corresponding to A39 of SEQ ID NO:2 is C, and the amino acid corresponding to F112 of SEQ ID NO:2 is C; and (m) the amino acid corresponding to Q56 of SEQ ID NO:2 is C, and the amino acid corresponding to K80 of SEQ ID NO:2 is C.

[0105] In some embodiments, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or all amino acids corresponding to positions 12, 16, 19, 23, 105, 114, 118, 121, 122, 124, 125, and 128 of the engineered IL-21 peptide are free of mutations. For example, in some embodiments, the amino acid corresponding to position 12 (e.g., M12) of SEQ ID NO:2 in the engineered IL-21 peptide described herein is Met; the amino acid corresponding to position 16 (e.g., R16) of SEQ ID NO:2 in the engineered IL-21 peptide described herein is Arg; the amino acid corresponding to position 19 (e.g., I19) of SEQ ID NO:2 in the engineered IL-21 peptide described herein is Ile; the amino acid corresponding to position 23 (e.g., D23) of SEQ ID NO:2 in the engineered IL-21 peptide described herein is Asp; the amino acid corresponding to position 105 (e.g., E105) of SEQ ID NO:2 in the engineered IL-21 peptide described herein is Glu; the amino acid corresponding to position 114 (e.g., E114) of SEQ ID NO:2 in the engineered IL-21 peptide described herein is Glu; and the amino acid corresponding to position 12 (e.g., E114) of SEQ ID NO:2 in the engineered IL-21 peptide described herein is Glu; and the amino acid corresponding to position 16 (e.g., R16) of SEQ ID NO:2 in the engineered IL-21 peptide described herein is Arg; the amino acid corresponding to position 19 (e.g., I19) of SEQ ID NO:2 in the engineered IL-21 peptide described herein is Ile; the amino acid corresponding to position 114 (e.g., E114) of SEQ ID NO:2 in the engineered IL-21 peptide described herein is Glu; and the amino acid corresponding to position 12 (e.g., R16) of SEQ ID NO:2 in the engineered IL-21 peptide described herein is Arg; and the amino acid corresponding to position 19 (e.g., I19) of SEQ ID NO:2 in the engineered The amino acid corresponding to position 118 of NO:2 (e.g., Q121) is Gln; the amino acid corresponding to position 122 of SEQ ID NO:2 (e.g., K122) of the engineered IL-21 peptide described herein is Lys; the amino acid corresponding to position 124 of SEQ ID NO:2 (e.g., I124) of the engineered IL-21 peptide described herein is Ile; the amino acid corresponding to position 125 of SEQ ID NO:2 (e.g., H125) of the engineered IL-21 peptide described herein is His; and / or the amino acid corresponding to position 128 of SEQ ID NO:2 (e.g., L128) of the engineered IL-21 peptide described herein is Leu.

[0106] In some embodiments, the engineered IL-21 polypeptide comprises or consists of at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the engineered IL-21 peptide described herein comprises or consists of the following: an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO:1 or SEQ ID NO:2, wherein the amino acid sequence comprises one or more mutations among those described herein. In some embodiments, the engineered IL-21 peptide described herein comprises at least one, at least two, at least three, at least four, or at least five pairs of cysteine ​​mutations as described in Table 1.

[0107] In some embodiments, the engineered IL-21 polypeptide may have at least one (e.g., at least two, three, four, five, six, seven, eight, nine, ten, twenty, thirty, or forty) amino acid insertions, deletions, or substitutions, compared to any of SEQ ID NO:2-15.

[0108] The engineered IL-21 peptide may have further modifications. In some embodiments, the engineered IL-21 peptide may have an Fc CH2 domain and / or a CH3 domain. In some embodiments, the engineered IL-21 peptide may be linked to the N-terminus of the CH2 domain (e.g., via an optional hinge region or GS linker). In some embodiments, the engineered IL-21 peptide may be linked to the C-terminus of the CH3 domain (e.g., via an optional GS linker). In some embodiments, the hinge region is an IgG hinge region (e.g., an IgG4 hinge region). In some embodiments, the CH2 domain is an IgG CH2 domain (e.g., an IgG4 CH2 domain). In some embodiments, the CH3 domain is an IgG CH3 domain (e.g., an IgG4 CH3 domain).

[0109] In some embodiments, the engineered IL-21 peptides described herein may also include a tag (e.g., a His tag) to facilitate screening and / or detection. In some embodiments, the tag has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any of the peptides in SEQ ID NO:22. In some embodiments, the tag is attached to the N-terminus or C-terminus of any engineered IL-21 peptide described herein.

[0110] In some embodiments, the engineered IL-21 peptide described herein can be expressed in Expi293 or CHO-S cells.

[0111] IL-21 protein construct

[0112] This disclosure provides protein constructs (e.g., fusion proteins or protein complexes) comprising engineered IL-21 variants described herein (e.g., any engineered IL-21 peptide described herein). In some embodiments, the protein construct further comprises human serum albumin (HSA) fused to the engineered IL-21 peptide. In some embodiments, the fusion protein may be expressed in Expi293 or CHO-S cells. In some embodiments, the HSA is fused to the engineered IL-21 peptide via a adaptor peptide (e.g., a flexible adaptor). In some embodiments, the HSA is linked to the N-terminus of the engineered IL-21 peptide via the adaptor peptide. In some embodiments, the HSA is linked to the C-terminus of the engineered IL-21 peptide via the adaptor peptide. In some embodiments, the protein construct described herein has an N-terminal His tag. In some embodiments, the protein construct described herein has a C-terminal His tag.

[0113] In some embodiments, the protein construct described herein comprises, from the N-terminus to the C-terminus: (a) optionally a His tag; (b) an HSA; (c) a linker peptide; and (d) an engineered IL-21 polypeptide (e.g., any engineered IL-21 polypeptide described herein). In some embodiments, the His tag comprises at least 6, at least 7, or at least 8 consecutive His residues. In some embodiments, the His tag comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:22. In some embodiments, the HSA is a wild-type HSA or a fragment thereof. In some embodiments, the HSA comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:26. In some embodiments, the adaptor peptide comprises at least one, two, three, four, five, six, seven, or eight repeating sequences of GGGGS (SEQ ID NO:27). In some embodiments, the adaptor peptide is a flexible adaptor. Detailed information on flexible adaptors can be found, for example, Chen, X. et al., “Fusion protein linkers: property, design and functionality”, Advanced Drug Delivery Reviews 65.10(2013):1357-1369, which is incorporated herein by reference in its entirety. In some embodiments, the adaptor peptide comprises or consists of an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:25. In some embodiments, the engineered IL-21 peptide comprises or consists of at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 97%, 98%, 99%, or 100% identical amino acid sequences to SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the engineered IL-21 peptide described herein contains at least one, at least two, at least three, at least four, or at least five pairs of cysteine ​​mutations as described in Table 1.

[0114] In some embodiments, the engineered IL-21 protein construct may include any engineered IL-21 variant as described herein. In some embodiments, the protein construct described herein comprises or consists of at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 17, 18, 19, 20, or 21.

[0115] In some embodiments, the protein constructs provided herein comprise a His tag (e.g., any His tag described herein) and an engineered IL-21 polypeptide (e.g., any engineered IL-21 polypeptide described herein) from the N-terminus to the C-terminus. In some embodiments, the protein constructs described herein comprise or consist of the following: an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:24.

[0116] This disclosure also provides a nucleic acid comprising a polynucleotide encoding a polypeptide, the polypeptide comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequences to any sequence in SEQ ID NO:2-15; or any sequence in SEQ ID NO:17-21.

[0117] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. The molecules are identical at said position when a position in the first sequence is occupied by the same amino acid residue or nucleotide as its corresponding position in the second sequence. The percentage of identity between two sequences is a function of the number of shared identical positions, taking into account the number of vacancies and the length of each vacancy, and this function needs to be introduced for optimal alignment of the two sequences. For example, sequence comparison and determination of the percentage of identity between two sequences can be accomplished using a Blossum 62 scoring matrix where the vacancy penalty is 12, the vacancy extension penalty is 4, and the frameshift vacancy penalty is 5.

[0118] Engineered IL-21 variants (e.g., any engineered IL-21 peptide described herein) and protein constructs may further include an Fc region of an antibody. These antibodies may belong to any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class, or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE1, IgE2). In some embodiments, the Fc region is derived from human IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the Fc region is the IgG4 Fc region (e.g., the human IgG4 Fc region).

[0119] In some embodiments, the engineered IL-21 variant is linked to the Fc region via an antibody hinge region (e.g., an IgG or IgE hinge region). Additionally, the Fc region may be modified to provide desired effector function or serum half-life.

[0120] In some embodiments, the protein construct as described herein includes a functional Fc region. In some embodiments, the Fc region is human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the effector function of the functional Fc region is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector functions of the functional Fc region are both ADCC and phagocytosis. In some embodiments, the protein construct as described herein has an Fc region that does not have an effector function. In some embodiments, the Fc is human IgG4 Fc. In some embodiments, the Fc does not have a functional Fc region. For example, the Fc region has an LALA mutation (EU designation L234A and L235A mutations) or an LALA-PG mutation (EU designation L234A, L235A, P329G mutation).

[0121] In some embodiments, an engineered IL-21 variant (e.g., any engineered IL-21 variant described herein) is linked to the N-terminus or C-terminus of the Fc region. In some embodiments, the engineered IL-21 variant is linked to the Fc region via a linker peptide (e.g., any linker peptide described herein).

[0122] In some embodiments, the protein constructs provided herein comprise, from the N-terminus to the C-terminus, a human IgG4 hinge region and an Fc region (e.g., SEQ ID NO:28), a adaptor peptide (e.g., any adaptor peptide described herein), and an engineered IL-21 polypeptide (e.g., any engineered IL-21 polypeptide described herein). In some embodiments, the engineered IL-21 polypeptide comprises or consists of the following: at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical sequence to any of SEQ ID NO:2-15. In some embodiments, the protein constructs described herein comprise or consist of the following: an amino acid sequence that is at least 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:16.

[0123] Other modifications can be made to the Fc region. For example, cysteine ​​residues can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The resulting homodimeric fusion protein may thus have any increased half-life in vitro and / or in vivo. In some embodiments, IgG4 has an S228P mutation (EU number). The S228P mutation prevents IgG4 Fab arm exchange in vivo and in vitro.

[0124] In some embodiments, the Fc region is provided as a carbohydrate structure lacking fucose (directly or indirectly) linked to the Fc region. For example, the amount of fucose in such Fc region compositions may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid, and high-mannose structures) linked to Asn297 as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 (EU number of the Fc region residue; or position 314 in the Kabat number) in the Fc region; however, due to minor sequence variations in the Fc region sequence, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC functionality. In some embodiments, to reduce glycan heterogeneity, the Fc region may be further engineered to replace the asparagine at position 297 with alanine (N297A).

[0125] In some embodiments, this disclosure relates to a protein construct comprising an engineered IL-21 peptide as described herein. In some embodiments, the protein construct comprises two or more engineered IL-21 peptides. In some embodiments, at least two of the engineered IL-21 peptides are identical. In some embodiments, at least two of the engineered IL-21 peptides are different. In some embodiments, the protein construct further comprises an Fc region. In some embodiments, the Fc region is an IgG4 Fc region. In some embodiments, the Fc region is an IgG1 Fc region (e.g., having a LALA mutation or a LALA-PG mutation). In some embodiments, the engineered IL-21 peptide is linked to the C-terminus of the Fc region. In some embodiments, the engineered IL-21 peptide is linked to the C-terminus of the Fc region via a adaptor peptide (e.g., any adaptor peptide described herein).

[0126] This disclosure provides protein complexes comprising engineered IL-21 variants described herein (e.g., any engineered IL-21 polypeptide described herein). In some embodiments, this disclosure relates to a protein complex comprising a first polypeptide and a second polypeptide. In some embodiments, the first polypeptide optionally comprises, or consists of, the following from its N-terminus to its C-terminus: an optional first hinge region (e.g., a human IgG4 hinge region), a first Fc region (e.g., a human IgG4 Fc region), an optional adaptor peptide (e.g., any adaptor peptide described herein), and an engineered IL-21 polypeptide (e.g., any engineered IL-21 polypeptide described herein). In some embodiments, the second polypeptide optionally comprises, or consists of, the following from its N-terminus to its C-terminus: an optional second hinge region (e.g., a human IgG4 hinge region) and a second Fc region (e.g., a human IgG4 Fc region). In some embodiments, the first Fc region and / or the second Fc region described herein do not contain a hinge region. In some embodiments, the first Fc region and / or the second Fc region described herein consist of a CH2 domain (e.g., human IgG4 CH2 domain) and a CH3 domain (e.g., human IgG4 CH3 domain).

[0127] In some embodiments, the first hinge region and / or the second hinge region comprises all or a portion of the hinge region of an immunoglobulin, such as the human IgG4 hinge region (SEQ ID NO: 31). In some embodiments, the first hinge region and / or the second hinge region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to that of SEQ ID NO: 31. In some embodiments, the first hinge region and the second hinge region are identical. In some embodiments, the first hinge region and the second hinge region are different.

[0128] In some embodiments, the first Fc region and / or the second Fc region are identical and may form an Fc homodimer. In some embodiments, the first Fc region and / or the second Fc region comprises all or part of the Fc region of an immunoglobulin, such as the human IgG4 Fc region (SEQ ID NO: 40). In some embodiments, the first Fc region and / or the second Fc region are different. In some embodiments, the first Fc region and / or the second Fc region may form an Fc heterodimer by introducing one or more mutations. In some cases, the first Fc region and / or the second Fc region may contain one or more kilomeroid (KIH) mutations. For example, the first Fc region may contain cysteine ​​at position 349, serine at position 366, alanine at position 368, and valine at position 407 according to EU numbers; and the second Fc region may contain cysteine ​​at position 354 and tryptophan at position 366 according to EU numbers. In some embodiments, the first Fc region comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO:33, and the second Fc region comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to that of SEQ ID NO:32. In some embodiments, the first Fc region and / or the second Fc region may be formed into an Fc heterodimer using other techniques. Details of KIH mutations and other heterodimer Fc technologies can be found, for example, Ha et al., “Immunoglobulin Fc heterodimer platform technology: from design to applications in therapeutic antibodies and proteins,” Frontiers in Immunology 7 (2016): 394, which is incorporated herein by reference in its entirety. In some embodiments, the first Fc region and / or the second Fc region described herein are derived from human IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the first Fc region and / or the second Fc region is the IgG4 Fc region (e.g., the human IgG4 Fc region). In some embodiments, the first Fc region and / or the second Fc region is an IgG Fc region whose effector function is silenced (e.g., the human IgG1 Fc region).Details of methods for modulating Fc effector function can be found, for example, Liu, R. et al., “Fc-engineering for modulated effector functions—improving antibodies for cancer treatment”, Antibodies 2020; 9:64; and Saunders, KO, “Conceptual approaches to modulating antibody effector functions and circulation half-life”, Frontiers in Immunology 10:1296. (2019); each of these references is incorporated herein by reference in its entirety.

