Manipulated IL-21 variant and its usage

Abstract

This disclosure relates to manipulated IL-21 variants and methods of using them. In some embodiments, the manipulated IL-21 variant includes a non-natural disulfide bond.

Description

[Technical Field]

[0001] Cross-reference of related applications This disclosure claims priority and interest in U.S. Provisional Patent Application No. 63 / 453,399, filed on March 20, 2023, which is incorporated herein by reference in its entirety.

[0002] Sequence List This application includes a sequence listing submitted electronically as an XML file named 52246-0012WO1_SL_ST26.xml. The XML file was created on March 18, 2024, and has a size of 47,998 bytes. The data within the XML file is incorporated herein by reference in its entirety.

[0003] Technical field This disclosure relates to a manipulated IL-21 variant and a method for using it. [Background technology]

[0004] background Cytokines belonging to the common gamma 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 therefore are of great interest for their use in anti-cancer therapy.

[0005] Among these, IL-21 exerts potent antitumor effects through its ability to induce and proliferate cytotoxic CD8+ T cells, NK cells, and NKT cells, as well as its ability to suppress FOXP3 expression and regulatory T cell (Treg) proliferation. In addition, IL-21 has been associated with clinical antitumor activity. However, at high concentrations, IL-21 can also cause dose-limiting side effects, including grade 3 / 4 granulocytopenia and hepatotoxicity. Furthermore, by driving the inflammatory response sustained by IL-6 and IL-17, IL-21 may also contribute to tumorigenesis, as it occurs in chronic colitis.

[0006] Recombinant IL-21 has been tested as an antitumor agent in various clinical trials. These trials have demonstrated promising antitumor activity and acceptable toxicity. However, due to its short half-life, IL-21's in vivo levels are reduced, requiring frequent administration and limiting its clinical applications.

[0007] Therefore, there is a need to develop cancer therapies that target the IL-21 pathway with a longer half-life, enhanced antitumor efficacy, and limited toxicity. [Overview of the Initiative]

[0008] overview 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 include unnatural disulfide bonds formed by mutating one, two, three, four, five, six, or seven or more residues (e.g., a pair of residues) of wild-type IL-21 to cysteine ​​without interfering with the overall structure or residues important for IL-21 receptor interaction. As a result, the variants or protein constructs can be more stable than wild-type IL-21. In some embodiments, the protein constructs have enhanced thermal stability (e.g., T) compared to wild-type IL-21. agg and T onset It showed an increase in (and) a greater immunosuppressive efficacy (e.g., suppression of Treg-mediated T cells) than wild-type IL-21. Therefore, the manipulated IL-21 variants and their protein constructs described herein can be used in cancer treatment with minimal toxicity.

[0009] Furthermore, methods for screening cytokines (e.g., IL-21) with increased stability and / or antitumor efficacy are also provided herein.

[0010] In one embodiment, the disclosure relates to an engineered IL-21 polypeptide, and in some embodiments, the engineered IL-21 polypeptide contains a non-natural disulfide bond. In some embodiments, the engineered IL-21 polypeptide contains an amino acid sequence that is at least 80% identical to SEQ ID NO: 2. In some embodiments, the engineered IL-21 polypeptide can bind to a complex formed by the human IL-21 receptor (IL-21R) and the common cytokine γ chain (γc).

[0011] In some embodiments, the manipulated 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.

[0012] In some embodiments, the manipulated 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 manipulated IL-21 polypeptide is as follows: (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: However, it includes one or more of the following: (h) the amino acid corresponding to position 63 of SEQ ID NO: 2 is C, and 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.

[0013] In some embodiments, the manipulated 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.

[0014] In some embodiments, the manipulated IL-21 polypeptide described herein comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the manipulated IL-21 polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NOs. In some embodiments, the manipulated IL-21 polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NOs. In some embodiments, the manipulated IL-21 polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NOs. In some embodiments, the manipulated IL-21 polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NOs. In some embodiments, the manipulated IL-21 polypeptide comprises an amino acid sequence that is at least 90% identical to SEQ ID NOs.

[0015] In some embodiments, the manipulated IL-21 polypeptide can induce proliferation of immune cells (e.g., T cells or NK cells). In some embodiments, the manipulated IL-21 polypeptide can induce phosphorylation of STAT-3.

[0016] In one embodiment, this disclosure relates to a fusion protein comprising an engineered IL-21 polypeptide as described herein. In some embodiments, the fusion protein described herein further comprises human serum albumin (HSA). In some embodiments, the HSA comprises 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 engineered IL-21 polypeptide is ligated to the C-terminal side of the HSA via a linker peptide. In some embodiments, the linker peptide comprises 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 fusion protein optionally further comprises a His tag at the N-terminus. In some embodiments, the fusion protein contains an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs. 17, 18, 19, 20, or 21.

[0017] In one embodiment, the present disclosure relates to a fusion protein comprising, optionally, 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. In some embodiments, the His tag comprises 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 comprises 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 linker peptide comprises 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 manipulated IL-21 polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0018] In some embodiments, the fusion protein described herein further comprises an Fc region.

[0019] In some embodiments, the fusion protein can mitigate Treg-mediated T cell suppression. In some embodiments, the fusion protein can induce cytotoxicity of primary NK cells.

[0020] In one embodiment, the present disclosure relates to a protein complex comprising (a) a first polypeptide comprising an optional first hinge region, a first Fc region, an optional linker peptide, and an engineered IL-21 polypeptide as described herein, extending from the N-terminus to the C-terminus; and (b) a second polypeptide comprising an optional second hinge region, a second Fc region, extending from the N-terminus to the C-terminus. 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 contain one or more knob-into-hole (KIH) mutations. In some embodiments, the first Fc region includes a sequence that is at least 80%, 90%, 95%, or 100% of SEQ ID NO: 33, and the second Fc region includes a sequence that is at least 80%, 90%, 95%, or 100% of SEQ ID NO: 32. In some embodiments, the first hinge region and / or the second hinge region includes a sequence that is at least 80%, 90%, 95%, or 100% of SEQ ID NO: 31. In some embodiments, the linker peptide includes a sequence that is at least 80%, 90%, 95%, or 100% of SEQ ID NO: 34.In some embodiments, the protein complex described herein is as follows: (1) the first polypeptide comprises a sequence of at least 80%, 90%, 95%, or 100% of SEQ ID NO: 35, and the first polypeptide comprises a sequence of at least 80%, 90%, 95%, or 100% of SEQ ID NO: 29; (2) the first polypeptide comprises a sequence of at least 80%, 90%, 95%, or 100% of SEQ ID NO: 36, and the first polypeptide comprises a sequence of at least 80%, 90%, 95%, or 100% of SEQ ID NO: 29; (3) the first polypeptide comprises at least 80%, 90%, 95%, or 10% of SEQ ID NO: 37 (4) The first polypeptide includes a sequence that is 0%, and the first polypeptide includes a sequence that is at least 80%, 90%, 95%, or 100% of sequence number 29, or (5) the first polypeptide includes a sequence that is at least 80%, 90%, 95%, or 100% of sequence number 38, and the first polypeptide includes a sequence that is at least 80%, 90%, 95%, or 100% of sequence number 29, or (6) the first polypeptide includes a sequence that is at least 80%, 90%, 95%, or 100% of sequence number 39, and the first polypeptide includes a sequence that is at least 80%, 90%, 95%, or 100% of sequence number 29.

[0021] In some embodiments, the protein complex can induce phosphorylation of STAT-3 and / or mitigate Treg-mediated T cell suppression. In some embodiments, the protein complex can induce proliferation of immune cells (e.g., T cells or NK cells) and / or induce cytotoxicity of primary NK cells.

[0022] In one embodiment, the present disclosure relates to a pharmaceutical composition comprising an engineered IL-21 polypeptide, fusion protein, or protein complex described herein and a pharmaceutically acceptable carrier.

[0023] In one embodiment, this disclosure relates to nucleic acids encoding the manipulated IL-21 polypeptide, fusion protein, or protein complex described herein. In one embodiment, this disclosure relates to vectors comprising the nucleic acids described herein.

[0024] In one embodiment, this disclosure relates to cells comprising nucleic acids or vectors as described herein. In some embodiments, the cells are Expi293 cells or CHO-S cells.

[0025] In one embodiment, the present disclosure relates to a method for producing an engineered IL-21 polypeptide or a fusion protein comprising an engineered IL-21 polypeptide, the method comprising (a) culturing the cells described herein under conditions sufficient to cause the cells to produce an engineered IL-21 polypeptide or a fusion protein, and (b) recovering the engineered IL-21 polypeptide, fusion protein, or protein complex produced by the cells.

[0026] In one embodiment, the disclosure relates to a method for treating a subject having cancer, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an engineered IL-21 polypeptide, fusion protein, or protein complex as described herein. In some embodiments, the subject has a solid tumor or hematological cancer. In some embodiments, the cancer is melanoma, renal cell carcinoma (RCC), lymphoma, esophageal adenocarcinoma, lung cancer, hepatocellular carcinoma, cholangiocarcinoma, gallbladder cancer, gastric cancer, pancreatic cancer, colorectal cancer, endometrial cancer, ovarian cancer, bladder cancer, or prostate cancer.

[0027] In one embodiment, the disclosure relates to a method for reducing the rate of tumor growth, the method comprising contacting tumor cells with an effective amount of a composition comprising an engineered IL-21 polypeptide, fusion protein, or protein complex as described herein.

[0028] In one embodiment, the 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 polypeptide, fusion protein, or protein complex as described herein.

[0029] In one embodiment, the present disclosure relates to a method for improving the stability of a protein (e.g., cytokine), comprising (a) providing a 3D structure of the protein (e.g., cytokine) and (b) one or more amino acid residues in the 3D structure. アルファ The process includes (c) measuring the distance between atoms and (c) selecting two amino acid residues from one or more amino acid residues, and in some embodiments, the C of the two selected amino acid residues アルファ The atoms are within 3 to 7 angstroms (e.g., 4.5 to 6.5 angstroms). In some embodiments, the methods described herein further include expressing a protein variant (e.g., a cytokine variant), in some embodiments the protein variant includes a non-native disulfide bond formed by mutating two selected amino acid residues to cysteine. In some embodiments, mutating two selected amino acid residues to cysteine ​​does not substantially alter the 3D structure of the protein (e.g., cytokine).

[0030] In one embodiment, the present disclosure relates to a method for screening cytokine variants having improved antitumor efficacy, comprising (a) providing a 3D structure of a cytokine and (b) one or more amino acid residues in the 3D structure. アルファ The process includes (c) measuring the distance between atoms and (c) selecting two amino acid residues from one or more amino acid residues, and in some embodiments, the C of the two selected amino acid residues アルファThe atoms are within 3 to 7 angstroms (e.g., 4.5 to 6.5 angstroms). In some embodiments, the methods described herein further include (d) expressing a cytokine variant, wherein in some embodiments the variant comprises a non-natural disulfide bond formed by mutating 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).

[0031] In some embodiments, the protein or cytokine described herein is IL-21 (e.g., human IL-21).

[0032] In some embodiments, the protein or cytokine has 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 or fewer amino acid residues.

[0033] As used herein, the term “unnatural disulfide bond” refers to a disulfide bond that is not naturally present in the wild-type protein. In some embodiments, the unnatural disulfide bond is formed by two cysteine ​​residues, at least one of which is a mutation. In some embodiments, two of which are mutations. In some embodiments, at least one or two cysteine ​​residues are introduced by insertion. In some embodiments, deletion then alters the distance between two existing cysteine ​​residues that form a disulfide bond not present in the wild-type protein.

[0034] As used herein, the term “engineered IL-21 polypeptide” refers to a polypeptide derived from or a portion thereof of the wild-type IL-21 polypeptide that optionally contains one or more mutations (e.g., insertions, deletions, or substitutions). In some embodiments, the engineered IL-21 polypeptide contains or comprises 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 polypeptide has one or more mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) (e.g., amino acids are substituted by cysteine).

[0035] As used herein, the term “protein construct” refers to a complex having one or more polypeptides. In some embodiments, the protein construct is a fusion protein comprising a fusion protein, e.g., HSA and an engineered IL-21 polypeptide (e.g., any of the engineered IL-21 polypeptides described herein). In some embodiments, the protein construct has two or more polypeptides that can associate with each other to form a dimer or a polymer (e.g., a trimer). In some embodiments, the protein construct is a heterodimer Fc-fusion IL-21 (e.g., any of the heterodimer Fc-fusion IL-21s described herein or their variants).

[0036] As used herein, the term “cancer” refers to cells that have the ability to proliferate autonomously and uncontrollably. Examples of such cells include cells that have an abnormal state or condition characterized by rapid cell proliferation. The term means cancerous proliferation, whether of histopathological type or stage of invasiveness, including tumors, carcinogenic processes, metastatic tissues, and cells, tissues, or organs that have been transformed to be malignant. It also includes malignant tumors of various organ systems, such as the respiratory, cardiovascular, renal, reproductive, hematological, nervous, hepatic, gastrointestinal, and endocrine systems, as well as adenocarcinomas, including most colon cancers, renal cell carcinomas, prostate cancers and / or testicular tumors, non-small cell lung carcinomas, and small intestine cancers. "Spontaneous" cancers include any cancers that are not experimentally induced by transplantation of cancer cells into a subject, and include, for example, cancers that occur spontaneously, cancers caused by a patient's exposure to a carcinogen, cancers caused by transgenic oncogene insertions or tumor suppressor gene knockouts, and cancers caused by infections, such as viral infections. The term "carcinoma" is recognized in the art and refers to a malignant tumor of epithelial or endocrine tissue. This term also includes carcinosarcoma, which includes malignant tumors consisting of cancerous and sarcoma tissues. "Adenocarcinoma" refers to a carcinoma that originates from glandular tissue or in which tumor cells form recognizable glandular structures. The term "sarcoma" is recognized in the art and refers to a malignant tumor of mesenchymal origin. The term "hematopoietic neoplasia" includes diseases involving hematopoietic hyperplasia / tumor cells. Hematopoietic neoplasia may originate from myeloid, lymphoid, or erythrocyte lineages, or their progenitor cells. Hematological cancers are cancers that originate in hematopoietic tissues such as bone marrow, or in cells of the immune system. Examples of blood cancers include leukemia, lymphoma, and multiple myeloma.

[0037] Where used herein, the terms “subject” and “patient” are interchangeable throughout this specification and describe animals, humans, or non-humans to which treatment by the methods of the present invention is provided. Veterinary and non-veterinary uses are contemplated in this disclosure. Human patients may be adult humans or young humans (e.g., humans under 18 years of age). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. For example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, pigs (e.g., pigs, miniature pigs), equids, canids, felids, cats, and other domestic animals, livestock, and zoo animals.

[0038] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers of amino acids of any length, including at least two amino acids.

[0039] As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably herein to refer to polymers of nucleotides of any length, including but not limited to DNA, RNA, DNA / RNA hybrids, and their modifications.

[0040] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains. Methods and materials are described herein for use in the present invention, and other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative and not intended to limit the scope. All publications, patent applications, patents, sequences, database entries, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, this specification shall prevail, including definitions.

[0041] Other features and advantages of the present invention will become apparent from the following detailed description and drawings, as well as from the claims. [Brief explanation of the drawing]