[0129] In some embodiments, the adaptor peptide described herein comprises at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO:34. In some embodiments, the adaptor peptide described herein comprises at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) repeating sequences of GGGGS (SEQ ID NO:41).

[0130] In some embodiments, the first polypeptide described herein comprises at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO:30, 35, 36, 37, 38, or 39; and the second polypeptide described herein comprises at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO:29.

[0131] Characterization of engineered IL-21 variants or protein constructs

[0132] In some embodiments, the engineered IL-21 variants described herein (e.g., any engineered IL-21 peptide described herein) or their protein constructs (e.g., fusion proteins or protein complexes) may bind to a complex formed by the human IL-21 receptor (IL-21R) and the common cytokine γ chain (γc). Because the newly introduced non-natural disulfide bonds stabilize IL-21 (e.g., the core structure formed by four helical bundles) without causing substantial conformational changes in the protein structure, and because the residues essential for IL-21R or γc binding (e.g., residues corresponding to M12, R16, I19, D23, E105, E114, S118, Q121, K122, I124, H125 and / or L128 in SEQ ID NO:2) are unmutated, engineered IL-21 variants or their protein constructs can induce downstream signaling pathways, such as the JAK-STAT, MAPK, and / or PI3K pathways, by binding to the IL-21R / γc complex expressed on immune cells (e.g., T cells, B cells, and / or NK cells).

[0133] In some embodiments, the introduction of non-natural disulfide bonds or cysteine ​​mutations (e.g., any cysteine ​​mutation described herein) may result in a protein conformational change where the RMSD (root mean square deviation of atomic positions) value is less than [value missing]. Less than Less than Less than Less than Less than Less than Less than Less than or less In some embodiments, the RMSD value is calculated by structurally comparing the wild-type protein and the protein variant. In some embodiments, only C α Atoms are used to determine conformational changes.

[0134] The biological effects of engineered IL-21 variants or their protein constructs include immunomodulatory and immunostimulatory effects. Exemplary immunomodulatory effects include, for example, increasing the immune response; increasing B cell proliferation; inducing plasma cell differentiation; increasing immunoglobulin production; and increasing T follicle-helping (T follicle-assisted ovulation). FHCell differentiation and / or proliferation; increased proliferation, survival, and / or antitumor activity of cytotoxic T lymphocytes (CTLs); increased expression of CD28 and L-selectin; increased NK cell proliferation, antitumor activity, and / or ADCC activity; increased differentiation, proliferation, and / or IL-23R expression of Th17 cells; and inhibition of Treg cell survival and / or production. Exemplary immunomodulatory effects include, for example, inhibition of differentiation, proliferation, and / or IL-10 production of type 1 regulatory T (Tr1) cells; inhibition of apoptosis, and / or inhibition of APC function of dendritic cells (DC cells); and inhibition of differentiation, proliferation, and / or IL-10 production of B-10 or B-reg cells. Further details can be found, for example, Croce, M. et al., “IL-21: a pleiotropic cytokine with potential applications in oncology”, Journal of Immunological Research 2015 (2015), which is incorporated herein by reference in its entirety.

[0135] In some embodiments, engineered IL-21 variants (e.g., any engineered IL-21 peptide described herein) or their protein constructs described herein (e.g., any of the HSA-fused IL-21 variants or heterodimeric Fc-fused IL-21 variants described herein) have comparable potency in inducing T cell responses. In some embodiments, engineered IL-21 variants or their protein constructs may induce STAT3 phosphorylation in immune cells (e.g., activated CD3+ or CD8+ T cells) with at least about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, or about 140% potency compared to wild-type IL-21 or its protein constructs. In some embodiments, EC50 values ​​of proliferation curves may be determined, for example, less than 0.1 nM, less than 0.09 nM, less than 0.08 nM, less than 0.07 nM, or less than 0.06 nM.

[0136] In some embodiments, the engineered IL-21 variants or protein constructs described herein (e.g., any one of the HSA-fused IL-21 variants or the heterodimeric Fc-fused IL-21 variants described herein) can induce the proliferation of immune cells (e.g., activated CD3+ or CD8+ T cells) with at least about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200% potency compared to wild-type IL-21 or its protein constructs. In some embodiments, the EC50 value of the proliferation curve may be determined, for example, less than 1 nM, less than 0.5 nM, less than 0.4 nM, or less than 0.3 nM. In some embodiments, the EC50 value of the proliferation curve of activated CD8+ T cells treated with the engineered IL-21 variant or protein construct thereof described herein is less than 50%, less than 40%, less than 30%, or less than 20% of the EC50 value of the proliferation curve of activated CD8+ T cells treated with wild-type IL-21 or protein construct thereof.

[0137] In some embodiments, the engineered IL-21 variants described herein comprise at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO: 3, 5, 9, 10, or 13. In some embodiments, the protein constructs described herein (HSA-fused IL-21 variants) comprise at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO: 17, 18, 19, 20, or 21. In some embodiments, the protein construct (e.g., a heterodimeric Fc-fused IL-21 variant) comprises a first polypeptide having at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO: 35, 36, 37, 38, or 39; and a second polypeptide having at least 80%, 85%, 90%, 95%, or 100% of the same amino acid sequence as SEQ ID NO: 29.

[0138] In some embodiments, the engineered IL-21 variants or protein constructs described herein (e.g., any one of the HSA-fused IL-21 variants or the heterodimeric Fc-fused IL-21 variants described herein) can induce NK cell (e.g., NK-92 cells) proliferation with about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, or about 200% potency compared to wild-type IL-21 or its protein constructs. In some embodiments, NK cell proliferation is determined after starvation for at least 12, 18, 24, 30, or 36 hours. In some embodiments, the EC50 value of the proliferation curve may be determined, for example, less than 0.2 nM, less than 0.15 nM, less than 0.14 nM, less than 0.13 nM, less than 0.12 nM, less than 0.11 nM, less than 0.1 nM, less than 0.09 nM, less than 0.08 nM, less than 0.07 nM, less than 0.06 nM, or less than 0.05 nM. In some embodiments, the EC50 value of the proliferation curve of NK cells treated with the engineered IL-21 variant or its protein construct described herein is less than 90%, less than 80%, less than 70%, or less than 60% of the EC50 value of the proliferation curve of NK cells treated with wild-type IL-21 or its protein construct.

[0139] In some embodiments, the engineered IL-21 variants or protein constructs thereof described herein (e.g., any one of the HSA-fused IL-21 variants or the heterodimeric Fc-fused IL-21 variants described herein) can alleviate Treg-mediated suppression of T cells (e.g., activated CD8+ T cells). In some embodiments, after treatment with the engineered IL-21 variant or its protein construct described herein and in the presence of Treg cells, T cell (e.g., activated CD8+ T cells) proliferation may be increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, or at least 300%, compared to wild-type IL-21 or its protein construct.

[0140] In some embodiments, the engineered IL-21 variants or protein constructs described herein (e.g., any one of the HSA-fused IL-21 variants or the heterodimeric Fc-fused IL-21 variants described herein) can induce NK cell (e.g., primary NK cells) cytotoxicity by about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, or about 140% compared to wild-type IL-21 or its protein constructs. In some embodiments, the percentage of specific cleavage of NK cells against target tumor cells (e.g., K-562 tumor cells) can be increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to wild-type IL-21 or its protein constructs.

[0141] In some embodiments, engineered IL-21 variants or protein constructs thereof, as described herein, can increase the immune response, activity, or number of immune cells (e.g., T cells or NK cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

[0142] In some implementations, engineered IL-21 variants or their protein constructs can be less than 0.1s. -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 Or less than 0.00001s -1 dissociation rate (k off ) binds to IL-21R, γc, or complexes thereof (e.g., human IL-21R / γc complex). In some embodiments, the dissociation rate (k off (greater than 0.01s) -1 Greater than 0.001s -1 Greater than 0.0001s -1 Greater than 0.00001s -1 or greater than 0.000001s -1 .

[0143] In some embodiments, the dynamic association rate (k on Greater than 1×10 2 / Ms, greater than 1×10 3 / Ms, greater than 1×10 4 / Ms, greater than 1×105 / Ms or greater than 1×10 6 / Ms. In some embodiments, the kinetic association rate (k on Less than 1×10 5 / Ms, less than 1×10 6 / Ms or less than 1×10 7 / Ms.

[0144] Affinity can be derived from the quotient of the kinetic rate constant (KD = k off / k on Derivation. In some embodiments, KD is less than 1 × 10⁻⁶. -6 M, less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M or less than 1×10 -10 M. In some embodiments, KD is less than 300 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. In some embodiments, KD is greater than 1 × 10⁻⁶. -7 M, greater than 1×10 -8 M, greater than 1×10 -9 M, greater than 1×10 -10 M, greater than 1×10 -11 M or greater than 1×10 -12 M.

[0145] Common techniques for measuring affinity include, for example, ELISA, radioimmunoassay (RIA), and surface plasmon resonance (SPR). In some embodiments, affinity is determined by cell-based assays.

[0146] In some embodiments, the engineered IL-21 protein constructs described herein (e.g., any HSA-fused IL-21 variants of the HSA-fused IL-21 variants described herein) can be expressed and purified using methods commonly used in the art (e.g., affinity chromatography). In some cases, the protein constructs can be purified by a combination of size exclusion chromatography (SEC) and HPLC. In some embodiments, the percentage of the main peak in the SEC-HPLC analysis results is at least 80%, at least 90%, at least 95%, at least 96%, at least 96%, at least 97%, at least 98%, or at least 99%. In some embodiments, the percentages of high molecular weight peaks (HMW%) and / or low molecular weight peaks (LMW%) are less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0147] In some embodiments, the thermal stability of the engineered IL-21 variants or their protein constructs described herein (e.g., any HSA-fused IL-21 variants described herein) is determined. The thermal stability (Tm) of the engineered IL-21 variants and their protein constructs described herein can be greater than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C. In some embodiments, Tm is less than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C. In some embodiments, the aggregation (Tm) of the engineered IL-21 variants and their protein constructs described herein. 聚集 ) and start (T) 起始 Temperature can be measured via DLS / SLS-based application on a thermal ramp (e.g., 25–85 °C). DLS / SLS is a well-known technique for determining sample interactions, particle size, and aggregation of molecules dispersed or dissolved in solution. The process begins with the unfolding of the sample (T0). 起始 ) and aggregation (T 聚集 Temperature is considered a key predictor of stability. In some embodiments, the T value of the engineered IL-21 variant or its protein construct described herein is... 聚集The temperatures are at least 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, or 65°C. In some embodiments, the T-cell temperature of the engineered IL-21 variant or its protein construct described herein is... 起始 The temperature must be at least 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, or 66°C.

[0148] In some embodiments, cysteine ​​mutations (e.g., any cysteine ​​mutation or combination thereof described herein) can cause aggregation (T... 聚集 ) and / or start (T 起始 The temperature increases by at least 0.5°C, at least 1°C, at least 1.5°C, at least 2°C, at least 2.5°C, at least 3°C, at least 3.5°C, at least 4°C, at least 4.5°C, or at least 5°C.

[0149] The radius of the engineered IL-21 variants or their protein constructs described herein (e.g., any HSA-fused IL-21 variants described herein) can also be determined by DSL / SLS. In some embodiments, the radius is measured under isothermal conditions (e.g., at about 25°C) as about 1-10 nm, about 3-8 nm, about 4-7 nm, about 4-6 nm, about 4-5 nm, about 4.5-7 nm, about 4.5-6 nm, about 4.5-5 nm, about 5-7 nm, about 5-6 nm, or about 6-7 nm. In some embodiments, under thermal ramp conditions (e.g., at 25-85°C), the radius is measured as about 1-20 nm, about 3-15 nm, about 5-13 nm, about 5-10 nm, about 5-8 nm, about 5-7 nm, about 5-6 nm, about 6-13 nm, about 6-10 nm, about 6-7 nm, about 7-13 nm, about 10-13 nm, or about 12-13 nm.

[0150] The polydispersity (PD%) of the engineered IL-21 variants or their protein constructs described herein (e.g., any HSA-fused IL-21 variants described herein) can also be determined by DSL / SLS. In some embodiments, the PD% is less than about 30%, about 28%, about 25%, about 20%, about 15%, about 13%, about 10%, about 8%, or about 5% under isothermal conditions (e.g., at about 25°C).

[0151] The molecular weight (MW-S) of the engineered IL-21 variants or their protein constructs described herein (e.g., any HSA-fused IL-21 variants of HSA-fused IL-21 described herein) can also be determined by DLS / SLS. In some embodiments, under isothermal conditions (e.g., at about 25°C) or thermal ramp conditions (e.g., under thermal ramp conditions of 25-85°C), MW-S is about 50-200 kDa, about 50-130 kDa, about 50-100 kDa, about 50-90 kDa, about 50-80 kDa, about 50-70 kDa, about 60-100 kDa, about 60-90 kDa, about 60-80 kDa, about 70-100 kDa, about 70-90 kDa, about 70-80 kDa, about 80-100 kDa, about 90-100 kDa, about 100-200 kDa, about 100-150 kDa, or about 150-200 kDa.

[0152] In some embodiments, the engineered IL-21 variants or protein constructs thereof described herein (e.g., any one of the HSA-fused IL-21 variants or the heterodimeric Fc-fused IL-21 variants described herein) can inhibit tumor growth, for example, when administered in tumor-bearing animals. In some cases, the percentage of tumor growth inhibition (TGI%) of the engineered IL-21 variants or protein constructs thereof is greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. In some embodiments, the percentage of tumor growth inhibition of the engineered IL-21 variant or its protein construct described herein is less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI% can be determined after treatment initiation, for example, at 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, or 30 days. As used herein, the percentage of tumor growth inhibition (TGI%) is calculated using the following formula:

[0153] TGI(%) = [1 - average(T)] 最终 -T 初始 ) / average (C 最终 -C 初始 )]×100

[0154] T 最终 This is the average tumor volume in the treatment group on the last day. (T) 初始 This is the average tumor volume in the treatment group on day 0. (C) 最终 This is the average tumor volume in the control group on the last day. (C) 起始 This is the average tumor volume in the control group on day 0.

[0155] In some embodiments, the TGI% of the engineered IL-21 variant or its protein construct described herein is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 2 times, at least 3 times, at least 4 times, at least 5 times, or at least 10 times greater than that of wild-type IL-21 or its protein construct.

[0156] In some embodiments, relative to the total number of mice vaccinated on day 0, the percentage of tumor-bearing mice whose tumor volume is less than half the average tumor volume of untreated tumor-bearing mice after treatment with the engineered IL-21 variant or its protein construct described herein (e.g., any one of the HSA-fused IL-21 variant or the heterodimeric Fc-fused IL-21 variant described herein) at 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days after tumor inoculation is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the mice treated on day 0, at 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days after tumor inoculation is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, the percentage is at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% relative to the total number of live mice in the same number of days.

[0157] In some embodiments, tumor-bearing mice, after 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days following tumor inoculation, exhibited survival rates of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% following treatment with the engineered IL-21 variant or its protein construct described herein (e.g., any one of the HSA-fused IL-21 variant or the heterodimeric Fc-fused IL-21 variant described herein).

[0158] In some embodiments, the engineered IL-21 variants or protein constructs thereof described herein (e.g., any one of the HSA-fused IL-21 variants or the heterodimeric Fc-fused IL-21 variants described herein) can induce robust tumor-specific memory responses. In some embodiments, the engineered IL-21 variants or protein constructs thereof described herein can inhibit tumor growth at least 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 days after tumor re-examination (e.g., reseeding of the same or different tumor cells).

[0159] In some embodiments, the body weight of tumor-bearing mice treated with engineered IL-21 variants or protein constructs thereof (e.g., any one of the HSA-fused IL-21 variants or heterodimeric Fc-fused IL-21 variants described herein) is at least 70%, at least 80%, or at least 90% compared to the body weight of tumor-bearing mice after treatment with wild-type IL-21 or its protein constructs.

[0160] In some embodiments, the half-life of the engineered IL-21 variants or protein constructs described herein may be increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20-fold, or at least 100-fold compared to the half-life of wild-type IL-21 or its protein constructs. In some embodiments, the half-life is determined by measuring the in vivo concentration of the molecule over time after administration to a subject.

[0161] Methods for preparing engineered IL-21 variants and protein constructs

[0162] The engineered IL-21 variants or protein constructs thereof described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding the IL-21 peptide or a portion thereof, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acid sequence. In some embodiments, selective cysteine ​​mutations can be introduced into one or more pairs of C... α Atoms in the 3D structure of human IL-21 Residues in ENERAL. In some embodiments, the 3D structure of human IL-21 has a PDB (Protein Database) ID of 3TGX.

[0163] Screening can be performed. Within this population of variants, some engineered IL-21 variants can be expressed and purified using methods known in the art. Furthermore, the potency of some engineered IL-21 variants in T cell responses can be determined by STAT-3 phosphorylation and / or T cell proliferation. Based on the above experimental results, engineered IL-21 variants with good expression / purification profiles and comparable T cell response potency can be selected to generate HSA-fused IL-21 variants (e.g., any HSA-fused IL-21 variant among the HSA-fused IL-21 variants described herein).

[0164] Screening of IL-21 variants fused to HSA can be performed. For example, their expression and purification profiles can be compared using various analytical methods (e.g., SDS-PAGE, HPLC-SEC, and / or DSL / SLS). The results can indicate whether the non-natural disulfide bonds formed by cysteine ​​mutations in engineered IL-21 variants and / or HSA fusions can stabilize the HSA fusion protein. For example, some HSA fused IL-21 variants may exhibit increased aggregation (T2) compared to wild-type IL-21 fused to HSA. 聚集 ) and start (T) 起始 )temperature.

[0165] Furthermore, the potency of the protein constructs described herein (e.g., any of the HSA-fused IL-21 variants or heterodimeric Fc-fused IL-21 variants described herein) can be determined, for example, by STAT-3 phosphorylation, T cell proliferation, and / or NK cell proliferation. Specifically, some HSA-fused IL-21 variants may have similar or relatively weaker potency in inducing STAT-3 phosphorylation and T cell proliferation compared to wild-type HSA-fused IL-21. Some HSA-fused IL-21 variants may have similar or relatively weaker potency in inducing NK cell proliferation compared to wild-type HSA-fused IL-21. Some heterodimeric Fc-fused IL-21 variants may have similar potency in inducing STAT-3 phosphorylation compared to wild-type heterodimeric Fc-fused IL-21. Compared to wild-type IL-21 fused with heterodimer Fc, some heterodimer Fc-fused IL-21 variants may have better potency in inducing T cell and / or NK cell proliferation.

[0166] Furthermore, the efficacy of the protein constructs described herein (e.g., either the HSA-fused IL-21 variant or the heterodimeric Fc-fused IL-21 variant described herein) in alleviating Treg-mediated T cell suppression can be determined. Specifically, some HSA-fused IL-21 variants may exhibit a significantly enhanced effect in alleviating Treg-mediated T cell suppression compared to wild-type HSA-fused IL-21. The efficacy of the HSA-fused IL-21 variants in inducing NK cell cytotoxicity can also be determined. Specifically, some HSA-fused IL-21 variants may have similar or relatively weaker efficacy in inducing NK cell cytotoxicity compared to wild-type HSA-fused IL-21. Some heterodimeric Fc-fused IL-21 variants may have slightly better efficacy in inducing NK cell cytotoxicity compared to wild-type heterodimeric Fc-fused IL-21.

[0167] Based on the above experimental results regarding HSA-fused IL-21 variants, some HSA-fused IL-21 variants with good expression / purification profiles, comparable immune cell response efficacy, enhanced efficacy in alleviating Treg-mediated T cell suppression, and / or comparable or reduced efficacy in inducing NK cell toxicity can be selected to evaluate their antitumor efficacy and in vivo toxicity, for example, in tumor-bearing animal models.

[0168] Based on the above experimental results regarding heterodimeric Fc fusion IL-21 variants, some heterodimeric Fc fusion IL-21 variants with comparable or better immune cell response efficacy and / or comparable or better efficacy in inducing NK cell toxicity can be selected to evaluate their antitumor efficacy and in vivo toxicity, for example, in tumor-bearing animal models.

[0169] In some embodiments, engineered IL-21 variants or protein constructs thereof (e.g., any of the HSA-fused IL-21 variants or heterodimeric Fc-fused IL-21 variants described herein) may have comparable or increased affinity for the IL-21R / γc complex. Any combination of deletions, insertions, and / or combinations may be made to obtain variants with increased binding affinity to binding partners (e.g., IL-21R). Amino acid changes introduced into variants may also alter the peptide or introduce new post-translational modifications into the peptide, such as altering (e.g., increasing or decreasing) the number of glycosylation sites, altering the type of glycosylation sites (e.g., altering the amino acid sequence to allow different sugars to be linked by enzymes present in the cell), or introducing new glycosylation sites.

[0170] Engineered IL-21 variants can be derived from any animal species, including mammals. Non-limiting examples of IL-21 variants include those derived from humans, primates (e.g., monkeys and apes), cows, pigs, horses, sheep, camels (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits).

[0171] This disclosure also provides recombinant vectors (e.g., expression vectors) containing isolated polynucleotides disclosed herein (e.g., polynucleotides encoding polypeptides disclosed herein), host cells incorporating recombinant vectors (i.e., vectors that cause host cells to contain polynucleotides and / or include polynucleotides), and the generation of recombinant polypeptides or fragments thereof by recombinant techniques.

[0172] As used herein, a “vector” is any construct capable of delivering one or more polynucleotides of interest to a host cell when introduced into the host cell. An “expression vector” is capable of delivering and expressing the one or more polynucleotides of interest as encoded polypeptides in a host cell in which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operatively linked within the vector or in the genome of the host cell at or near the integration site of the polynucleotide of interest, or flanking it, to enable translation of the polynucleotide of interest in the host cell in which the expression vector has been introduced.

[0173] Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-glucan), transformation, transfection, and infection and / or transduction (e.g., using recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, granules, phage vectors, and DNA or RNA expression vectors associated with cationic condensers.

[0174] In some embodiments, the polynucleotides disclosed herein (e.g., polynucleotides encoding the polypeptides disclosed herein) are introduced using a viral expression system (e.g., vaccinia or other poxviruses, retroviruses, or adenoviruses). This may involve the use of a non-pathogenic (deficient), replicating virus, or a replication-deficient virus. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. The DNA can also be "naked." The uptake of naked DNA can be increased by coating the DNA onto biodegradable beads, which are efficiently transported into cells.

[0175] For expression, a DNA insert comprising a polynucleotide encoding a polypeptide disclosed herein can be operatively linked to a suitable promoter (e.g., a heterologous promoter), such as the bacteriophage λPL promoter, E. coli lac, trp and tac promoters, SV40 early and late promoters, and promoters of retroviral LTRs, to name just a few. Other suitable promoters are known to those skilled in the art. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. In some embodiments, the promoter is a human promoter, such as the uHS or HS promoter. Human promoters can improve the expression of proteins derived from humans. Detailed information on such human promoters can be found, for example, in the following: Antoniou, M. et al., “Transgenes encompassing dual-promoter CpG islands from the human TBP and HNRPA2B1 loci are resistant to heterochromatin-mediated silencing,” *Genomics* 82.3(2003):269-279; and Zhang, F. et al., “A ubiquitous chromatinopening element (UCOE) confers resistance to DNA methylation–mediated silencing of lentiviral vectors,” *Molecular Therapy*. Therapy) 18.9(2010):1640-1649; each of the references mentioned is incorporated herein by reference in its entirety. The expression construct may further contain transcription initiation and termination sites, and ribosome binding sites for translation within the transcriptional region. The coding portion of the mature transcript expressed by the construct may contain translation initiated at the initiation site and a stop codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide to be translated.

[0176] As indicated, the expression vector may contain at least one optional biomarker. Such biomarkers include dihydrofolate reductase resistance or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in *E. coli* and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as *E. coli*, *Streptomyces*, and *Salmonella typhimurium* cells; fungal cells such as yeast cells; insect cells such as Drosophila S2 and Noctuidae Sf9 cells; animal cells such as CHO, COS, Bowes melanoma, and HEK 293 cells; and plant cells. Suitable culture media and conditions for the host cells described herein are known in the art.

[0177] Non-restrictive vectors for bacteria include: pQE70, pQE60, and pQE-9, available from Qiagen; pBS, Phagescript, Bluescript, pNH8A, pNH16a, pNH18A, and pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5, available from Pharmacia. Non-restrictive eukaryotic vectors include: pWLNEO, pSV2CAT, pOG44, pXT1, and pSG, available from Stratagene; and pSVK3, pBPV, pMSG, and pSVL, available from Pharmacia. Other suitable vectors will be readily apparent to those skilled in the art.

[0178] Suitable non-restrictive bacterial promoters include the Escherichia coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoters, λPR and PL promoters, and trp promoters. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, promoters of retroviral LTRs such as the Raúl's sarcoma virus (RSV) promoter, and metallothionein promoters such as the mouse metallothionein-I promoter.

[0179] In the yeast Saccharomyces cerevisiae, many vectors containing constitutive or inducible promoters such as α-factor, alcohol oxidase and PGH can be used.

[0180] Constructs can be introduced into host cells via calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. These methods are described in many standard laboratory manuals, such as Davis et al., *Basic Methods in Molecular Biology* (1986), which is incorporated herein by reference in its entirety.

[0181] In some embodiments, the host cell is a human cell suitable for protein expression, such as HEK293 cells or CHO cells (e.g., CHO-S cells). In some embodiments, the host cell is Expi293 cells. Compared to other transient 293 expression systems that may require two weeks or longer, the Expi293 expression system is designed to deliver up to 6 times more protein in just one week. This is partly due to the fact that Expi293F cells are adapted to achieve higher pg / cell / day productivity than standard HEK 293 cells, and that the Expifectamine 293 transfection reagent and enhancer enable highly efficient transfection and expression levels in high-density HEK 293 cultures. Additionally, the Expi293 expression system requires less plastic equipment, meaning less waste and more incubator space.

[0182] Transcription of DNA encoding the disclosed polypeptides by higher eukaryotic cells can be enhanced by inserting enhancer sequences into vectors. Enhancers are cis-acting elements of DNA, typically from about 10 bp to 300 bp, used to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer located posterior to the origin of replication at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer located posterior to the origin of replication, and the adenovirus enhancer.

[0183] To induce the secretion of translated proteins into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment, an appropriate secretion signal can be incorporated into the expressed polypeptide. This signal can be endogenous to the polypeptide or it can be a heterologous signal.

[0184] Peptides (e.g., engineered IL-21 variants) can be in modified forms, such as fusion proteins (e.g., HSA-fusions or GST-fusions), or expressed with histidine tags, and can contain not only secretion signals but also additional heterologous functional regions. For example, additional amino acid regions, particularly charged amino acid regions, can be added to the N-terminus of the peptide to improve stability and persistence in host cells during purification or during subsequent processing and storage. Similarly, peptide moieties can be added to the peptide to facilitate purification. Such regions can be removed prior to the final preparation of the peptide. Adding peptide moieties to peptides to induce secretion or excretion, improve stability, and facilitate purification (among other things) are well-known and conventional techniques in the art.

[0185] Treatment methods

[0186] The engineered IL-21 variants and their protein constructs disclosed herein can be used for a variety of therapeutic purposes.

[0187] On the one hand, this disclosure provides methods for treating cancer in a subject, methods for reducing the rate of increase in the volume of a subject's tumor over time, methods for reducing the risk of metastasis, or methods for reducing the risk of further metastasis in a subject. In some embodiments, the treatment may stop, slow, delay, or inhibit the progression of cancer. In some embodiments, the treatment may reduce the number, severity, and / or duration of one or more symptoms of the subject's cancer.