[0042] [Figure 1] This table summarizes the purification profiles of IL-21 variants in Expi293 and CHO-S cells. "H8" represents the 8×His tag. [Figure 2A] This demonstrates the efficacy of the IL-21 variant in inducing STAT-3 phosphorylation. [Figure 2B] This demonstrates the efficacy of IL-21 variants in inducing T cell proliferation. [Figure 3A] The results of SDS-PAGE for the selected HSA-fused IL-21 variants are shown. NR: Unreduced form. R: Reduced form. [Figure 3B] This table summarizes the HPLC-SEC analysis results of the HSA-fused IL-21 variant. [Figure 3C] This table summarizes the results of DLS / SLS analysis of HSA-fused IL-21 variants. [Figure 4A] This demonstrates the efficacy of the HSA-fused IL-21 variant in inducing STAT-3 phosphorylation. [Figure 4B] This study demonstrates the efficacy of HSA-fused IL-21 variants in inducing T cell proliferation. [Figure 5] This study demonstrates the efficacy of an HSA-fused IL-21 variant in inducing NK-92 cell proliferation. [Figure 6] This study demonstrates the efficacy of HSA-fused IL-21 variants in alleviating Treg-mediated T cell suppression. [Figure 7] This study demonstrates the efficacy of an HSA-fused IL-21 variant in inducing cytotoxicity in primary NK cells. [Figure 8A] This table shows the treatment plan and administration schedule for HSA-fused IL-21 and its variants in the CT26-supported BLAB / c mouse model. [Figure 8B]The mean tumor growth curves of CT26-supported BALB / c mice treated with HSA-fused IL-21 and its variants are shown. After grouping (day 0), HSA-fused IL-21 and its variants were injected into the mice on days 4, 7, 11, 14, and 18 (indicated by arrows). [Figure 8C] Figures 8C to 8F show the individual tumor growth curves of CT26-carrying BALB / c mice treated with vehicle (Figure 8C), HSA-IL21-WT (Figure 8D), HSA-IL21-C01 (Figure 8E), and HSA-IL21-C08 (Figure 8F), respectively. [Figure 8D] See the explanation for Figure 8C. [Figure 8E] See the explanation for Figure 8C. [Figure 8F] See the explanation for Figure 8C. [Figure 8G] The individual tumor volumes of each CT26-carrying BALB / c mouse treated with HSA-fused IL-21 and its variants on day 21 (or day 25 after vaccination) are shown. [Figure 8H] This shows the change in mean body weight of CT26-supported BALB / c mice treated with HSA-fused IL-21 and its variants. [Figure 8I] This table shows the tumor volume (TV), mean percentage of tumor growth inhibition (TGI%), and corresponding p-values ​​at day 21 (or day 25 post-inoculation) of CT26-carrying BALB / c mice treated with HSA-fused IL-21 and its variants. P-values ​​were determined using one-way ANONA analysis. [Figure 9A] This study demonstrates the efficacy of an HSA-fused IL-21 variant in alleviating Treg-mediated T cell suppression. hSIRPα-Fc-mt10 (SEQ ID NO: 42) was used as a negative control. [Figure 9B] This shows the percentage of viable CD8+ CFSE-low cells after incubation of activated CD8+ T cells (Teff) and Treg cells pretreated with 30 nM HSA-IL21-WT ("WT"), HSA-IL21-C07 ("C07"), or HSA-IL21-C08 ("C08"). [Figure 10]This study demonstrates the efficacy of an HSA-fused IL-21 variant in inducing cytotoxicity in primary NK cells. [Figure 11-1] Figure 11A shows the treatment plan and administration schedule for HSA-fused IL-21 and its variants in the CT26-supported BLAB / c mouse model. [Figure 11-2] Figure 11B shows the mean tumor growth curves of CT26-supported BALB / c mice treated with HSA-fused IL-21 and its variants. After grouping (day 0), HSA-fused IL-21 and its variants were injected into the mice on days 4, 8, 11, 14, 18, and 21 (indicated by arrows). [Figure 11-3] Figures 11C to 11F show the individual tumor growth curves of CT26-carrying BALB / c mice treated with vehicle (Figure 11C), HSA-IL21-WT (Figure 11D), HSA-IL21-C01 (Figure 11E), and HSA-IL21-C08 (Figure 11F), respectively. [Figure 11-4] See the explanation in Figure 11-3. [Figure 11-5] Figure 11G shows the change in mean body weight of CT26-supported BALB / c mice treated with HSA-fused IL-21 and its variants. [Figure 11-6] Figure 11H shows the survival curves of CT26-supported BALB / c mice treated with HSA-fused IL-21 and its variants. [Figure 11-7] Figures 11I–11K show individual tumor growth curves of CT26-carrying BALB / c mice that were rechallenged by inoculation with CT26 cells. Six mice injected with the vehicle were used as controls for tumor rechallenge (Figure 11I). Ten mice (n=10) with tumor sizes less than 50 mm³ that had previously been inoculated with CT26 and then treated with HSA-IL21-C01 were selected for tumor rechallenge (Figure 11J). Eleven mice (n=11) with tumor sizes less than 50 mm³ that had previously been inoculated with CT26 and then treated with HSA-IL21-C08 were selected for tumor rechallenge (Figure 11K). [Figure 11-8] See the explanation in Figure 11-7. [Figure 12] This study demonstrates the efficacy of a heterodimer Fc-fused IL-21 variant in inducing STAT-3 phosphorylation. Human recombinant IFN-gamma protein (BioLegend, catalog number: 570208) was used as a negative control. [Figure 13A] This study demonstrates the efficacy of a heterodimer Fc-fused IL-21 variant in inducing T cell proliferation. hSIRPα-Fc-mt10 (SEQ ID NO: 42) was used as a negative control. [Figure 13B] This study demonstrates the efficacy of a heterodimer Fc-fused IL-21 variant in inducing NK-92 cell proliferation. hSIRPα-Fc-mt10 (SEQ ID NO: 42) was used as a negative control. [Figure 14] This study demonstrates the efficacy of a heterodimer Fc-fused IL-21 variant in inducing cytotoxicity in primary NK cells. [Figure 15A] This table shows the treatment plan and administration schedule for heterodimer Fc-fused IL-21 and its variants in the CT26-supported BLAB / c mouse model. [Figure 15B] Figures 15B-15G show the individual tumor growth curves of CT26-supported BALB / c mice treated with the vehicle (Figure 15B), heterodimer Fc-IL21-WT (Figure 15C), heterodimer Fc-IL21-C01 (Figure 15D), heterodimer Fc-IL21-C03 (Figure 15E), heterodimer Fc-IL21-C07 (Figure 15F), and heterodimer Fc-IL21-C08 (Figure 15G), respectively. After grouping (day 0), the mice were injected with heterodimer Fc-fused IL-21 and its variants on days 4, 7, 10, 13, 17, and 20 (indicated by arrows). [Figure 15C] See the explanation in Figure 15B. [Figure 15D] See the explanation in Figure 15B. [Figure 15E] See the explanation in Figure 15B. [Figure 15F] See the explanation in Figure 15B. [Figure 15G] See the explanation in Figure 15B. [Figure 15H] This shows the change in mean body weight of CT26-supported BALB / c mice treated with heterodimer Fc-fused IL-21 and its variants. [Figure 16-1] The amino acid sequences discussed in this disclosure are listed below. [Figure 16-2] The amino acid sequences discussed in this disclosure are listed below. [Figure 16-3] The amino acid sequences discussed in this disclosure are listed below. [Figure 16-4] The amino acid sequences discussed in this disclosure are listed below. [Figure 16-5] The amino acid sequences discussed in this disclosure are listed below. [Figure 16-6] The amino acid sequences discussed in this disclosure are listed below. [Modes for carrying out the invention]

[0043] Detailed explanation Interleukin-21 (also known as IL-21, IL21, Za11, or CVID11) is a multifaceted cytokine composed of four α-helical bundles, primarily affecting natural killer T (NKT) cells and T follicular helpers (T FH IL-21 is produced by TH17 cells and is also produced in low levels by a large number of other lymphopiopoietic cell populations. 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 via its receptor complex, which consists of an inherent chain IL-21Rα and a common chain γC, the latter of which is shared by five other cytokines: IL-2, IL-4, IL-7, IL-9, and IL-15. Together, these cytokines 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 topology of four helical bundles.

[0044] Functional IL-21R is widely expressed in lymphopiohematopoietic populations, including bone marrow cells. Correspondingly, IL-21 exerts its effects on a wide range of cell types. Given the breadth of its immunomodulatory targets and the multifaceted effects of IL-21, IL-21 and IL-21R are attractive targets for therapeutic manipulation. In fact, antibodies against IL-21, IL-21R, and IL-21 antagonists have been developed.

[0045] IL-21 transmits signals via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, and the phosphoinositide 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 more potently and persistently 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), the IL-21 response element was shown to consist of a bifidative element that binds to both interferon regulator 4 (IRF4) and STAT3. Unexpectedly, analysis by chromatin immunoprecipitation (ChIP-seq) linked to next-generation sequencing revealed that such bifidative response elements were found throughout the genome and were collectively involved in the regulation of many IL-21-responsive genes.

[0046] For detailed descriptions of IL-21 and its function, see, 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." Journal of Immunology Research 2015(2015), and Stolfi, C., et al. "Interleukin-21 in cancer immunotherapy: Friend or foe?". Oncoimmunology This can be found in 1.3(2012):351–354 (each of which is incorporated by reference).

[0047] This disclosure provides engineered IL-21 variants having at least one non-native 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, which can form a non-native disulfide bond. In some embodiments, the mutation does not substantially alter the overall structure of IL-21, e.g., the relative positions of the four alpha helices of IL-21. In some embodiments, the non-native 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., phosphorylation of STAT3) and / or proliferation of immune cells (e.g., T cells or NK cells). In some embodiments, the engineered IL-21 variant comprises or consists of any of the engineered IL-21 polypeptides described herein.

[0048] Also provided herein are protein constructs (e.g., fusion proteins) further comprising human serum albumin (HSA) fused to the manipulated IL-21 variant described herein. In some embodiments, the HSA can stabilize the manipulated IL-21 variant. In some embodiments, the protein constructs can promote an immune response (e.g., mitigating Treg-mediated T cell suppression and / or inducing cytotoxicity of primary NK cells).

[0049] Also provided herein are protein constructs (e.g., protein complexes) in which an engineered IL-21 variant (e.g., any of the engineered IL-21 variants described herein) is ligated to the C-terminus of human IgG4 Fc to form a heterodimer (e.g., any of the heterodimeric Fc-fused IL-21 variants described herein). In some embodiments, the protein complex can promote an immune response (e.g., by inducing IL-21-STAT3 signaling, by inducing the proliferation of immune cells, and / or by inducing cytotoxicity of primary NK cells).

[0050] This disclosure also provides a method for screening cytokine (e.g., IL-21) variants that have higher stability and / or improved antitumor efficacy.

[0051] Manipulated IL-21 variant IL-21 is expressed in activated human CD4+ T cells but not in most other tissues. In addition, IL-21 expression is upregulated in Th2 and Th17 subsets of T helper cells, as well as in T follicular cells. Indeed, it has been shown that peripheral T follicular helper cells can be identified using IL-21. Furthermore, IL-21 is expressed in NK T cells, which regulate the function of these cells.

[0052] Human IL-21 contains 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 as SEQ ID NO: 2. The average sequence homology among the γc cytokines IL-21, IL-2, and IL-4 is only about 17%, but the topology of the four helical bundles that form the core structure of these γc cytokines is highly conserved. Specifically, the first alpha helix corresponds to M12-N30 of SEQ ID NO: 2, the second alpha helix corresponds to E48-A58 of SEQ ID NO: 2, the third alpha helix corresponds to N67-K78 of SEQ ID NO: 2, and the fourth alpha helix corresponds to P109-L128 of SEQ ID NO: 2. According to the model of the IL-21 / IL-21R / γc complex, the following residues of IL-21: the amino acids corresponding to positions 12, 16, 19, 23, 105, 114, 118, 121, 122, 124, 125, and 128 of SEQ ID NO: 2 were identified as potentially important positions for IL-21R or γc binding. Specifically, these residues are M12, R16, I19, D23, E105, E114, S118, Q121, K122, I124, H125, and L128 of SEQ ID NO: 2. Details can be found, for example, in 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 (which is incorporated herein by reference in its entirety).

[0053] Based on the 3D structure of human IL-21, the C アルファ atomic distances of one or more amino acid residues (e.g., those in proximity) in the 3D structure can be determined. C アルファTwo residues with atoms within 3–7 angstroms (e.g., 4.5–6.5 angstroms) can be selectively mutated to cysteine ​​without disrupting the overall structure of IL-21 (e.g., the core structure formed by four helical bundles). The newly introduced cysteine ​​is intended to form a non-natural disulfide bond with wild-type IL-21, which can stabilize IL-21 and / or improve its functional potency.

[0054] Therefore, in one embodiment, the engineered IL-21 variant (e.g., engineered IL-21 polypeptide) includes a first cysteine ​​mutation and a second cysteine ​​mutation so that two cysteines can form a non-natural disulfide bond. In some embodiments, the first cysteine ​​mutation occurs at the amino acid residue 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 the amino acid residue 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 two selected amino acid residues アルファ The atoms are within 5 Å, 4.9 Å, 4.8 Å, 4.7 Å, 4.6 Å, 4.5 Å, 4.4 Å, 4.3 Å, 4.2 Å, 4.1 Å, 4 Å, 3.9 Å, 3.8 Å, 3.7 Å, 3.6 Å, 3.5 Å, 3.4 Å, 3.3 Å, 3.2 Å, 3.1 Å, 3 Å, 2.9 Å, 2.8 Å, 2.7 Å, 2.6 Å, 2.5 Å, 2.4 Å, 2.3 Å, 2.2 Å, 2.1 Å, or 2 Å. In some embodiments, one of the first cysteine ​​mutations is C of two selected amino acid residues. アルファ If the atomic distances are within the above range, they can pair with any of the second cysteine ​​mutations described herein. In some embodiments, the non-natural disulfide bond can 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.

[0055] In some embodiments, the engineered IL-21 polypeptide IL21-C01 is provided herein, comprising a first cysteine ​​residue at the position corresponding to position 8 of SEQ ID NO: 2 (e.g., Q8) and a second cysteine ​​residue at the position corresponding to position 86 of SEQ ID NO: 2 (T86). The sequence of IL21-C01 is described in SEQ ID NO: 3.

[0056] In some embodiments, the engineered IL-21 polypeptide IL21-C02 is provided herein, comprising a first cysteine ​​residue at the position corresponding to position 19 of SEQ ID NO: 2 (e.g., I19) and a second cysteine ​​residue at the position corresponding to position 117 of SEQ ID NO: 2 (K117). The sequence of IL21-C02 is described in SEQ ID NO: 4.

[0057] In some embodiments, the engineered IL-21 polypeptide IL21-C03 is provided herein, comprising a first cysteine ​​residue at the position corresponding to position 19 (e.g., V29) of SEQ ID NO: 2 and a second cysteine ​​residue at the position corresponding to position 110 (K110) of SEQ ID NO: 2. The sequence of IL21-C03 is described in SEQ ID NO: 5.

[0058] In some embodiments, the engineered IL-21 polypeptide IL21-C04 is provided herein, comprising a first cysteine ​​residue at the position corresponding to position 31 (e.g., D31) of SEQ ID NO: 2 and a second cysteine ​​residue at the position corresponding to position 62 (S62) of SEQ ID NO: 2. The sequence of IL21-C04 is described in SEQ ID NO: 6.

[0059] In some embodiments, the engineered IL-21 polypeptide IL21-C05 is provided herein, comprising a first cysteine ​​residue at the position corresponding to position 31 (e.g., D31) of SEQ ID NO: 2 and a second cysteine ​​residue at the position corresponding to position 63 (A63) of SEQ ID NO: 2. The sequence of IL21-C05 is described in SEQ ID NO: 7.

[0060] In some embodiments, the engineered IL-21 polypeptide IL21-C06 is provided herein, comprising a first cysteine ​​residue at the position corresponding to position 33 (e.g., V33) of SEQ ID NO: 2 and a second cysteine ​​residue at the position corresponding to position 61 (K61) of SEQ ID NO: 2. The sequence of IL21-C06 is described in SEQ ID NO: 8.

[0061] In some embodiments, the engineered IL-21 polypeptide IL21-C07 is provided herein, comprising a first cysteine ​​residue at the position corresponding to position 33 of SEQ ID NO: 2 (e.g., V33) and a second cysteine ​​residue at the position corresponding to position 63 of SEQ ID NO: 2 (A63). The sequence of IL21-C07 is described in SEQ ID NO: 9.

[0062] In some embodiments, the engineered IL-21 polypeptide IL21-C08 is provided herein, comprising a first cysteine ​​residue at the position corresponding to position 36 of SEQ ID NO: 2 (e.g., F36) and a second cysteine ​​residue at the position corresponding to position 106 of SEQ ID NO: 2 (K106). The sequence of IL21-C08 is described in SEQ ID NO: 10.

[0063] In some embodiments, the engineered IL-21 polypeptide IL21-C09 is provided herein, comprising a first cysteine ​​residue at the position corresponding to position 36 of SEQ ID NO: 2 (e.g., F36) and a second cysteine ​​residue at the position corresponding to position 107 of SEQ ID NO: 2 (K107). The sequence of IL21-C09 is described in SEQ ID NO: 11.

[0064] In some embodiments, the engineered IL-21 polypeptide IL21-C10 is provided herein, comprising a first cysteine ​​residue at the position corresponding to position 39 (e.g., A39) of SEQ ID NO: 2 and a second cysteine ​​residue at the position corresponding to position 105 (E105) of SEQ ID NO: 2. The sequence of IL21-C10 is described in SEQ ID NO: 12.

[0065] In some embodiments, the engineered IL-21 polypeptide IL21-C11 is provided herein, comprising a first cysteine ​​residue at the position corresponding to position 39 (e.g., A39) of SEQ ID NO: 2 and a second cysteine ​​residue at the position corresponding to position 107 (K107) of SEQ ID NO: 2. The sequence of IL21-C11 is described in SEQ ID NO: 13.

[0066] In some embodiments, the engineered IL-21 polypeptide IL21-C12 is provided herein, comprising a first cysteine ​​residue at the position corresponding to position 39 of SEQ ID NO: 2 (e.g., A39) and a second cysteine ​​residue at the position corresponding to position 112 of SEQ ID NO: 2 (F112). The sequence of IL21-C12 is described in SEQ ID NO: 14.

[0067] In some embodiments, the engineered IL-21 polypeptide IL21-C13 is provided herein, comprising a first cysteine ​​residue at the position corresponding to position 56 of SEQ ID NO: 2 (e.g., Q56) and a second cysteine ​​residue at the position corresponding to position 80 of SEQ ID NO: 2 (K80). The sequence of IL21-C13 is described in SEQ ID NO: 15.

[0068] In some embodiments, the following applies: (a) the amino acid corresponding to Q8 in SEQ ID NO: 2 is C, and the amino acid corresponding to T86 in SEQ ID NO: 2 is C; (b) the amino acid corresponding to I19 in SEQ ID NO: 2 is C, and the amino acid corresponding to K117 in SEQ ID NO: 2 is C; (c) the amino acid corresponding to V29 in SEQ ID NO: 2 is C, and the amino acid corresponding to K110 in SEQ ID NO: 2 is C; (d) the amino acid corresponding to D31 in SEQ ID NO: 2 is C, and the amino acid corresponding to S62 in SEQ ID NO: 2 is C; (e) the amino acid corresponding to D31 in SEQ ID NO: 2 is C, and the amino acid corresponding to A63 in SEQ ID NO: 2 is C; (f) the amino acid corresponding to V33 in SEQ ID NO: 2 is C, and the amino acid corresponding to K61 in SEQ ID NO: 2 is C; (g) the amino acid corresponding to V33 in SEQ ID NO: 2 is C, and the amino acid corresponding to A63 in SEQ ID NO: 2 is C The following modified IL-21 polypeptides are provided herein, comprising one or more of the following: (h) the amino acid corresponding to F36 in SEQ ID NO: 2 is C, and the amino acid corresponding to K106 in SEQ ID NO: 2 is C, (i) the amino acid corresponding to F36 in SEQ ID NO: 2 is C, and the amino acid corresponding to K107 in SEQ ID NO: 2 is C, (j) the amino acid corresponding to A39 in SEQ ID NO: 2 is C, and the amino acid corresponding to E105 in SEQ ID NO: 2 is C, (k) the amino acid corresponding to A39 in SEQ ID NO: 2 is C, and the amino acid corresponding to K107 in SEQ ID NO: 2 is C, (l) the amino acid corresponding to A39 in SEQ ID NO: 2 is C, and the amino acid corresponding to F112 in SEQ ID NO: 2 is C, and (m) the amino acid corresponding to Q56 in SEQ ID NO: 2 is C, and the amino acid corresponding to K80 in SEQ ID NO: 2 is C.