[0188] On one hand, this disclosure is characterized by a method of administering a therapeutically effective amount of the engineered IL-21 variant and protein construct disclosed herein to a subject in need (e.g., a subject with cancer or identified or diagnosed with cancer), said cancer being, for example, breast cancer (e.g., triple-negative breast cancer), carcinoid, cervical cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematologic malignancies. In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-resistant prostate cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is head and neck squamous cell carcinoma (SCCHN), renal cell carcinoma (RCC), triple-negative breast cancer (TNBC), or colorectal cancer. In some embodiments, the subject has Hodgkin's lymphoma. In some embodiments, the subject has triple-negative breast cancer (TNBC), gastric cancer, urothelial carcinoma, Merkel-cellcarcinoma, or head and neck cancer.

[0189] In some embodiments, the cancers described herein are esophageal adenocarcinoma, lung cancer, hepatocellular carcinoma, bile duct cancer, gallbladder cancer, gastric cancer, pancreatic cancer, colorectal cancer, endometrial cancer, ovarian cancer, bladder cancer, prostate cancer, or T-cell lymphoma. In some embodiments, the cancer is kidney cancer.

[0190] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of developing cancer. Patients with cancer can be identified using a variety of methods known in the art.

[0191] On the one hand, this disclosure provides methods for treating, preventing, or reducing the risk of developing conditions associated with abnormal or undesirable immune responses, such as autoimmune diseases, for example by administering a therapeutically effective amount of the engineered IL-21 variants and protein constructs disclosed herein to a subject in need. These autoimmune diseases include, but are not limited to, rheumatoid arthritis, Crohn's disease, systemic lupus erythematosus, ankylosing spondylitis, inflammatory bowel disease (IBD), ulcerative colitis, or scleroderma. In some embodiments, autoimmune diseases include allergies, asthma, and / or atopic dermatitis. Therefore, the engineered IL-21 variants and protein constructs disclosed herein can be used to suppress immune responses. In some embodiments, the immune disorders described herein are allergies, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological disorders.

[0192] As used herein, “effective amount” means an amount or dose sufficient to produce a beneficial or desired outcome, including stopping, slowing, delaying, or inhibiting the progression of a disease, such as cancer. Effective amounts will vary depending on, for example, the age and weight of the subject to whom the engineered IL-21 variant and protein construct, including a carrier encoding a polynucleotide of the engineered IL-21 variant and protein construct, and / or a combination thereof, the severity of symptoms, and the route of administration, and therefore administration can be determined on an individual basis.

[0193] Effective amounts can be administered in a single or multiple doses. For example, an effective amount of an engineered IL-21 variant and / or protein construct is sufficient to improve, stop, stabilize, reverse, inhibit, slow, and / or delay the progression of a patient's cancer in vitro, or is sufficient to improve, stop, stabilize, reverse, slow, and / or delay the proliferation of cells (e.g., biopsy cells, any cancer cells or cell lines (e.g., cancer cell lines)) in vitro. As is understood in the art, effective amounts can vary, depending in particular on patient history and other factors such as the type (and / or dosage) of the engineered IL-21 variant and protein construct used.

[0194] The effective amount and schedule for administering the engineered IL-21 variant or protein construct thereof, the polynucleotide encoding the engineered IL-21 variant or protein construct thereof, and / or the composition thereof disclosed herein can be determined empirically, and making such a determination is within the scope of the art. Those skilled in the art will understand that the dosage that must be administered will vary depending, for example, on the mammal to which the engineered IL-21 variant or protein construct thereof, the polynucleotide and / or the composition thereof disclosed herein will be received, the route of administration, the specific type of polynucleotide and / or the composition thereof disclosed herein used, and other drugs administered to the mammal.

[0195] The typical daily dose of an effective amount of an engineered IL-21 variant or its protein construct is 0.1 mg / kg to 200 mg / kg (mg / kg patient body weight). In some embodiments, the dose may be less than 150 mg / kg, 140 mg / kg, 130 mg / kg, 120 mg / kg, 110 mg / kg, 100 mg / kg, 90 mg / kg, 80 mg / kg, 70 mg / kg, 60 mg / kg, 50 mg / kg, 40 mg / kg, 30 mg / kg, 20 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dose may be greater than 150 mg / kg, 140 mg / kg, 130 mg / kg, 120 mg / kg, 110 mg / kg, 100 mg / kg, 90 mg / kg, 80 mg / kg, 70 mg / kg, 60 mg / kg, 50 mg / kg, 40 mg / kg, 30 mg / kg, 20 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, 1 mg / kg, 0.5 mg / kg, or 0.1 mg / kg. In some embodiments, the dosage is approximately 150 mg / kg, 140 mg / kg, 130 mg / kg, 120 mg / kg, 110 mg / kg, 100 mg / kg, 90 mg / kg, 80 mg / kg, 70 mg / kg, 60 mg / kg, 50 mg / kg, 40 mg / kg, 30 mg / kg, 20 mg / kg, 10 mg / kg, 9 mg / kg, 8 mg / kg, 7 mg / kg, 6 mg / kg, 5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, or 1 mg / kg.In some embodiments, the dosage is about 1 to 150 mg / kg, about 1 to 100 mg / kg, about 1 to 80 mg / kg, about 1 to 50 mg / kg, about 1 to 30 mg / kg, about 1 to 20 mg / kg, about 1 to 10 mg / kg, about 1 to 5 mg / kg, about 5 to 150 mg / kg, about 5 to 100 mg / kg, about 5 to 80 mg / kg, about 5 to 50 mg / kg, about 5 to 30 mg / kg, about 5 to 20 mg / kg, about 5 to 10 mg / kg, about 10 to 150 mg / kg, about 10 to 100 mg / kg, about 10 to 80 mg / kg, about 10 to 50 mg / kg, about 10 to 30 mg / kg, about 10 to 20 mg / kg, about 20 to 150 mg / kg, about 20 to 100 mg / kg, about 20 to 80 mg / kg, about 20 to 50 mg / kg, about 20 to 30 mg / kg, about 30 to 150 mg / kg, about 30 to 100 mg / kg, about 30 to 80 mg / kg, about 30 to 50 mg / kg, about 50 to 150 mg / kg, about 50 to 100 mg / kg, about 50 to 80 mg / kg, about 80 to 150 mg / kg, about 80 to 100 mg / kg or about 100 to 150 mg / kg.

[0196] In any of the methods described herein, the engineered IL-21 variant or its protein construct may be administered to the subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day).

[0197] In some embodiments, one or more additional therapeutic agents may be administered to the subject before or after administration of the engineered IL-21 variant or its protein construct. In some embodiments, the one or more additional therapeutic agents are administered to the subject such that the one or more additional therapeutic agents overlap with the period of biological activity of the engineered IL-21 variant or its protein construct in the subject.

[0198] In some embodiments, one or more additional therapeutic agents may be administered to the subject. These additional therapeutic agents may include one or more inhibitors selected from the group consisting of: B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, PI3K / mTOR dual inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and / or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the additional therapeutic agent is an indoleamine 2,3-dioxygenase-1 (IDO1) inhibitor (e.g., epacadostat).

[0199] In some embodiments, additional therapeutic agents may include one or more inhibitors selected from the group consisting of: HER3 inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, inhibitors of activated hedgehog signaling pathways, and agents that selectively degrade estrogen receptors.

[0200] In some embodiments, additional therapeutic agents may include one or more therapeutic agents selected from the group consisting of: Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, and sorafenib. The following are listed: Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, Hsp90 inhibitors, Ad-GM-CSF, Temozolomide, IL-2, IFNa, Vincristine, Thalidomide, Dacarbazine, Cyclophosphamide, Lenalidomide, Azacytidine, Bortezomid, Amrubicin, Carfilzomib, Pralatrexate, and Enzastaurin.

[0201] In some embodiments, additional therapeutic agents may include one or more therapeutic agents selected from the group consisting of: adjuvants, TLR agonists, tumor necrosis factor (TNF)α, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, therapeutically targeted CX3CL1, therapeutically targeted CXCL9, therapeutically targeted CXCL10, therapeutically targeted CCL5, LFA-1 agonists, ICAM1 agonists, and selectin agonists.

[0202] In some embodiments, the subject is administered carboplatin, nalbupivacaine, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI.

[0203] In some embodiments, additional therapeutic agents are anti-OX40 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-SIRPα antibodies, anti-CD47 antibodies, anti-LAG3 antibodies, anti-TIGIT antibodies, anti-BTLA antibodies, anti-CTLA-4 antibodies, or anti-GITR antibodies. In some embodiments, additional therapeutic agents are anti-CD20 antibodies (e.g., rituximab) or anti-EGF receptor antibodies (e.g., cetuximab).

[0204] Pharmaceutical Compositions and Routes of Administration

[0205] This document also provides pharmaceutical compositions containing the engineered IL-21 variant or a protein construct thereof as described herein. The pharmaceutical compositions may be formulated in any manner known in the art.

[0206] The pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The composition may contain a sterile diluent (e.g., sterile water or saline), a fixing oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvents, an antibacterial or antifungal agent such as benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; and an isotonic agent such as a sugar (e.g., dextran), a polyol (e.g., mannitol or sorbitol), or a salt (e.g., sodium chloride) or any combination thereof. Liposome suspensions may also be used as pharmaceutically acceptable carriers. Formulations of the composition may be formulated and packaged in ampoules, disposable syringes, or multi-dose vials. Where necessary (e.g., in injectable formulations), adequate flowability can be maintained, for example, by using a coating such as lecithin or a surfactant. Drug absorption can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved via implants and microencapsulated delivery systems that may contain biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid).

[0207] Compositions containing the engineered IL-21 variant or its protein construct as described herein can be formulated for parenteral administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) in dose units (i.e., physically discrete units containing a predetermined amount of the active compound for easy administration and uniform dosage).

[0208] Pharmaceutical compositions intended for parenteral administration are preferably sterile and substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. The pharmaceutical composition may be provided in unit dosage forms (i.e., a single-dose dose). The pharmaceutical composition may be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the chosen route of administration. For injection, engineered IL-21 variants or protein constructs thereof may be formulated in aqueous solutions, preferably in physiologically compatible buffers to minimize injection site discomfort. The solution may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, engineered IL-21 variants or protein constructs thereof may be in lyophilized form for use with a suitable mediator (e.g., sterile pyrogen-free water) prior to use.

[0209] The toxicity and therapeutic efficacy of the composition can be determined using standard pharmaceutical procedures in cell cultures or laboratory animals (e.g., monkeys). For example, the LD50 (the dose that is lethal to 50% of the population) and ED50 (the dose that is therapeutically effective in 50% of the population) can be determined: the therapeutic index is the ratio of LD50:ED50. Agents exhibiting a high therapeutic index are preferred. In cases where the agent exhibits undesirable side effects, care should be taken to minimize potential harm (i.e., reduce undesirable side effects). Toxicity and therapeutic efficacy can also be determined using other standard pharmaceutical procedures.

[0210] Exemplary doses comprise milligrams or micrograms per kilogram of the body weight of any engineered IL-21 variant or protein construct described herein (e.g., about 1 μg / kg to about 500 mg / kg; about 100 μg / kg to about 500 mg / kg; about 100 μg / kg to about 50 mg / kg; about 10 μg / kg to about 5 mg / kg; about 10 μg / kg to about 0.5 mg / kg; about 1 μg / kg to about 50 μg / kg; about 1 mg / kg to about 10 mg / kg; or about 1 mg / kg to about 5 mg / kg). While these doses cover a broad range, those skilled in the art will understand that the potency of the therapeutic agent can vary, and the effective amount can be determined by methods known in the art. Typically, a relatively low dose is initially administered, and the attending healthcare professional or veterinary professional (in the case of therapeutic applications) or researcher (when still working in the research and development phase) may subsequently and gradually increase the dose until an appropriate response is obtained. In addition, it should be understood that the specific dose level for any particular subject will depend on a wide range of factors, including the activity of the specific compound used on the subject, age, weight, general health status, sex and diet, time of administration, route of administration, excretion rate, and the half-life in vivo of the engineered IL-21 variant or its protein construct.

[0211] The pharmaceutical composition may be included in a container, package, or dispenser along with instructions for use. This disclosure also provides methods for manufacturing engineered IL-21 variants or protein constructs thereof for the various uses described herein.

[0212] Methods for engineering cytokines

[0213] This article provides a method for improving the properties (e.g., stability, activity, or affinity) of a protein (e.g., a cytokine), the method comprising one or more of the following steps: (a) providing a 3D structure of the protein (e.g., a cytokine); (b) measuring the C10 of one or more amino acid residues in the 3D structure. α (c) the distance between atoms; and (d) selecting two amino acid residues from the one or more amino acid residues, wherein the C0 of the two selected amino acid residues is... α Atoms in In some embodiments, the C of the two selected amino acid residues... α Atoms in For example, about or In some embodiments, the two selected amino acid residues are derived from C. α To C β The orientation of atoms favors the formation of non-natural disulfide bonds. For example, C β Atoms are pointing in the direction in which disulfide bonds may form (from C). α To C β In some cases, the C of the first selected amino acid residue... β The orientation of the atoms (from the C of the first selected amino acid residue) α To C β ) and the C of the second selected amino acid residue β The orientation of the atoms (from the C of the second selected amino acid residue) α To C β The angle between them is less than 120 degrees. In some cases, C β Atomic points do not point in directions away from each other. In some embodiments, the 3D structure of a protein (e.g., a cytokine) is derived from the PDB structure of a protein (e.g., a cytokine) bound to its binding partner, a fragment thereof, or a protein (e.g., a cytokine) complex. In some embodiments, the spatial coordinates of all atoms in the 3D structure can be loaded into software (e.g., software for modeling and simulation), and the distance between any two atoms in the 3D structure can be determined.