[0069] 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 of the amino acids corresponding to positions 12, 16, 19, 23, 105, 114, 118, 121, 122, 124, 125, and 128 of SEQ ID NO: 2 in the manipulated IL-21 polypeptide are not mutated. For example, in some embodiments, the amino acid corresponding to position 12 (e.g., M12) of SEQ ID NO: 2 in the manipulated IL-21 polypeptide described herein is Met, the amino acid corresponding to position 16 (e.g., R16) of SEQ ID NO: 2 in the manipulated IL-21 polypeptide described herein is Arg, the amino acid corresponding to position 19 (e.g., I19) of SEQ ID NO: 2 in the manipulated IL-21 polypeptide described herein is Ile, the amino acid corresponding to position 23 (e.g., D23) of SEQ ID NO: 2 in the manipulated IL-21 polypeptide described herein is Asp, the amino acid corresponding to position 105 (e.g., E105) of SEQ ID NO: 2 in the manipulated IL-21 polypeptide described herein is Glu, and the sequence number in the manipulated IL-21 polypeptide described herein is The amino acid corresponding to position 114 of SEQ ID NO. 2 (e.g., E114) is Glu, the amino acid corresponding to position 118 of SEQ ID NO. 2 in the manipulated IL-21 polypeptide described herein (e.g., Q121) is Gln, the amino acid corresponding to position 122 of SEQ ID NO. 2 in the manipulated IL-21 polypeptide described herein (e.g., K122) is Lys, the amino acid corresponding to position 124 of SEQ ID NO. 2 in the manipulated IL-21 polypeptide described herein (e.g., I124) is Ile, the amino acid corresponding to position 125 of SEQ ID NO. 2 in the manipulated IL-21 polypeptide described herein (e.g., H125) is His, and / or the amino acid corresponding to position 128 of SEQ ID NO. 2 in the manipulated IL-21 polypeptide described herein (e.g., L128) is Leu.

[0070] In some embodiments, the manipulated IL-21 polypeptide contains or consists of amino acid sequences that are 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 NOs. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the manipulated IL-21 polypeptide described herein comprises or consists of 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: 1 or SEQ ID NO: 2, the amino acid sequence comprising one or more of the mutations described herein. In some embodiments, the manipulated IL-21 polypeptide described herein comprises at least one, at least two, at least three, at least four, or at least five pairs of cysteine ​​mutations as listed in Table 1.

[0071] In some embodiments, the manipulated IL-21 polypeptide may have at least or about one (e.g., at least or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40) amino acid insertions, deletions, or substitutions compared to any one of SEQ ID NOs. 2-15.

[0072] The manipulated IL-21 polypeptide may have additional modifications. In some embodiments, the manipulated IL-21 polypeptide may have a CH2 domain and / or a CH3 domain of Fc. In some embodiments, the manipulated IL-21 polypeptide 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 manipulated IL-21 polypeptide 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).

[0073] In some embodiments, the manipulated IL-21 polypeptides described herein may also include a tag (e.g., a His tag) to facilitate screening and / or detection. In some embodiments, the tag has a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs. In some embodiments, the tag is attached to the N-terminus or C-terminus of any manipulated IL-21 polypeptide described herein.

[0074] In some embodiments, the manipulated IL-21 polypeptide described herein can be expressed in Expi293 or CHO-S cells.

[0075] IL-21 protein construct This disclosure provides protein constructs (e.g., fusion proteins or protein complexes) comprising an engineered IL-21 variant as described herein (e.g., any of the engineered IL-21 polypeptides described herein). In some embodiments, the protein construct further comprises human serum albumin (HSA) fused to the engineered IL-21 polypeptide. In some embodiments, the fusion protein can be expressed in Expi293 or CHO-S cells. In some embodiments, the HSA is fused to the engineered IL-21 polypeptide via a linker peptide (e.g., a flexible linker). In some embodiments, the HSA is ligated to the N-terminus of the engineered IL-21 polypeptide via the linker peptide. In some embodiments, the HSA is ligated to the C-terminus of the engineered IL-21 polypeptide via the linker peptide. In some embodiments, the protein constructs described herein have an N-terminal His tag. In some embodiments, the protein constructs described herein have a C-terminal His tag.

[0076] In some embodiments, the protein constructs described herein include, in the direction 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 of the engineered IL-21 polypeptides described herein). In some embodiments, the His tag includes at least six, at least seven, or at least eight consecutive His residues. In some embodiments, the His tag includes 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 includes 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 linker peptide comprises at least one, two, three, four, five, six, seven, or eight repeats of GGGGS (SEQ ID NO: 27). In some embodiments, the linker peptide is a flexible linker. Details of flexible linkers can be found, for example, in Chen, X., et al. “Fusion protein linkers: property, design and functionality.” Advanced Drug Delivery Reviews 65.10(2013):1357-1369 (the entire text is incorporated herein by reference). In some embodiments, the linker 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 manipulated IL-21 polypeptide contains or consists of 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 NOs.In some embodiments, the manipulated IL-21 polypeptides described herein include at least one, at least two, at least three, at least four, or at least five pairs of cysteine ​​mutations as listed in Table 1.

[0077] In some embodiments, the manipulated IL-21 protein construct may include any manipulated IL-21 variant described herein. In some embodiments, the protein construct described herein contains or comprises 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 NOs.

[0078] In some embodiments, protein constructs are provided herein that include a His tag (e.g., any of the His tags described herein) and an engineered IL-21 polypeptide (e.g., any of the engineered IL-21 polypeptides described herein) in the direction from the N-terminus to the C-terminus. In some embodiments, the protein constructs described herein include or consist of 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.

[0079] The disclosure also provides nucleic acids comprising polynucleotides encoding a polypeptide containing a sequence that is 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 to any sequence of SEQ ID NOs.

[0080] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Then, amino acid residues or nucleotides are compared at their corresponding amino acid or nucleotide positions. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of those two sequences and the length of each gap. For example, the comparison of sequences and determination of the percentage of identity between two sequences can be achieved using a Blossum62 scoring matrix with a 12-gap penalty, a 4-gap expansion penalty, and a 5-frameshift gap penalty.

[0081] The manipulated IL-21 variant (e.g., any of the manipulated IL-21 polypeptides described herein) and protein constructs may further comprise the Fc region of an antibody. These antibodies may be of 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).

[0082] In some embodiments, the manipulated IL-21 variant is linked to the Fc region via an antibody hinge region (e.g., an IgG or IgE hinge region). In addition, the Fc region can be modified to provide a desired effector function or serum half-life.

[0083] In some embodiments, the protein constructs described herein include a functional Fc. 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 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 ADCC and phagocytosis. In some embodiments, the protein constructs described herein have an Fc region without effector function. In some embodiments, Fc is human IgG4 Fc. In some embodiments, Fc does not have a functional Fc region. For example, the Fc region has an LALA mutation (L234A and L235A mutations in EU numbering) or an LALA-PG mutation (L234A, L235A, P329G mutations in EU numbering).

[0084] In some embodiments, the manipulated IL-21 variant (e.g., any of the manipulated IL-21 variants described herein) is ligated to the N-terminus or C-terminus of the Fc region. In some embodiments, the manipulated IL-21 variant is ligated to the Fc region via a linker peptide (e.g., any of the linker peptides described herein).

[0085] In some embodiments, protein constructs are provided herein that, from the N-terminus to the C-terminus, comprise a human IgG4 hinge region and Fc region (e.g., SEQ ID NO: 28), a linker peptide (e.g., any of the linker peptides described herein), and an engineered IL-21 polypeptide (e.g., any of the engineered IL-21 polypeptides described herein). In some embodiments, the engineered IL-21 polypeptide comprises or consists of a 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 any one of SEQ ID NOs: 2-15. In some embodiments, the protein constructs described herein contain or consist of 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.

[0086] Several other modifications to the Fc region can be made. For example, a cysteine ​​residue can be introduced into the Fc region, thereby enabling interchain disulfide bond formation in this region. The homodimeric fusion protein thus produced may have any increased half-life in vitro and / or in vivo. In some embodiments, IgG4 has the S228P mutation (EU numbering). The S228P mutation prevents IgG4 Fab-arm exchange in vivo and in vitro.

[0087] In some embodiments, the Fc region has a carbohydrate structure lacking fucose (directly or indirectly) bound to the Fc region. For example, the amount of fucose in such an Fc region composition 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 in the glycans at Asn297 for the sum of all sugar structures (e.g., complexes, hybrids, and high-mannose structures) bound to Asn297, measured by MALDI-TOF mass spectrometry, as described, for example, in WO2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (position 314 in the EU numbering or Kabat numbering of the Fc region residue), although slight sequence variations in the Fc region sequence may also cause Asn297 to be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. In some embodiments, to reduce glycan heterogeneity, the Fc region can be further manipulated to replace the asparagine at position 297 with alanine (N297A).