[0214] In some embodiments, the C of the first selected amino acid residueβ The orientation of the atom is related to the C of the second selected amino acid residue. β The angles between the directions of atoms are less than 120 degrees, for example, less than 120 degrees, 119 degrees, 118 degrees, 117 degrees, 116 degrees, 115 degrees, 114 degrees, 113 degrees, 112 degrees, 111 degrees, 110 degrees, 109 degrees, 108 degrees, 107 degrees, 106 degrees, 105 degrees, 104 degrees, 103 degrees, 102 degrees, 101 degrees, 100 degrees, 9 9 degrees, 98 degrees, 97 degrees, 96 degrees, 95 degrees, 94 degrees, 93 degrees, 92 degrees, 91 degrees, 90 degrees, 89 degrees, 88 degrees, 87 degrees, 86 degrees, 85 degrees, 84 degrees, 83 degrees, 82 degrees, 81 degrees, 80 degrees, 79 degrees, 78 degrees, 77 degrees, 76 degrees, 75 degrees, 74 degrees, 73 degrees, 72 degrees, 71 degrees, 70 degrees, 69 degrees, 68 degrees, 67 degrees 66 degrees, 65 degrees, 64 degrees, 63 degrees, 62 degrees, 61 degrees, 60 degrees, 59 degrees, 58 degrees, 57 degrees, 56 degrees, 55 degrees, 54 degrees, 53 degrees, 52 degrees, 51 degrees, 50 degrees, 49 degrees, 48 ​​degrees, 47 degrees, 46 degrees, 45 degrees, 44 degrees, 43 degrees, 42 degrees, 41 degrees, 40 degrees, 39 degrees, 38 degrees, 37 degrees, 36 degrees, 35 degrees 34 degrees, 33 degrees, 32 degrees, 31 degrees, 30 degrees, 29 degrees, 28 degrees, 27 degrees, 26 degrees, 25 degrees, 24 degrees, 23 degrees, 22 degrees, 21 degrees, 20 degrees, 19 degrees, 18 degrees, 17 degrees, 16 degrees, 15 degrees, 14 degrees, 13 degrees, 12 degrees, 11 degrees, 10 degrees, 9 degrees, 8 degrees, 7 degrees, 6 degrees, 5 degrees, 4 degrees, 3 degrees, 2 degrees, or 1 degree.

[0215] In some embodiments, the C of the two selected amino acid residues described herein α The distance between atoms is approximately 3 to approximately Approximately 3 to approximately Approximately 3 to approximately Approximately 3 to approximately Approximately 3 to approximately Approximately 3 to approximately Approximately 3 to approximately Approximately 3 to approximately Approximately 3.5 to approximately Approximately 3.5 to approximately Approximately 3.5 to approximately Approximately 3.5 to approximately Approximately 3.5 to approximately Approximately 3.5 to approximately Approximately 3.5 to approximately Approximately 4 to approximately Approximately 4 to approximately Approximately 4 to approximately Approximately 4 to approximately Approximately 4 to approximately Approximately 4 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 5 to approximately Approximately 5 to approximately Approximately 5 to approximately Approximately 5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 6 to approximately Approximately 6 to approximately Or approximately 6.5 to approximately

[0216] In some embodiments, the C of the two selected amino acid residues described herein α The distance between atoms is approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.6 to approximately Approximately 5.6 to approximately Approximately 5.6 to approximately Approximately 5.6 to approximately Approximately 5.6 to approximately Approximately 5.6 to approximately Approximately 5.6 to approximately Approximately 5.6 to approximately Approximately 5.6 to approximately Approximately 5.7 to approximately Approximately 5.7 to approximately Approximately 5.7 to approximately Approximately 5.7 to approximately Approximately 5.7 to approximately Approximately 5.7 to approximately Approximately 5.7 to approximately Approximately 5.7 to approximately Approximately 5.8 to approximately Approximately 5.8 to approximately Approximately 5.8 to approximately Approximately 5.8 to approximately Approximately 5.8 to approximately Approximately 5.8 to approximately Approximately 5.8 to approximately Approximately 5.9 to approximately Approximately 5.9 to approximately Approximately 5.9 to approximately Approximately 5.9 to approximately Approximately 5.9 to approximately Approximately 5.9 to approximately Approximately 6.0 to approximately Approximately 6.0 to approximately Approximately 6.0 to approximately Approximately 6.0 to approximately Approximately 6.0 to approximately Approximately 6.1 to approximately Approximately 6.1 to approximately Approximately 6.1 to approximately Approximately 6.1 to approximately Approximately 6.2 to approximately Approximately 6.2 to approximately Approximately 6.2 to approximately Approximately 6.3 to approximately Approximately 6.3 to approximately or about to approximately In some embodiments, the range described herein includes end values, that is, values ​​at the range boundaries are included within the range.

[0217] In some embodiments, the C of the two selected amino acid residues described herein β The distance between atoms is approximately 3 to approximately Approximately 3 to approximately Approximately 3 to approximately Approximately 3 to approximately Approximately 3 to approximately Approximately 3 to approximately Approximately 3 to approximately From approximately 3 to approximately 4. Approximately 3 to approximately Approximately 3 to approximately 3. Approximately 3.5 to approximately Approximately 3.5 to approximately Approximately 3.5 to approximately Approximately 3.5 to approximately Approximately 3.5 to approximately Approximately 3.5 to approximately Approximately 3.5 to approximately Approximately 3.5 to approximately 4. Approximately 3.5 to approximately Approximately 4 to approximately Approximately 4 to approximately Approximately 4 to approximately Approximately 4 to approximately Approximately 4 to approximately Approximately 4 to approximately Approximately 4 to approximately Approximately 4 to approximately 4. Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 5 to approximately Approximately 5 to approximately Approximately 5 to approximately Approximately 5 to approximately Approximately 5 to approximately Approximately 5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 6 to approximately Approximately 6 to approximately Approximately 6 to approximately Approximately 6 to approximately Approximately 6.5 to approximately Approximately 6.5 to approximately 7. Approximately 6.5 to approximately Approximately 7 to approximately Approximately 7 to approximately Or approximately 7.5 to approximately

[0218] In some embodiments, the C of the two selected amino acid residues described herein β The distance between atoms is approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.5 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.6 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.7 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.8 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 4.9 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.0 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.1 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.2 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.3 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.4 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.5 to approximately Approximately 5.6 to approximately Approximately 5.6 to approximately Approximately 5.6 to approximately Approximately 5.6 to approximately Approximately 5.6 to approximately Approximately 5.6 to approximately Approximately 5.6 to approximately Approximately 5.6 to approximately Approximately 5.6 to approximately Approximately 5.7 to approximately Approximately 5.7 to approximately Approximately 5.7 to approximately Approximately 5.7 to approximately Approximately 5.7 to approximately Approximately 5.7 to approximately Approximately 5.7 to approximately Approximately 5.7 to approximately Approximately 5.8 to approximately Approximately 5.8 to approximately Approximately 5.8 to approximately Approximately 5.8 to approximately Approximately 5.8 to approximately Approximately 5.8 to approximately Approximately 5.8 to approximately Approximately 5.9 to approximately Approximately 5.9 to approximately Approximately 5.9 to approximately Approximately 5.9 to approximately Approximately 5.9 to approximately Approximately 5.9 to approximately Approximately 6.0 to approximately Approximately 6.0 to approximately Approximately 6.0 to approximately Approximately 6.0 to approximately Approximately 6.0 to approximately Approximately 6.1 to approximately Approximately 6.1 to approximately Approximately 6.1 to approximately Approximately 6.1 to approximately Approximately 6.2 to approximately Approximately 6.2 to approximately Approximately 6.2 to approximately Approximately 6.3 to approximately Approximately 6.3 to approximately or about to approximately In some embodiments, the range described herein includes end values, that is, values ​​at the range boundaries are included within the range.

[0219] In some embodiments, the C of the two selected amino acid residues αThe atoms are close enough to form a non-natural disulfide bond. In some embodiments, the two selected amino acid residues do not participate in the interaction between the protein (e.g., a cytokine) and its binding partner (e.g., a receptor (e.g., a cytokine) that binds to the protein). In some embodiments, the method further includes expressing a protein variant (e.g., a cytokine variant) comprising a non-natural disulfide bond formed by mutating the two selected amino acid residues to cysteine. In some embodiments, mutating the two selected amino acid residues to cysteine ​​does not substantially alter the 3D structure of the protein (e.g., a cytokine). For example, a cysteine ​​mutation does not substantially interfere with the overall structure of the protein (e.g., a cytokine). In some cases, the mutated residues do not exhibit any spatial conflict with the unmutated residues. In some cases, those skilled in the art can simulate the structure of the mutated protein (e.g., a mutated cytokine) in computer simulations and determine the associated conformational changes caused by the cysteine ​​mutation. In some embodiments, the associated conformational changes are minimal, for example, having RMSD values ​​that are considered insignificant by those skilled in the art.

[0220] This article also provides a method for screening cytokine variants with improved antitumor efficacy, the method comprising (a) providing the 3D structure of the cytokine; and (b) measuring the C-value of one or more amino acid residues in the 3D structure. α (c) the distance between atoms; and (d) selecting two amino acid residues from the one or more amino acid residues, wherein the C0 of the two selected amino acid residues is... α Atoms in or Inside, Within, or within any range described herein.

[0221] In some embodiments, the protein or cytokine has no more than 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 amino acid residues.

[0222] In some embodiments, the method further includes: (d) expressing a cytokine variant, wherein the variant comprises a non-natural disulfide bond formed by mutating the two selected amino acid residues to cysteine; (e) administering the cytokine variant to a tumor-bearing animal; and (f) determining tumor growth in the tumor-bearing animal (e.g., by measuring tumor volume). In some embodiments, administration of the cytokine variant does not cause substantial toxicity to the animal. For example, the weight of the animal administered the cytokine variant does not decrease significantly compared to the weight of a reference animal (e.g., an animal administered with a causative agent).

[0223] In some embodiments, the cytokine described herein is IL-21 (e.g., human IL-21). In some embodiments, the cytokine is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, or IL-36. In some embodiments, the cytokine is IL-2, IL-7, IL-10, IL-15, IL-21, IFNα, GM-CSF, or FLT-3.

[0224] In some embodiments, functional assays are performed to compare one or more expressed protein variants (e.g., cytokine variants).

[0225] Example

[0226] The invention is further described in the following examples, which do not limit the scope of the invention as described in the claims.

[0227] Example 1. Design of an IL-21 variant by introducing artificial disulfide bonds

[0228] Interleukin-21 (IL-21) is a pleiotropic cytokine composed of four α-helical bundles and is primarily produced by natural killer T (NKT) cells, T follicular helper (TFH) cells, and TH17 cells. IL-21 signals via IL-21R (IL-21 receptor) and utilizes the JAK-STAT, MAPK, and PI3K pathways. The wild-type human IL-21 protein (SEQ ID NO:1) has 162 amino acids, of which residues 1-24 constitute the signal peptide.

[0229] To screen for IL-21 variants with enhanced stability and different functional efficiencies (e.g., T cell proliferation and STAT-3 signaling), the C-terminus of two residues within the 3D structure of IL-21 (e.g., PDB ID: 2OQP) was considered. α Atom distance and C β The orientation of atoms was used to design variants of wild-type IL-21 (SEQ ID NO:2; signal-free peptide) by selectively mutating two spatially close residues to cysteine. It is conceivable that the two newly introduced cysteines could form artificial disulfide bonds, thereby stabilizing IL-21 and / or altering its functional potency.

[0230] The sequences of wild-type IL-21 and its variants are shown in the table below.

[0231] Table 1

[0232]

[0233]

[0234] An IL-21 variant fused with a His tag (SEQ ID NO:22) was expressed in Expi293 and CHO-S cells. This was achieved using Ni-HisTrap. TM Affinity chromatography and its use with column (Cytiva, catalog number: 17371206) The expressed protein was purified by size exclusion chromatography (SEC) using a 75 column (Stenofan, catalog number: 29148721). The purified protein was then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0235] like Figure 1 As shown, the results indicated that H8-IL21-C04, H8-IL21-C06, and H8-IL21-C13 were expressed in Expi293 or CHO-S cells, while H8-IL21-C02, H8-IL21-C05, H8-IL21-C09, H8-IL21-C10, and H8-IL21-C12 were not expressed in either Expi293 or CHO-S cells. In contrast, H8-IL21-C01, H8-IL21-C03, H8-IL21-C07, H8-IL21-C08, and H8-IL21-C011 were expressed in both Expi293 and CHO-S cells.

[0236] Example 2. Determination of its efficacy in inducing STAT-3 phosphorylation and T cell proliferation.

[0237] The potency of IL-21 variants to T cell responses was determined by STAT-3 phosphorylation and T cell proliferation. Specifically, for the STAT-3 phosphorylation assay, CD3+ T cells were isolated from hPBMC (human peripheral blood mononuclear cell) donors according to the manufacturing protocol. The CD3+ T cells were then converted from CD3 / CD28... Activation was performed for 3 days at a cell-to-bead ratio of 1:4. The activated T cells were then left to rest for 24 hours, and then 5 × 10⁻⁶ cells were added. 4 CD3+ T cells in each well were incubated with a His-labeled IL-21 variant at a specified concentration (2 nM, 5-fold serial dilution, 6 spots) at 37°C for 30 min. After incubation, CD3+ T cells were harvested and permeabilized. Cells were further stained with 2 μl pSTAT3(Y705)-PE antibody and then analyzed by flow cytometry. For T cell proliferation assays, CD3+ T cells were isolated from hPBMC donors according to the manufacturing protocol. CD3+ T cells were converted from CD3 / CD28... Activation was performed for 3 days at a cell-to-bead ratio of 1:4. The activated T cells were then left to rest for 24 hours, and then 5 × 10⁻⁶ cells were added. 4 CD3+ T cells in wells were incubated for 3 days with a His-labeled IL-21 variant at a specified concentration of 1 μg / ml anti-CD3 antibody in the presence of pre-coated antibody. Following incubation, [the cells were then]... T cell proliferation was determined by luminescent cell viability assay (Promega, catalog number: G7573).

[0238] like Figure 2A As shown, all IL-21 variants exhibited relatively low potency in inducing STAT-3 phosphorylation, with little difference between them. Figure 2B As shown, H8-IL21-C11 exhibited the highest potency in inducing T cell proliferation. H8-IL21-C03 showed a relatively weaker ability to induce T cell proliferation. Compared with the wild-type control (H8-IL21-WT), H8-IL21-C01, H8-IL21-C07, and H8-IL21-C08 showed similar potency in inducing T cell proliferation. His-labeled mesothelin extracellular domain (MSLN-His) was used as a negative control.

[0239] Based on the protein expression and T cell response results of IL-21 variants, the IL-21 variants IL21-C01 (SEQ ID NO:3), IL21-C03 (SEQ ID NO:5), IL21-C07 (SEQ ID NO:9), IL21-C08 (SEQ ID NO:10), and IL21-C11 (SEQ ID NO:13) were selected for subsequent experiments.

[0240] Example 3. Expression and purification of HSA-fused IL-21 variant

[0241] To enhance the stability of the IL-21 variant, a human promoter uHS or HS( encoding wild-type IL-21 (SEQ ID NO:2) was constructed. A single expression vector of puromycin, CET 1019HS-puro-SceI (Merck, catalog number: UC0E01), and plasmids of selected IL-21 variants (IL21-C01, IL21-C03, IL21-C07, and IL21-C08) fused with HSA (human serum albumin). Expi293 cells were transfected to express the HSA-fused IL-21 HSA-IL21-WT (SEQ ID NO:17) and its variants HSA-IL21-C01 (SEQ ID NO:18), HSA-IL21-C03 (SEQ ID NO:19), HSA-IL21-C07 (SEQ ID NO:20), and HSA-IL21-C08 (SEQ ID NO:21). The HSA-fused IL-21 variants were purified and characterized as follows.

[0242] First, the purity of the HSA-fused IL-21 variant was analyzed by SDS-PAGE and HPLC-SEC (High Performance Liquid Chromatography-Size Exclusion Chromatography), respectively. Figure 3A As shown, by non-reducing (NR) or reducing (R) SDS-PAGE, all selected HSA fusions of IL-21 variants did not show aggregation or small fragments. Figure 3B As shown, HPLC-SEC analysis results indicate that the purity (as indicated by "major %") of all selected HSA-fused IL-21 variants is above 95%.

[0243] In addition, the aggregation (T) of the IL-21 variant fused to HSA was analyzed by dynamic and static light scattering (DLS / SLS). 聚集 ) and start (T) 起始 Temperature. (e.g.) Figure 3C As shown, compared to HSA-IL21-WT, all selected HSA-fused IL-21 variants exhibited significantly increased Tw. 聚集 and T 起始 Temperature. The results showed that the selected IL-21 variant had better stability than wild-type IL-21.

[0244] Example 4. Determination of the potency of HSA-fused IL-21 variants in inducing STAT-3 phosphorylation and T cell proliferation.

[0245] The potency of the HSA-fused IL-21 variant to T cell responses was determined by STAT-3 phosphorylation and T cell proliferation. Specifically, for the STAT-3 phosphorylation assay, CD8+ T cells were isolated from hPBMC donors according to the manufacturing protocol. The CD8+ T cells were then converted from CD3 / CD28... Activation was performed at a cell-to-bead ratio of 1:5 for 3 days. The activated T cells were then left to rest for 24 hours, and then 5 × 10⁻⁶ cells were added. 4 CD8+ T cells were incubated at 37°C for 30 min with a His-labeled HSA fusion IL-21 variant at a specified concentration (1 nM, 20-fold serial dilution, 4 spots). After incubation, CD8+ T cells were harvested and permeabilized. Cells were further stained with 2 μl of pSTAT3(Y705)-PE antibody and then analyzed by flow cytometry. For T cell proliferation assays, CD8+ T cells were isolated from hPBMC donors according to the manufacturing protocol. CD8+ T cells were converted from CD3 / CD28... Activation was performed for 3 days at a cell-to-bead ratio of 1:8. The activated T cells were then left to rest for 24 hours, and then 5 × 10⁶ cells were injected with pre-coated 1 μg / ml anti-CD3 antibody. 4 CD8+ T cells in each well were incubated for 6 days with a His-labeled HSA-fused IL-21 variant at a specified concentration (10 nM, 5-fold serial dilution, 6 spots). Following incubation, [the cells were then]... Luminescent cell viability assay (Promega, catalog number: G7573) to determine T cell proliferation.

[0246] like Figure 4A As shown, HSA-IL21-CO3, HSA-IL21-CO7, and HSA-IL21-CO8 exhibited relatively weaker potency in inducing STAT-3 phosphorylation compared to HSA-IL21-WT. HSA-IL21-CO1 showed similar potency in inducing STAT-3 phosphorylation compared to HSA-IL21-WT. Figure 4B As shown, HSA-L21-C03 exhibited the highest potency in inducing T cell proliferation compared to HSA-IL21-WT and other HSA-fused IL-21 variants. However, HSA-IL21-C08 showed a relatively weaker potency in inducing T cell proliferation compared to HSA-IL21-WT, though the difference was small. His-labeled HSA (HSA-His), MSLN-His, and SIRPα-G4Fc (Trillium) (or SIRPα-G4Fc-WT (Trillium); SEQ ID NO:23) were used as negative controls.

[0247] The results also showed that, compared with wild-type IL-21 fused with HSA and its variants expressed in Expi293 cells, wild-type IL-21 fused with IgG4 Fc (G4Fc-IL21-WT; SEQ ID NO:16) and H8-IL21-WT expressed in CHO-S cells (H8-IL21-WT_CHOS) exhibited relatively weaker potency in inducing STAT-3 phosphorylation and T cell proliferation.

[0248] Example 5. Determination of the potency of HSA-fused IL-21 variants in inducing NK-92 cell proliferation

[0249] pass A luminescent cell viability assay (Promega, catalog number: G7573) was used to determine the potency of the HSA-fused IL-21 variant in inducing NK-92 cell proliferation. Specifically, NK-92 cells were starved for 24 hours in culture medium without IL-2 supplementation. After starvation, 5 × 10⁶ cells were... 4 / well NK-92 cells with the HSA-IL-21 variant at the specified concentration (100 nM, 10-fold serial dilution, 8 spots) were incubated for an additional 24 hours in complete medium (75% MEM-α, with ribonucleosides and deoxyribonucleosides (Gibco, catalog number: 12571-048) + 12.5% ​​horse serum + 12.5% ​​FBS + 150 U / mL IL-2). Proliferation was determined by luminescent cell viability assay.

[0250] The EC50 values ​​for each HSA fusion IL-21 variant are shown in the table below.

[0251] Table 2

[0252]

[0253] like Figure 5 As shown in the table above, HSA-IL21-C03 exhibited better potency in inducing NK-92 cell proliferation compared to HSA-IL21-WT. Among the HSA-fused IL-21 variants, HSA-IL21-C08 showed the lowest potency in inducing NK-92 cell proliferation. HSA-His protein (Acro) was used as a negative control.

[0254] Example 6. Determination of the efficacy of HSA-fused IL-21 variants in alleviating Treg-mediated T cell suppression.

[0255] The potency of the IL-21 variant fused to Treg function was determined by a Treg-mediated T cell suppression assay.

[0256] Specifically, CD4 is isolated from the hPBMC donor according to the manufacturing plan. + CD25 + CD127 低 Treg cells. Treg cells were converted from CD3 / CD28... Activation was performed for 4 days at a cell-to-bead ratio of 1:10. Following activation, CD8+ T cells were isolated from another hPBMC donor according to the manufacturing protocol and stained with 1 μM CFSE (carboxyfluorescein succinimide) dye. Treg and CFSE-labeled CD8+ T cells were incubated at a cell-to-cell ratio of 1:4 and then treated for 3 days with or without HSA-fused IL-21 and its variants at specified concentrations. Cells were then harvested and stained with PE-anti-CD8 antibody. T cell proliferation is mediated via CD8... + CFSE 低 The percentage of cells is used to determine this.

[0257] like Figure 6 As shown, all selected HSA-fusion IL-21 variants (HSA-IL21-C01, HSA-IL21-C03, HSA-IL21-C07, and HSA-IL21-C08) exhibited significantly enhanced efficacy in alleviating Treg-mediated T cell suppression (e.g., at approximately 0.0586 nM) compared to HSA-IL21-WT. Specifically, HSA-IL21-C08 showed lower potency in alleviating Treg-mediated T cell suppression compared to other HSA-fusion IL-21 variants. HSA-IL21-C01, HSA-IL21-C03, and HSA-IL21-C07 showed similar efficacy in Treg function. SIRPα-G4Fc-WT (Trillium) was used as a negative control.

[0258] The results also showed that HSA-IL21-WT was more effective in alleviating Treg-mediated T cell suppression compared to H8-IL21-WT (SEQ ID NO:24).

[0259] Example 7. Determination of the potency of HSA-fused IL-21 variants in inducing primary NK cell cytotoxicity

[0260] pass EuTDA cytotoxic reagent (PerkinElmer) was used to determine the potency of HSA-fused IL-21 variants in inducing primary NK cell cytotoxicity. Specifically, primary NK cells were isolated from hPBMC donors according to the manufacturing protocol and then incubated for 24 hours with or without HSA-fused IL-21 at a specified concentration. K-562 tumor cells were treated with 20 ng / ml IFN-γ for 24 hours prior to incubation with primary NK cells, and then... EuTDA cytotoxic reagent (PerkinElmer) was manufactured using a fluorescently enhanced ligand, BATDA (2,2':6',2'-terpyridine-6,6'-dicarboxylic acid bis(acetoxymethyl) ester). Next, effector cells (primary NK cells) and target cells (BATDA-labeled K-562) were co-incubated at 37°C for 4 hours for the next 4 hours in the presence of 1-100 nM HSA-fused IL-21 variants in 96-well U-shaped cell culture plates at a cell-to-cell ratio of 4:1. Cell supernatant was carefully collected from the assay plate to avoid cell interference. 20 μl of the clarified supernatant sample was mixed with 200 μl of europium solution in the provided [prepared / prepared] solution. Mix in the strips. Use on a compatible reader. Time-resolved fluorescence (TRF) was used to set the detection signal (excitation at 340 nm and emission at 615 nm). The specific percentage of release was determined using the following formula:

[0261] (Experimental release - spontaneous release) / (maximum release - spontaneous release) × 100.

[0262] like Figure 7 As shown, HSA-IL21-C01 and HSA-IL21-C08 exhibited lower potency in inducing primary NK cell cytotoxicity compared to HSA-IL21-WT. The results indicate that the selected IL-21 variants can induce specific lysis of K-562 tumor cells mediated by primary NK cells.

[0263] Based on the above characterization and in vitro assay results, HSA-IL21-C01 and HSA-IL21-C08 are lead candidates and have been selected for subsequent experiments.

[0264] Example 8. Evaluation of the antitumor efficacy and in vivo toxicity of HSA-fused IL-21 variants

[0265] The antitumor efficacy of HSA-fused IL-21 and its variants was evaluated in a BALB / c mouse model carrying CT26 tumors. Treatment protocol and dosing schedule are as follows: Figure 8AAs shown. Specifically, 5-6 week old BALB / c mice were selected and subcutaneously (sc) inoculated with 2×10 5 CT26 colon cancer cells in mice. When the tumor volume reaches approximately 100-200 mm... 3 Four days later, mice were randomly assigned to three treatment groups and one control group (n=10 per group). Treatment mice were intraperitoneally (ip) injected with 10 mg / kg HSA-IL21-WT (Group 2), HSA-IL21-C01 (Group 3), or HSA-IL21-C08 (Group 4). Control mice (Group 1) were injected with 10 mL / kg of a carrier (isotonic sodium chloride solution). Treatment began on grouping day 0 and was administered twice weekly for a total of three weeks.

[0266] like Figure 8B As shown, HSA-IL21-C01 and HSA-IL21-C08 exhibited significant tumor growth inhibition compared to HSA-IL21-WT. Individual tumor growth curves and tumor volumes for each mouse on day 21 are shown in the figure. Figure 8C-8F and Figure 8G The results showed that HSA-IL21-C01 and HSA-IL21-C08 induced significant tumor shrinkage in the CT26 syngeneic mouse model. Figure 8H The results of body weight changes indicated that treatment with HSA-fused IL-21 and its variants did not induce significant toxicity in CT26 syngeneic mice. The overall summary of tumor growth inhibition percentage (TGI%) is expressed as... Figure 8I The results showed that in the CT26 syngeneic mouse model, the TGI% of HSA-IL21-C01 and HSA-IL21-C08 were 91% and 99%, respectively. The corresponding P-values ​​were determined to be less than 0.01.

[0267] Example 9. Determination of the efficacy of HSA-fused IL-21 variants in alleviating Treg-mediated T cell suppression.

[0268] CD4 was isolated from the hPBMC donor according to the manufacturing plan. + CD25 + CD127 低 Treg cells. Treg cells were converted from CD3 / CD28... (Thermo Fisher Scientific, Catalog No.: 11132D) Activated at a cell-to-bead ratio of 1:10 and treated for 4 days with or without HSA-fused IL-21 and its variants at specified concentrations. After 4 days of treatment, CD3 / CD28... The culture medium containing IL-21 was discarded. Additionally, CD8 was isolated from another hPBMC donor according to the manufacturing protocol. + T cells were then stained with 1 μM CFSE (Thermo Fisher Scientific, catalog number: C34554) dye. Treg cells and CFSE-labeled CD8 cells were then... + T cells were incubated at a 1:8 cell-to-cell ratio for 6 days. Cells were then collected and stained with PE-anti-CD8 antibody (BioLegend, catalog number: 980902). T cell proliferation is mediated by CD8. + CFSE 低 The percentage of cells is used to determine this.

[0269] like Figures 9A-9B As shown, all IL-21 variants can alleviate CD8+ induced by Treg cells. + T cell suppression. Compared with HSA-IL21-WT, all selected HSA fusion IL-21 variants (HSA-IL21-C01, HSA-IL21-C03, HSA-IL21-C07, and HSA-IL21-C08) showed significant enhancement in alleviating Treg-mediated T cell suppression.

[0270] Example 10. Determination of the potency of HSA-fused IL-21 variants in inducing primary NK cell cytotoxicity

[0271] The efficacy of the HSA-fused IL-21 variant in inducing NK cell cytotoxicity on K-562 cells was tested as described in Example 7.

[0272] like Figure 10 As shown, IL21-WT and its variants exhibited potency in inducing NK cell cytotoxicity. Compared to HSA-IL21-WT, all selected HSA-fused IL-21 variants (HSA-IL21-C01, HSA-IL21-C03, HSA-IL21-C07, and HSA-IL21-C08) showed similar potency in inducing primary NK cell cytotoxicity. These results indicate that the selected IL-21 variants can induce specific lysis of K-562 tumor cells mediated by primary NK cells.