[0088] In some embodiments, this disclosure relates to protein constructs comprising the engineered IL-21 polypeptides described herein. In some embodiments, the protein construct comprises two or more engineered IL-21 polypeptides. In some embodiments, at least two of the engineered IL-21 polypeptides are identical. In some embodiments, at least two of the engineered IL-21 polypeptides 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 an LALA mutation or an LALA-PG mutation). In some embodiments, the engineered IL-21 polypeptide is ligated to the C-terminus of the Fc region. In some embodiments, the engineered IL-21 polypeptide is ligated to the C-terminus of the Fc region via a linker peptide (e.g., one of the linker peptides described herein).

[0089] This disclosure provides protein complexes comprising an engineered IL-21 variant as described herein (e.g., any of the engineered IL-21 polypeptides 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 comprises, or comprises, an optionally selected first hinge region (e.g., a human IgG4 hinge region), a first Fc region (e.g., a human IgG4 Fc region), an optionally selected linker peptide (e.g., any of the linker peptides described herein), and an engineered IL-21 polypeptide (e.g., any of the engineered IL-21 polypeptides described herein), optionally from the N-terminus to the C-terminus. In some embodiments, the second polypeptide comprises, or comprises, an optionally selected second hinge region (e.g., a human IgG4 hinge region) and a second Fc region (e.g., a human IgG4 Fc region), optionally from the N-terminus to the C-terminus. In some embodiments, the first Fc region and / or the second Fc region described herein does not include a hinge region. In some embodiments, the first Fc region and / or the second Fc region described herein consists of a CH2 domain (e.g., a human IgG4 CH2 domain) and a CH3 domain (e.g., a human IgG4 CH3 domain).

[0090] In some embodiments, the first and / or second hinge regions include all or part of an immunoglobulin hinge region, for example, the human IgG4 hinge region (SEQ ID NO: 31). In some embodiments, the first and / or second hinge regions include an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 31. In some embodiments, the first and second hinge regions are identical. In some embodiments, the first and second hinge regions are different.

[0091] In some embodiments, the first and / or second Fc regions are identical and can form an Fc homodimer. In some embodiments, the first and / or second Fc regions include all or part of an immunoglobulin Fc region, e.g., the human IgG4 Fc region (SEQ ID NO: 40). In some embodiments, the first and / or second Fc regions are different. In some embodiments, the first and / or second Fc regions can form an Fc heterodimer by introducing one or more mutations. In some cases, the first and / or second Fc regions can contain one or more knob-into-hole (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 numbering, and the second Fc region may contain cysteine ​​at position 354 and tryptophan at position 366 according to EU numbering. In some embodiments, the first Fc region includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 33, and the second Fc region includes an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 32. In some embodiments, the first and / or second Fc regions can form an Fc heterodimer using other techniques. Details of KIH mutations and other heterodimer Fc technologies can be found in Ha, et al. “Immunoglobulin Fc heterodimer platform technology: from design to applications in therapeutic antibodies and proteins.” Frontiers In Immunology 7(2016):394 (the entire article is incorporated herein by reference). In some embodiments, the first and / or second Fc regions described herein are derived from human IgG (e.g., IgG1, IgG2, IgG3, or IgG4).In some embodiments, the first and / or second Fc region is an IgG4 Fc region (e.g., a human IgG4 Fc region). In some embodiments, the first and / or second Fc region is an IgG Fc region (e.g., a human IgG1 Fc region) whose effector function is silenced. Details of methods for modulating Fc effector function can be found, for example, in 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.” Front Immunol 10:1296.” 2019 (the entire text of which is incorporated herein by reference).

[0092] In some embodiments, the linker peptides described herein include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 34. In some embodiments, the linker peptides described herein include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) repeats of GGGGS (SEQ ID NO: 41).

[0093] In some embodiments, the first polypeptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 30, 35, 36, 37, 38, or 39, and the second polypeptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 29.

[0094] Characterization of manipulated IL-21 variants or protein constructs In some embodiments, the manipulated IL-21 variants described herein (e.g., any of the manipulated IL-21 polypeptides described herein) or their protein constructs (e.g., fusion proteins or protein complexes) can bind to a complex formed by the human IL-21 receptor (IL-21R) and the common cytokine γ chain (γc). The newly introduced non-native disulfide bond can stabilize IL-21 (e.g., the core structure formed by four helical bundles) without causing substantial conformational changes to the protein structure, and since the residues crucial for IL-21R or γc binding (e.g., M12, R16, I19, D23, E105, E114, S118, Q121, K122, I124, H125, and / or L128 in SEQ ID NO: 2) are nonmutated, the engineered IL-21 variant or its protein construct can induce downstream signaling pathways (e.g., 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).

[0095] In some embodiments, the introduction of a non-natural disulfide bond or cysteine ​​mutation (e.g., any of the cysteine ​​mutations described herein) may result in protein conformational changes with RMSD (Mean Squared Deviation of Atomic Position) values ​​of less than 10 Å, less than 9 Å, less than 8 Å, less than 7 Å, less than 6 Å, less than 5 Å, less than 4 Å, less than 3 Å, less than 2 Å, or less than 1 Å. In some embodiments, the RMSD value is calculated by structurally aligning the wild-type protein and the protein variant. In some embodiments, to determine the conformational change, C アルファ Only atoms are used.

[0096] The biological effects of manipulated IL-21 variants or their protein constructs include immunoenhancing and immunomodulatory effects. Exemplary immunoenhancing effects include, for example, increased immune response, increased B cell proliferation, induction of plasma cell differentiation, increased immunoglobulin production, and increased T follicular helper (T) cell proliferation. FH Examples of immunomodulatory effects include increased cell differentiation and / or proliferation, increased proliferation, survival, and / or antitumor activity of cytotoxic T lymphocytes (CTLs), increased CD28 and L-selectin expression, increased proliferation, antitumor activity, and / or ADCC activity of NK cells, increased differentiation, proliferation, and / or IL-23R expression by Th17 cells, and inhibition of Treg cell survival and / or production. Exemplary immunomodulatory effects include, for example, suppression of differentiation, proliferation, and / or IL-10 production by type 1 regulatory T (Tr1) cells, suppression of apoptosis and / or APC function of dendritic cells (DC cells), and suppression of differentiation, proliferation, and / or IL-10 production by B-10 or B-regulatory (Breg) cells. Further details can be found, for example, in Croce, M. et al. “IL-21: a pleiotropic cytokine with potential applications in oncology.” Journal of Immunology Research 2015 (2015) (the entire article is incorporated herein by reference).

[0097] In some embodiments, the engineered IL-21 variants described herein (e.g., any of the engineered IL-21 polypeptides described herein) or their protein constructs (e.g., any of the HSA-fused IL-21 variants described herein or the heterodimer Fc-fused IL-21 variants described herein) have equivalent efficacy in inducing a T cell response. In some embodiments, the engineered IL-21 variants or their protein constructs can 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% of the efficacy of wild-type IL-21 or its protein constructs. In some embodiments, the EC50 value of the growth curve can be determined, for example, to 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.

[0098] In some embodiments, the manipulated IL-21 variants or their protein constructs described herein (e.g., either the HSA-fused IL-21 variant or the heterodimer Fc-fused IL-21 variant described herein) can induce 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% of the potency of wild-type IL-21 or its protein constructs. In some embodiments, the EC50 value of the proliferation curve can 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 values ​​of the growth curves of activated CD8+ T cells treated with the manipulated IL-21 variant or its protein construct described herein are less than 50%, less than 40%, less than 30%, or less than 20% of the growth curves of activated CD8+ T cells treated with wild-type IL-21 or its protein construct.

[0099] In some embodiments, the manipulated IL-21 variants described herein include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NOs. 3, 5, 9, 10, or 13. In some embodiments, the protein constructs described herein (e.g., HSA-fused IL-21 variants) include an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NOs. 17, 18, 19, 20, or 21. In some embodiments, the protein constructs (e.g., heterodimer Fc-fused IL-21 variants) include a first polypeptide having an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NOs. 35, 36, 37, 38, or 39, and a second polypeptide having an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NOs. 29.

[0100] In some embodiments, the manipulated IL-21 variants or their protein constructs described herein (e.g., either the HSA-fused IL-21 variant or the heterodimer Fc-fused IL-21 variant described herein) can induce proliferation of NK cells (e.g., NK-92 cells) 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% of the potency of wild-type IL-21 or its protein constructs. In some embodiments, NK cell proliferation is determined after at least 12, 18, 24, 30, or 36 hours of starvation. In some embodiments, the EC50 value of the growth curve can be determined, for example, to 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 growth curve can be determined, for example, to less than 8 nM, less than 7 nM, less than 6 nM, or less than 5 nM. In some embodiments, the EC50 value of the growth curve of NK cells treated with the manipulated IL-21 variant or its protein construct described herein is less than 90%, less than 80%, less than 70%, or less than 60% of that of NK cells treated with wild-type IL-21 or its protein construct.

[0101] In some embodiments, the manipulated IL-21 variants or their protein constructs described herein (e.g., either the HSA-fused IL-21 variant or the heterodimer Fc-fused IL-21 variant described herein) can mitigate Treg-mediated repression of T cells (e.g., activated CD8+ T cells). In some embodiments, treatment of Treg cells with the manipulated IL-21 variants or their protein constructs described herein can enhance T cell proliferation (e.g., activated CD8+ T cells) 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 constructs.