[0273] Example 11. Evaluation of the antitumor efficacy and memory effect of HSA-fused IL-21 variants

[0274] The antitumor efficacy and memory effect of HSA-fused IL-21 and its variants were evaluated in a BALB / c mouse model carrying CT26 tumors. Treatment plan and dosing schedule are as follows: Figure 11AAs shown. To determine the antitumor efficacy, 5-6 week old BALB / c mice were selected and subcutaneously (sc) inoculated with 2×10⁻⁶ mice. 5 CT26 colon cancer cells were collected from one side of all mice. The tumor volume reached approximately 100-200 mm. 3 Four days after tumor inoculation, mice were randomly assigned to three treatment groups (n=16 per group) and a control group (n=8 per group). Treatment mice were intraperitoneally (ip) injected with 10 mg / kg HSA-IL21-WT (Group 2), HSA-IL21-C01 (Group 3), or HSA-IL21-C08 (Group 4). Control mice (Group 1) were injected with 10 mL / kg of a carrier (isotonic sodium chloride solution). Treatment began on day 4 and was administered twice weekly for a total of three weeks.

[0275] After six administrations, mice were allowed to rest (no treatment after day 21), and tumor growth was observed. On day 40 post-initial inoculation, mice with tumors smaller than 50 mm were selected. 3 Mice carrying CT26 tumors in groups 3 and 4 underwent tumor re-provocation to determine the memory effect of IL-21 and its variants in HSA fusions. Ten mice from the original group 3 and eleven mice from the original group 4 were subepithelially (sc)-inoculated with 2 × 10⁻⁶ IL-2 ... 5 2 × 10 CT26 cells were injected subcutaneously into new control mice (group 5, 6 mice). 5 One CT26 cell was used as the re-excitation control group.

[0276] like Figure 11B As shown, HSA-IL21-C01 and HSA-IL21-C08 exhibited significant tumor growth inhibition compared to HSA-IL21-WT. Individual tumor growth curves for each mouse in groups 1-4 are shown below. Figure 11C-11F In these figures, the dashed line indicates that the mediator group (Group 1) had half the mean tumor volume on day 14, which was 458 mm. 3 The ratios following “D14” and “D40” show that, relative to the total number of mice in the original group, tumor sizes were less than 458 mm at days 14 and 40 after tumor inoculation. 3 The number of mice was [number missing]. The results showed that HSA-IL21-C01 and HSA-IL21-C08 induced significant tumor shrinkage in the CT26 syngeneic mouse model. Figure 11G The results of body weight changes indicated that treatment with HSA-fused IL-21 and its variants did not induce significant toxicity in CT26 syngeneic mice. Figure 11HThe mouse survival curves showed that, compared with HSA-IL21-WT, HSA-IL21-C01 and HSA-IL21-C08 significantly prolonged the survival time of mice. Figure 11I-11K As shown, HSA-IL21-C01 and HSA-IL21-C08 exhibited memory effects in a BALB / c syngeneic mouse model carrying CT26. These results indicate that IL-21 signaling is crucial for the generation of tumor-specific memory responses in response to tumor reactivation, and that the tested HSA-fused IL-21 variants induced robust tumor-specific memory responses.

[0277] Example 12. Expression and purification of Fc-fused IL-21 variants

[0278] To test other forms of IL-21 variants, heterodimeric Fc fusion-based IL-21 variants were designed. Specifically, each heterodimeric Fc fusion-based IL-21 variant contains two polypeptide chains: a first polypeptide chain (or "potassium chain") and a second polypeptide chain (or "pestle chain"). The first polypeptide chain, from its N-terminus to its C-terminus, includes: a human IgG4 hinge region (SEQ ID NO:31), a human IgG4 Fc region with a potassium mutation (SEQ ID NO:33), a linker peptide (SEQ ID NO:34), and an IL-21 variant (e.g., any of the IL-21 variants described above). The second polypeptide chain, from its N-terminus to its C-terminus, includes: a human IgG4 hinge region (SEQ ID NO:31) and a human IgG4 Fc region with a pestle mutation (SEQ ID NO:32). Plasmids expressing heterodimeric Fc fusion-based IL-21 and its variants were constructed. Expi293 cells were transfected to express the following molecules: heterodimer Fc-IL21-WT (mortar chain: SEQ ID NO:35; mortar chain: SEQ ID NO:29), heterodimer Fc-IL21-C01 (mortar chain: SEQ ID NO:36; mortar chain: SEQ ID NO:29), heterodimer Fc-IL21-C03 (mortar chain: SEQ ID NO:37; mortar chain: SEQ ID NO:29), heterodimer Fc-IL21-C07 (mortar chain: SEQ ID NO:38; mortar chain: SEQ ID NO:29), and heterodimer Fc-IL21-C08 (mortar chain: SEQ ID NO:39; mortar chain: SEQ ID NO:29).

[0279] To generate the heterodimeric Fc-fused IL-21 variant, the following reasons were considered. First, some reports indicate that HSA-fused proteins may experience precipitation issues at 37°C and acidic pH conditions. Second, it was hypothesized that the heterodimeric Fc-fused protein might be more stable than the HSA-fused protein. Finally, preliminary data showed that the heterodimeric Fc-IL21-WT exhibited stronger IL-21 signaling induction activity compared to the homodimeric Fc-IL21-WT (e.g., G4Fc-IL21-WT, which contains two identical polypeptide chains, each with an amino acid sequence as shown in SEQ ID NO:16).

[0280] The purified heterodimers Fc-IL21-WT, Fc-IL21-C01, Fc-IL21-C03, Fc-IL21-C07, and Fc-IL21-C08 were used in subsequent experiments.

[0281] Example 13. Determination of the potency of heterodimeric Fc fusion-derived IL-21 variants in inducing IL-21-STAT3 reporter gene signaling.

[0282] IL-21 reporter gene assays were performed to test the IL-21-phspho STAT3 (pSTAT3)-signaling induction activity of heterodimeric Fc fusion IL-21 and its variants. Specifically, 2.8 × 10⁻⁶ cells were used. 5 HEK-Blue cells / mL TM IL-21 (InvivoGen, catalog number: hkb-il21) was resuspended in pre-warmed assay medium (DMEM (Dupoisk modified E. Silva medium; Corning, catalog number: 10-013-CV), 10% (v / v) heat-inactivated FBS (Gibco, catalog number: A31606-01), and 100× penicillin-streptomycin solution (Corning, catalog number: 30-002-CI)). 180 μL of HEK-Blue was added... TM IL-21 cells were incubated with 20 μL of serially diluted heterodimer Fc-fused IL-21 and its variants at 37 °C for 22 h in 96-well TC-treated microplates (Corning, catalog number: 3599). Following incubation, 20 μL of induced HEK-Blue was added... TM IL-21 cell supernatant resuspended in 180 μL of QUANTI-Blue TMThe solution (InvivoGen, catalog number: rep-qbs2) was incubated at 37°C in a flat-bottomed 96-well plate (Paul Bottger, catalog number: 05-031-0100) for 2-3 hours. A spectrophotometer (Varioskan) was used. TM The activity of SEAP (secretory alkaline phosphatase) was measured at 630 nm using a LUK (Thermo Scientific, Model 3020).

[0283] The EC50 values ​​of the IL-21 variant fused to each heterodimer Fc are shown in the table below.

[0284] Table 3

[0285]

[0286] like Figure 12 As shown in the table above, the heterodimers Fc-IL21-C01, C03, C07 and C08 exhibit similar IL-21-STAT3 signaling induction activity compared to the heterodimer Fc-IL21-WT.

[0287] Example 14. Determination of the potency of heterodimeric Fc fusion-derived IL-21 variants in inducing T cell and NK-92 cell proliferation.

[0288] For T cell proliferation assays, CD8+ cells were isolated from hPBMC donors according to the manufacturing protocol. + T cells. CD8 + T cells are composed of CD3 / CD28 (Thermo Fisher Scientific, Catalog No.: 11132D) Activated for 2 days at a cell-to-bead ratio of 4:1. The activated T cells were then left to rest for 24 hours, and then 5 × 10⁻⁶ cells were added. 4 CD8 per cell / well + T cells fused with heterodimer Fc containing IL-21 and its variants were incubated for 5 days at a specified concentration (200 nM, 5-fold serial dilution, 10 spots) in the presence of 1 μg / ml soluble anti-CD3 antibody (Baijin Biotechnology Co., Ltd., catalog number: 317347). Following incubation, [the cells were then analyzed]. T cell proliferation was determined using a luminescent cell viability assay (Promega, catalog number: G7573). For NK-92 cell proliferation assay, NK-92 cells were starved for 24 hours in culture medium without IL-2 supplementation. After starvation, 5 × 10⁶ cells were... 4NK-92 cells / well were incubated for an additional 24 hours with IL-21 or its variants fused with heterodimer Fc at a specified concentration (200 nM, 5-fold serial dilution, 8 spots) in complete medium (75% MEM-α, with ribonucleosyl and deoxyribonucleosyl (Gibco, catalog number: 12571-048) + 12.5% ​​horse serum (Gibco, catalog number: 16050-122) + 12.5% ​​FBS (Gibco, catalog number: A31606-01) + 150 U / mL IL-2 (Baijin Biotechnology, catalog number: 589108)). Proliferation was determined by luminescent cell viability assay.

[0289] like Figure 13A As shown in the table below, compared with the heterodimer Fc-IL21-WT, the heterodimers Fc-IL21-CO1, CO3, CO7, and CO8 induced activated CD8+. + It showed relatively stronger efficacy in T cell proliferation.

[0290] Table 4

[0291]

[0292] like Figure 13B As shown in the table below, the heterodimers Fc-IL21-C01, C07, and C08 showed better efficacy in inducing NK-92 cell proliferation compared to the heterodimers Fc-IL21-WT and C03.

[0293] Table 5

[0294]

[0295] Example 15. Determination of the potency of heterodimeric Fc fusion-derived IL-21 variants in inducing primary NK cell cytotoxicity.

[0296] The potency of the heterodimeric Fc-fused IL-21 variant in inducing primary NK cell cytotoxicity was determined by flow cytometry. Specifically, primary NK cells were isolated from hPBMC donors according to the manufacturing protocol, and K-562 tumor cells were inoculated with 5 μM CellTrace TM- Staining with purple dye (Thermo Fisher Scientific, catalog number: C34557). Effector cells (primary NK cells) and target cells (purple-labeled K-562) at a cell-to-cell ratio of 8:1 were co-incubated for 24 hours at 37°C in the presence of 0.00001-100 nM heterodimer Fc-fused IL-21 or its variants in 96-well U-shaped cell culture plates. Cells were then harvested and stained with 7-AAD viability staining solution (Baijin Biotechnology, catalog number: 420404). NK cell cytotoxicity was determined by the percentage of dead K-562 tumor cells using the following formula: (CellTrace-purple) + 7-AAD + Cells / (CellTrace-purple) + cell) * 100% of the total.

[0297] like Figure 14 As shown, the heterodimer Fc-IL21-WT and its variants exhibited potency in inducing NK cell cytotoxicity. Compared to the heterodimer Fc-IL21-WT and other variants, the heterodimers Fc-IL21-C07 and C08 showed slightly stronger potency in inducing primary NK cell cytotoxicity. These results indicate that the selected heterodimer Fc-IL-21 variants can induce specific lysis of K-562 tumor cells mediated by primary NK cells.

[0298] Example 16. Evaluation of the antitumor efficacy of IL-21 variants fused with heterodimer Fc.

[0299] The antitumor efficacy of heterodimeric Fc fusion-based IL-21 and its variants was evaluated in a BALB / c mouse model carrying CT26 tumors. Treatment protocol and dosing schedule are as follows: Figure 15A As shown. Specifically, 5-week-old BALB / c mice were selected and subcutaneously (sc) inoculated with 2×10 5 Mice were injected with CT26 colon cancer cells from mice. The molecular treatments tested began on day 4 post-tumor inoculation. Mice in the treatment groups were intraperitoneally (ip) injected with 10 mg / kg of the heterodimers Fc-IL21-WT (Group 2), Fc-IL21-C01 (Group 3), Fc-IL21-C03 (Group 4), Fc-IL21-C07 (Group 5), and Fc-IL21-C08 (Group 6), respectively. Control mice (Group 1) were injected with 10 mL / kg of a carrier (isotonic sodium chloride solution). Injections were administered twice weekly for a total of 3 weeks.

[0300] The individual tumor growth curves for each mouse in groups 1 through 6 are shown below. Figure 15B-15GIn these figures, the dashed line indicates that the mean tumor volume in the treatment group (Group 1) was half of the mean tumor volume on day 13, which was 286 mm. 3 The ratios following "D13", "D20", and "D41" indicate tumor sizes smaller than 286 mm at days 13, 20, and 41 post-tumor inoculation, relative to the total number of live mice at the same number of days. 3 The number of mice was [number missing]. The results showed that, compared with the heterodimer Fc-IL21-WT and other variants, the heterodimer Fc-IL21-C01 significantly inhibited tumor growth. Furthermore, heterodimers Fc-IL21-C01, C03, and C08 induced significant tumor shrinkage in this CT26 syngeneic mouse model after treatment cessation (after day 20). Heterodimers Fc-IL21-C01, C03, and C08 may induce tumor-specific memory responses to inhibit tumor growth. Figure 15H The results of body weight changes indicated that treatment with heterodimer Fc fused IL-21 and its variants did not induce significant toxicity in CT26 syngeneic mice.

[0301] Other embodiments

[0302] It should be understood that although the invention has been described in conjunction with a detailed description, the foregoing description is intended to illustrate rather than limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An engineered IL-21 polypeptide, wherein the engineered IL-21 polypeptide comprises non-natural disulfide bonds.

2. The engineered IL-21 polypeptide according to claim 1, comprising at least 80% of the same amino acid sequence as SEQ ID NO:

2.

3. The engineered IL-21 peptide according to claim 1 or 2, wherein the engineered IL-21 peptide is capable of binding to a complex formed by the human IL-21 receptor (IL-21R) and the common cytokine γ chain (γc).