[0102] In some embodiments, the manipulated IL-21 variants or their protein constructs described herein (e.g., either the HSA-fused IL-21 variant or the heterodimer Fc-fused IL-21 variant described herein) can induce cytotoxicity in NK cells (e.g., primary NK cells) with about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, or about 140% of the potency of wild-type IL-21 or its protein constructs. In some embodiments, the rate of specific lysis of NK cells against target tumor cells (e.g., K-562 tumor cells) can be enhanced 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 construct.

[0103] In some embodiments, the manipulated IL-21 variants or protein constructs described herein can increase the immune response, the 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%, 2x, 3x, 5x, 10x, or 20x.

[0104] In some embodiments, the manipulated IL-21 variant or its protein construct is used for 0.1 seconds. -1 Less than 0.01s -1 Less than 0.001s -1 Less than 0.0001s -1 Less than, or 0.00001s -1 Dissociation rate less than (k off It can bind to IL-21R, γc, or their complex (e.g., human IL-21R / γc complex) at a dissociation rate (k). In some embodiments, the dissociation rate (k) is used. off ) is 0.01s -1 Super, 0.001s -1 Super, 0.0001s-1 Super, 0.00001s -1 Greater than, or 0.000001s -1 It's incredible.

[0105] In some embodiments, the dynamic association rate (k on ) is 1 × 10 2 / Ms super, 1×10 3 / Ms super, 1×10 4 / Ms super, 1×10 5 / Ms greater than, or 1 × 10⁻⁶ 6 In some embodiments, the dynamic association rate (k on ) is 1 × 10 5 / Ms less than 1 × 10 6 Less than / Ms, or 1 × 10⁻⁶ 7 It is less than / Ms.

[0106] Affinity is the dynamic rate constant (KD=k off / k on It can be estimated from the quotient of ). In some embodiments, KD is 1 × 10 -6 Less than M, 1 x 10 -7 Less than M, 1 x 10 -8 Less than M, 1 x 10 -9 Less than M, or 1 × 10 -10 It is less than M. In some embodiments, KD is 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 less than 10 pM. In some embodiments, KD is 1 × 10 -7 Super M, 1×10 -8 Super M, 1×10 -9 Super M, 1×10 -10 Super M, 1×10 -11 M or 1 × 10 -12 It is greater than M.

[0107] 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.

[0108] In some embodiments, the manipulated IL-21 protein construct described herein (e.g., any of the HSA-fused IL-21 variants described herein) can be expressed and purified by methods commonly used in the art (e.g., affinity chromatography). In some cases, the protein construct can be purified by size-exclusive chromatography (SEC) in combination with HPLC. In some embodiments, the percentage of major peaks 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 percentage of high molecular weight peaks (HMW%) and / or low molecular weight peaks (LMW%) is less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0109] In some embodiments, the thermal stability of the manipulated IL-21 variants or their protein constructs described herein (e.g., any of the HSA-fused IL-21 variants described herein) is determined. The manipulated IL-21 variants and their protein constructs described herein may have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95°C. In some embodiments, Tm is 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or less than 95°C. In some embodiments, aggregation (T) of the manipulated IL-21 variants and their protein constructs as described herein. agg ) and start (T onset The temperature can be measured by applying a heat lamp (e.g., 25-85°C) based on DLS / SLS. DLS / SLS is a well-known technique for determining sample interactions, particle size, and aggregation of molecules dispersed or dissolved in solution. (T) onset ) and aggregation (T agg The temperature at which the manipulated IL-21 variant or its protein construct described herein experiences is considered an important predictor of stability. In some embodiments, the T agg The temperature is at least 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65°C. In some embodiments, the temperature of the manipulated IL-21 variant or its protein construct described herein is T onset The temperature is at least 59, 60, 61, 62, 63, 64, 65, or 66°C.

[0110] In some embodiments, cysteine ​​mutations (e.g., any of the cysteine ​​mutations described herein or any combination thereof) cause aggregation (T agg ) and / or commencement (Tonset The temperature can be raised 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.

[0111] The radii of the manipulated IL-21 variants or their protein constructs described herein (e.g., any of the HSA-fused IL-21 variants described herein) can also be determined by DSL / SLS. In some embodiments, under isothermal conditions (e.g., at about 25°C), the radii are measured to be 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 thermolamp conditions (for example, with a heat lamp at 25-85°C), the radius is measured to be approximately 1-20 nm, approximately 3-15 nm, approximately 5-13 nm, approximately 5-10 nm, approximately 5-8 nm, approximately 5-7 nm, approximately 5-6 nm, approximately 6-13 nm, approximately 6-10 nm, approximately 6-7 nm, approximately 7-13 nm, approximately 10-13 nm, or approximately 12-13 nm.

[0112] The polydispersity (%PD) of the manipulated IL-21 variants or their protein constructs described herein (e.g., any of the HSA-fused IL-21 variants described herein) can also be determined by DSL / SLS. In some embodiments, the %PD is less than about 30%, less than about 28%, less than about 25%, less than about 20%, less than about 15%, less than about 13%, less than about 10%, less than about 8%, or less than 5% under isothermal conditions (e.g., about 25°C).

[0113] The molecular weight (MW-S) of the manipulated IL-21 variants or their protein constructs described herein (e.g., any of the HSA-fused IL-21 variants described herein) can also be determined by DLS / SLS. In some embodiments, under isothermal conditions (e.g., at about 25°C) or thermolamp conditions (e.g., with a heat lamp at 25-85°C), MW-S is about 50-200kDa, about 50-130kDa, about 50-100kDa, about 50-90kDa, about 50-80kDa, about 50-70kDa, about 60-100kDa, about 60-90kDa, about 60-80kDa, about 70-100kDa, about 70-90kDa, about 70-80kDa, about 80-100kDa, about 90-100kDa, about 100-200kDa, about 100-150kDa, or about 150-200kDa.

[0114] In some embodiments, the manipulated IL-21 variants or their protein constructs described herein (e.g., either the HSA-fused IL-21 variant or the heterodimer Fc-fused IL-21 variant described herein) can inhibit tumor growth when administered, for example, to tumor-carrying animals. In some cases, the manipulated IL-21 variants or their protein constructs have a tumor growth inhibition rate (TGI%) of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or more than 200%. In some embodiments, the manipulated IL-21 variants or their protein constructs described herein have a percentage of tumor growth inhibition of less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, or 200%. The TGI% can be determined, for example, 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 after the start of treatment. When used herein, the percentage of tumor growth inhibition (TGI%) is given by the following formula: TGI(%)=[1-Average(T final -T initial ) / Average(C final -C initial )] × 100 It is calculated using

[0115] T final This represents the mean tumor volume in the treatment group on the final day. initial This is the mean tumor volume in the treatment group on day 0. final This represents the average tumor volume in the control group on the final day. initial This represents the mean tumor volume in the control group on day 0.

[0116] In some embodiments, the TGI% of the manipulated 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 that of wild-type IL-21 or its protein construct.

[0117] In some embodiments, the proportion of tumor-bearing mice treated with the manipulated IL-21 variant or its protein construct described herein (e.g., either the HSA-fused IL-21 variant or the heterodimer Fc-fused IL-21 variant described herein) having a tumor volume less than half the average tumor volume of untreated tumor-bearing mice 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 total number of mice inoculated on day 0, 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. In some embodiments, the above proportions are at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the total number of surviving mice on the same day.

[0118] In some embodiments, the survival rate of tumor-bearing mice after treatment with the manipulated IL-21 variant or its protein construct described herein (e.g., either the HSA-fused IL-21 variant or the heterodimer Fc-fused IL-21 variant described herein) is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% 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.

[0119] In some embodiments, the manipulated IL-21 variants or their protein constructs described herein (e.g., either the HSA-fused IL-21 variant or the heterodimer Fc-fused IL-21 variant described herein) can induce a robust tumor-specific memory response. In some embodiments, the manipulated IL-21 variants or their protein constructs described herein can inhibit tumor growth after tumor rechallenge (e.g., reinoculation with the same or different tumor cells) 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.

[0120] In some embodiments, the body weight of tumor-bearing mice after treatment with the manipulated IL-21 variant or its protein construct described herein (e.g., either the HSA-fused IL-21 variant or the heterodimer Fc-fused IL-21 variant described herein) is at least 70%, at least 80%, or at least 90% of the body weight of tumor-bearing mice after treatment with wild-type IL-21 or its protein construct.

[0121] In some embodiments, the half-life of the manipulated IL-21 variant or its protein construct described herein can 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 construct. In some embodiments, the half-life is determined by measuring the in vivo concentration of the molecule over time after administration to the subject.

[0122] Method for producing manipulated IL-21 variants and protein constructs The manipulated IL-21 variants or protein constructs described herein can be prepared by introducing appropriate nucleotide changes into 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 in the amino acid sequence. In some embodiments, selective cysteine ​​mutations are C アルファ Atoms can be introduced into one or more pairs of residues within 3–7 Å-angstroms of the human IL-21 3D structure. In some embodiments, the human IL-21 3D structure has a PDB (Protein Databank) ID of 3TGX.