4. The engineered IL-21 peptide according to any one of claims 1 to 3, wherein the engineered IL-21 peptide comprises one or more of the following: (a) The amino acid corresponding to position 8 of SEQ ID NO:2 is cysteine ​​(C); (b) The amino acid corresponding to position 19 of SEQ ID NO:2 is C; (c) The amino acid corresponding to position 29 of SEQ ID NO:2 is C; (d) The amino acid corresponding to position 31 of SEQ ID NO:2 is C; (e) The amino acid corresponding to position 33 of SEQ ID NO:2 is C; (f) The amino acid corresponding to position 36 of SEQ ID NO:2 is C; (g) The amino acid corresponding to position 39 of SEQ ID NO:2 is C; and (h) The amino acid corresponding to position 56 of SEQ ID NO:2 is C.

5. The engineered IL-21 peptide according to any one of claims 1 to 4, wherein the engineered IL-21 peptide comprises one or more of the following: (a) The amino acid corresponding to position 61 of SEQ ID NO:2 is C; (b) The amino acid corresponding to position 62 of SEQ ID NO:2 is C; (c) The amino acid corresponding to position 63 of SEQ ID NO:2 is C; (d) The amino acid corresponding to position 80 of SEQ ID NO:2 is C; (e) The amino acid corresponding to position 86 of SEQ ID NO:2 is C; (f) The amino acid corresponding to position 105 of SEQ ID NO:2 is C; (g) The amino acid corresponding to position 106 of SEQ ID NO:2 is C; (h) The amino acid corresponding to position 107 of SEQ ID NO:2 is C; (i) The amino acid corresponding to position 110 of SEQ ID NO:2 is C; (j) The amino acid corresponding to position 112 of SEQ ID NO:2 is C; and (k) The amino acid corresponding to position 117 of SEQ ID NO:2 is C.

6. The engineered IL-21 peptide according to any one of claims 1 to 5, wherein the engineered IL-21 peptide comprises one or more of the following: (a) The amino acid corresponding to position 8 of SEQ ID NO:2 is C, and the amino acid corresponding to position 86 of SEQ ID NO:2 is C; (b) The amino acid corresponding to position 19 of SEQ ID NO:2 is C, and the amino acid corresponding to position 117 of SEQ ID NO:2 is C; (c) The amino acid corresponding to position 29 of SEQ ID NO:2 is C, and the amino acid corresponding to position 110 of SEQ ID NO:2 is C; (d) The amino acid corresponding to position 31 of SEQ ID NO:2 is C, and the amino acid corresponding to position 62 of SEQ ID NO:2 is C; (e) The amino acid corresponding to position 31 of SEQ ID NO:2 is C, and the amino acid corresponding to position 63 of SEQ ID NO:2 is C; (f) The amino acid corresponding to position 33 of SEQ ID NO:2 is C, and the amino acid corresponding to position 61 of SEQ ID NO:2 is C; (g) The amino acid corresponding to position 33 of SEQ ID NO:2 is C, and the amino acid corresponding to position 63 of SEQ ID NO:2 is C; (h) The amino acid corresponding to position 36 of SEQ ID NO:2 is C, and the amino acid corresponding to position 106 of SEQ ID NO:2 is C; (i) The amino acid corresponding to position 36 of SEQ ID NO:2 is C, and the amino acid corresponding to position 107 of SEQ ID NO:2 is C; (j) The amino acid corresponding to position 39 of SEQ ID NO:2 is C, and the amino acid corresponding to position 105 of SEQ ID NO:2 is C; (k) The amino acid corresponding to position 39 of SEQ ID NO:2 is C, and the amino acid corresponding to position 107 of SEQ ID NO:2 is C; (l) The amino acid corresponding to position 39 of SEQ ID NO:2 is C, and the amino acid corresponding to position 112 of SEQ ID NO:2 is C; and (m) The amino acid corresponding to position 56 of SEQ ID NO:2 is C, and the amino acid corresponding to position 80 of SEQ ID NO:2 is C.

7. The engineered IL-21 peptide according to any one of claims 1 to 6, wherein the engineered IL-21 peptide comprises one or more of the following: (a) The amino acid corresponding to position 12 of SEQ ID NO:2 is M; (b) The amino acid corresponding to position 16 of SEQ ID NO:2 is R; (c) The amino acid corresponding to position 19 of SEQ ID NO:2 is I; (d) The amino acid corresponding to position 23 of SEQ ID NO:2 is D; (e) The amino acid corresponding to position 105 of SEQ ID NO:2 is E; (f) The amino acid corresponding to position 114 of SEQ ID NO:2 is E; (g) The amino acid corresponding to position 118 of SEQ ID NO:2 is S; (h) The amino acid corresponding to position 121 of SEQ ID NO:2 is Q; (i) The amino acid corresponding to position 122 of SEQ ID NO:2 is K; (j) The amino acid corresponding to position 124 of SEQ ID NO:2 is I; (k) The amino acid corresponding to position 125 of SEQ ID NO:2 is H; and (l) The amino acid corresponding to position 128 of SEQ ID NO:2 is L.

8. The engineered IL-21 polypeptide according to any one of claims 1 to 7, comprising at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

9. The engineered IL-21 polypeptide of claim 8, wherein the engineered IL-21 polypeptide comprises at least 90% of the same amino acid sequence as SEQ ID NO:

3.

10. The engineered IL-21 polypeptide of claim 8, wherein the engineered IL-21 polypeptide comprises at least 90% of the same amino acid sequence as SEQ ID NO:

5.

11. The engineered IL-21 polypeptide of claim 8, wherein the engineered IL-21 polypeptide comprises at least 90% of the same amino acid sequence as SEQ ID NO:

9.

12. The engineered IL-21 polypeptide of claim 8, wherein the engineered IL-21 polypeptide comprises at least 90% of the same amino acid sequence as SEQ ID NO:

10.

13. The engineered IL-21 peptide according to any one of claims 1 to 12, wherein the engineered IL-21 peptide can induce the proliferation of immune cells (e.g., T cells or NK cells).

14. The engineered IL-21 polypeptide according to any one of claims 1 to 13, wherein the engineered IL-21 polypeptide can induce STAT-3 phosphorylation.

15. A fusion protein comprising an engineered IL-21 polypeptide according to any one of claims 1 to 14.

16. The fusion protein of claim 15, further comprising human serum albumin (HSA).

17. The fusion protein of claim 16, wherein the HSA comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:

26.

18. The fusion protein of claim 16 or 17, wherein the engineered IL-21 polypeptide is linked to the C-terminus of the HSA via a linker peptide.

19. The fusion protein of claim 18, wherein the adaptor peptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:

25.

20. The fusion protein according to any one of claims 15 to 19, wherein the fusion protein further optionally includes a His tag at the N-terminus.

21. The fusion protein according to any one of claims 15 to 20, wherein the fusion protein comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO: 17, 18, 19, 20, or 21.

22. A fusion protein, optionally comprising, from the N-terminus to the C-terminus: (a) Optional His tag; (b)HSA; (c) Connector peptides, and (d) Engineered IL-21 peptide.

23. The fusion protein of claim 22, wherein the His tag comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:22; wherein the HSA comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:26; wherein the adaptor peptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:25; and / or wherein the engineered IL-21 polypeptide comprises at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the same amino acid sequence as SEQ ID NO:2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

24. The fusion protein according to any one of claims 15 to 23, further comprising an Fc region.

25. The fusion protein according to any one of claims 15 to 24, wherein the fusion protein can alleviate Treg-mediated T cell suppression.

26. The fusion protein according to any one of claims 15 to 25, wherein the fusion protein can induce primary NK cell toxicity.

27. A protein complex comprising: (a) A first polypeptide, comprising, from the N-terminus to the C-terminus: an optional first hinge region, a first Fc region, an optional linker peptide, and an engineered IL-21 polypeptide according to any one of claims 1 to 14; and (b) A second polypeptide, the second polypeptide including, from the N-terminus to the C-terminus, an optional second hinge region and a second Fc region.

28. The protein complex of claim 27, wherein the first hinge region, the first Fc region, the second hinge region and / or the second Fc region are derived from human IgG4.

29. The protein complex of claim 27 or 28, wherein the first Fc region and / or the second Fc region comprises one or more kilometre (KIH) mutations.

30. The protein complex according to any one of claims 27 to 29, wherein the first Fc region comprises at least 80%, 90%, 95%, or 100% of the sequence identical to SEQ ID NO:33, and the second Fc region comprises at least 80%, 90%, 95%, or 100% of the sequence identical to SEQ ID NO:

32.

31. The protein complex according to any one of claims 27 to 30, wherein the first hinge region and / or the second hinge region comprises at least 80%, 90%, 95%, or 100% of the same sequence as SEQ ID NO:

31.

32. The protein complex according to any one of claims 27 to 31, wherein the adaptor peptide comprises at least 80%, 90%, 95%, or 100% of the same sequence as SEQ ID NO:

34.

33. The protein complex according to any one of claims 27 to 32, wherein: (1) The first polypeptide comprises at least 80%, 90%, 95% or 100% of the same sequence as SEQ ID NO:35, and the first polypeptide comprises at least 80%, 90%, 95% or 100% of the same sequence as SEQ ID NO:29; (2) The first polypeptide comprises at least 80%, 90%, 95% or 100% of the same sequence as SEQ ID NO:36, and the first polypeptide comprises at least 80%, 90%, 95% or 100% of the same sequence as SEQ ID NO:29; (3) The first polypeptide comprises at least 80%, 90%, 95% or 100% of the same sequence as SEQ ID NO:37, and the first polypeptide comprises at least 80%, 90%, 95% or 100% of the same sequence as SEQ ID NO:29; (4) The first polypeptide comprises at least 80%, 90%, 95%, or 100% of the sequence identical to SEQ ID NO:38, and the first polypeptide comprises at least 80%, 90%, 95%, or 100% of the sequence identical to SEQ ID NO:29; or (5) The first polypeptide comprises at least 80%, 90%, 95% or 100% of the same sequence as SEQ ID NO:39, and the first polypeptide comprises at least 80%, 90%, 95% or 100% of the same sequence as SEQ ID NO:

29.

34. The protein complex according to any one of claims 27 to 33, wherein the protein complex can induce STAT-3 phosphorylation and / or alleviate Treg-mediated T cell suppression.

35. The protein complex according to any one of claims 27 to 34, wherein the protein complex is capable of inducing the proliferation of immune cells (e.g., T cells or NK cells) and / or inducing primary NK cell toxicity.

36. A pharmaceutical composition comprising an engineered IL-21 polypeptide according to any one of claims 1 to 14, a fusion protein according to any one of claims 15 to 26, or a protein complex according to any one of claims 27 to 35; and a pharmaceutically acceptable carrier.

37. A nucleic acid encoding an engineered IL-21 polypeptide according to any one of claims 1 to 14, a fusion protein according to any one of claims 15 to 26, or a protein complex according to any one of claims 27 to 35.

38. A vector comprising the nucleic acid according to claim 37.

39. A cell comprising the nucleic acid according to claim 37 or the vector according to claim 38.

40. The cell of claim 39, wherein the cell is an Expi293 cell or a CHO-S cell.

41. A method for producing an engineered IL-21 peptide or a fusion protein comprising said engineered IL-21 peptide, said method comprising (a) The cells are cultured under conditions sufficient to produce the engineered IL-21 polypeptide or the fusion protein according to claim 39 or 40; and (b) Collect the engineered IL-21 polypeptide, the fusion protein, or the protein complex produced by the cells.

42. A method of treating a subject with cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an engineered IL-21 polypeptide according to any one of claims 1 to 14, a fusion protein according to any one of claims 15 to 26, or a protein complex according to any one of claims 27 to 35.

43. The method of claim 42, wherein the subject has a solid tumor or hematologic malignancy.

44. The method of claim 42, wherein the cancer is melanoma, renal cell carcinoma (RCC), lymphoma, esophageal adenocarcinoma, lung cancer, hepatocellular carcinoma, bile duct carcinoma, gallbladder cancer, gastric cancer, pancreatic cancer, colorectal cancer, endometrial cancer, ovarian cancer, bladder cancer, or prostate cancer.

45. A method for reducing tumor growth rate, the method comprising: Tumor cells are contacted with an effective amount of the composition, said composition comprising an engineered IL-21 polypeptide according to any one of claims 1 to 14, a fusion protein according to any one of claims 15 to 26, or a protein complex according to any one of claims 27 to 35.

46. ​​A method for killing tumor cells, the method comprising: Tumor cells are contacted with an effective amount of the composition, said composition comprising an engineered IL-21 polypeptide according to any one of claims 1 to 14, a fusion protein according to any one of claims 15 to 26, or a protein complex according to any one of claims 27 to 35.

47. A method for improving the stability of a protein (e.g., a cytokine), the method comprising: (a) Provide the 3D structure of the protein (e.g., cytokine); (b) Measuring the C10 of one or more amino acid residues in the 3D structure. α The distance between atoms; and (c) Selecting two amino acid residues from the one or more amino acid residues, wherein the C of the two selected amino acid residues is... α The atoms are located in the range of 3-7 angstroms.

48. The method of claim 47, further comprising expressing a protein variant (e.g., a cytokine variant), wherein the protein variant comprises a non-natural disulfide bond formed by mutating the two selected amino acid residues to cysteine.

49. The method of claim 47 or 48, wherein mutating the two selected amino acid residues to cysteine ​​does not substantially alter the 3D structure of the protein (e.g., cytokine).

50. The method according to any one of claims 47 to 49, wherein the protein has no more than 200 amino acid residues.

51. A method for screening cytokine variants with improved antitumor efficacy, the method comprising: (a) Provide the 3D structure of the cytokine; (b) Measuring the C10 of one or more amino acid residues in the 3D structure. α The distance between atoms; and (c) Selecting two amino acid residues from the one or more amino acid residues, wherein the C of the two selected amino acid residues is... α The atoms are located in the range of 3-7 angstroms.

52. The method of claim 51, further comprising: (d) Expressing cytokine variants, wherein the variants include non-natural disulfide bonds formed by mutating the two selected amino acid residues to cysteine; (e) administering the cytokine variant to tumor-bearing animals; and (f) Determine tumor growth in the tumor-bearing animal (e.g., by measuring tumor volume).

53. The method according to any one of claims 47 to 52, wherein the protein or cytokine is IL-21 (e.g., human IL-21).

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