[0123] Screening can be performed. In a population of such variants, several engineered IL-21 variants can be expressed and purified using methods known in the art. In addition, the efficacy of several engineered IL-21 variants on T cell response can be determined by STAT-3 phosphorylation and / or T cell proliferation. Based on the above experimental results, several engineered IL-21 variants with good expression / purification profiles and equivalent T cell response ability can be selected to generate HSA-fused IL-21 variants (e.g., any of the HSA-fused IL-21 variants described herein).

[0124] HSA-fused IL-21 variants can be screened. 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-native disulfide bonds formed by cysteine ​​mutations in the manipulated IL-21 variant and / or HSA fusion can stabilize the HSA-fused protein. For example, some HSA-fused IL-21 variants show increased aggregation (T) compared to HSA-fused wild-type IL-21. agg ) and start (T onset ) May have a temperature.

[0125] In addition, the potency of the protein constructs described herein (for example, either the HSA-fused IL-21 variant or the heterodimer Fc-fused IL-21 variant described herein) can be determined, for example, by STAT-3 phosphorylation, T cell proliferation, and / or NK cell proliferation. In particular, some HSA-fused IL-21 variants may have similar or relatively weaker potency than HSA-fused wild-type IL-21 in inducing STAT-3 phosphorylation and T cell proliferation. Some HSA-fused IL-21 variants may have similar or relatively weaker potency in inducing NK cell proliferation compared to HSA-fused wild-type IL-21. Some heterodimer Fc-fused IL-21 variants may have similar potency to heterodimer Fc-fused wild-type IL-21 in inducing STAT-3 phosphorylation. Some heterodimer Fc-fused IL-21 variants may have better potency than heterodimer Fc-fused wild-type IL-21 in inducing T cell proliferation and / or NK cell proliferation.

[0126] Furthermore, the efficacy of the protein constructs described herein (e.g., either the HSA-fused IL-21 variant or the heterodimer Fc-fused IL-21 variant described herein) in mitigating Treg-mediated T cell suppression can be determined. In particular, some HSA-fused IL-21 variants may show a significant enhancement in mitigating Treg-mediated T cell suppression compared to HSA-fused wild-type IL-21. The efficacy of HSA-fused IL-21 variants in inducing NK cell cytotoxicity can also be determined. In particular, some HSA-fused IL-21 variants may have similar or relatively weaker efficacy in inducing NK cell cytotoxicity compared to HSA-fused wild-type IL-21. Some heterodimer Fc-fused IL-21 variants may have slightly better efficacy in inducing NK cell cytotoxicity than heterodimer Fc-fused wild-type IL-21.

[0127] Based on the experimental results regarding the above-mentioned HSA-fused IL-21 variants, several HSA-fused IL-21 variants possessing favorable expression / purification profiles, comparable immune cell response capabilities, enhanced potency replicating Treg-mediated T cell suppression, and / or comparable or reduced potency in inducing NK cell cytotoxicity can be selected, and their antitumor efficacy and in vivo toxicity can be evaluated, for example, in tumor-bearing animal models.

[0128] Based on the experimental results regarding the heterodimer Fc-fusion IL-21 variants described above, several heterodimer Fc-fusion IL-21 variants with equivalent or better immune cell response and / or equivalent or better efficacy in inducing NK cell cytotoxicity can be selected, and their antitumor efficacy and in vivo toxicity can be evaluated, for example, in tumor-bearing animal models.

[0129] In some embodiments, the manipulated IL-21 variant or its protein construct (e.g., either the HSA-fused IL-21 variant or the heterodimer Fc-fused IL-21 variant described herein) may have equivalent or increased affinity for the IL-21R / γc complex. Any combination of deletions, insertions, and / or combinations can be performed to arrive at variants with increased binding affinity for the binding partner (e.g., IL-21R). Amino acid changes introduced into the variant can also modify or introduce new post-translational modifications to the polypeptide, such as changing the number of glycosylation sites (e.g., increasing or decreasing them), changing the type of glycosylation site (e.g., altering the amino acid sequence so that different sugars are bound by enzymes present in the cell), or introducing new glycosylation sites.

[0130] The manipulated IL-21 variant can originate from any animal species, including mammals. Non-limiting examples of IL-21 variants include those derived from humans, primates (e.g., monkeys and apes), cattle, pigs, horses, sheep, camelids (e.g., camels and llamas), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits).

[0131] This disclosure also provides a recombinant vector (e.g., an expression vector) containing an isolated polynucleotide (e.g., a polynucleotide encoding a polypeptide disclosed herein), a host cell into which the recombinant vector is introduced (i.e., such that the host cell contains the polynucleotide and / or the polynucleotide-containing vector), and the production of recombinant polypeptides or fragments thereof by recombinant technology.

[0132] As used herein, “vector” is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. “Expression vector” can deliver and express one or more polynucleotides of interest as a polypeptide encoded in the host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotides of interest are positioned to be expressed in the vector by being operably ligated to regulatory elements such as promoters, enhancers, and / or poly-A tails, either within the vector or in the genome of the host cell, at, near, or adjacent to an integration site of the polynucleotides of interest, such that the polynucleotides of interest are translated in the host cell into which the expression vector has been introduced.

[0133] Vectors can be introduced into host cells by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and / or transduction (e.g., with recombinant viruses). Therefore, non-limiting examples of vectors include viral vectors (which can be used to generate recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors conjugated with cationic condensants.

[0134] 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 poxvirus, retrovirus, or adenovirus), which may involve the use of non-pathogenic (defective) replicating viruses or replicating-deficient viruses. Techniques for incorporating DNA into such expression systems are well known to those skilled in the art. The DNA may also be “naked.” The uptake of naked DNA can be increased by coating the DNA onto biodegradable beads that are efficiently transported into cells.

[0135] For expression, DNA inserts containing polypeptide-coding polynucleotides disclosed herein can be operably ligated to suitable promoters (e.g., heterologous promoters), such as, to name a few, the phage-lambda PL promoter, the Escherichia coli (E. coli) lac, trp, and tac promoters, the SV40 early and late promoters, and the retroviral LTR promoter. Other suitable promoters are known to those skilled in the art. In some embodiments, the promoter is the cytomegalovirus (CMV) promoter. In some embodiments, the promoter is a human promoter, such as the uHS or HS promoter. Human promoters can enhance the expression of human-derived proteins. Details of such human promoters can be found, for example, in 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 chromatin opening element (UCOE) confers resistance to DNA methylation-mediated silencing of lentiviral vectors.” Molecular Therapy 18.9 (2010):1640-1649 (the entire text is incorporated herein by reference). The expression construct may further contain sites for transcription initiation, termination, and ribosome-binding sites for translation within the transcriptional region. The coding portion of the mature transcript expressed by the construct may include a translation initiation and a stop codon (UAA, UGA, or UAG) appropriately positioned at the end of the polypeptide being translated.

[0136] As shown, the expression vector may contain at least one selection marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in Escherichia coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium cells, fungal cells such as yeast cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as CHO, COS, Bowes melanoma, and HEK293 cells, and plant cells. Suitable culture media and conditions for the host cells described herein are known in the art.

[0137] Non-restrictive vectors for bacterial use include pQE70, pQE60, and pQE-9 from Qiagen, pBS vector, Phagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A, and pNH46A from Stratagene, and ptrc99a, pKK223-3, pKK233-3, pDR540, and pRIT5 from Pharmacia. Non-restrictive eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG from Stratagene, and pSVK3, pBPV, pMSG, and pSVL from Pharmacia. Other suitable vectors will readily become apparent to those skilled in the art.

[0138] Suitable non-limiting bacterial promoters for use include the E. coli lacI and lacZ promoters, T3 and T7 promoters, gpt promoter, lambda PR and PL promoters, and trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, HSV thymidine kinase promoter, early and late SV40 promoters, retroviral LTR promoters such as those for Rous sarcoma virus (RSV), and metallothionein promoters such as the mouse metallothionein-I promoter.

[0139] In the yeast Saccharomyces cerevisiae, numerous vectors containing alpha factor, alcohol oxidase, and constitutive or inducible promoters such as PGH can be used.

[0140] The introduction of constructs into host cells may be influenced by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which are incorporated herein by reference in their entirety.

[0141] In some embodiments, the host cells are human cells suitable for protein expression, e.g., HEK293 cells or CHO cells (e.g., CHO-S cells). In some embodiments, the host cells are Expi293 cells. The Expi293 expression system is designed to deliver up to six times more protein in just one week, compared to other transient 293 expression systems that may take two weeks or more. This is partly due to the fact that Expi293F cells are adapted to achieve higher pg / cell / day productivity than standard HEK293 cells, and that Expifectamine293 transfection reagents and enhancers enable highly efficient transfection and expression levels of high-density HEK293 cultures. Furthermore, the Expi293 expression system requires less plasticware, which means less waste and more incubator space.

[0142] Transcription of the DNA encoding the polypeptides of this disclosure by higher eukaryotes can be increased by inserting enhancer sequences into vectors. Enhancers are cis-acting elements of DNA, typically about 10–300 bp in length, that act to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include SV40 enhancers located 100–270 base pairs behind the origin of replication, cytomegalovirus early promoter enhancers, polyoma enhancers behind the origin of replication, and adenovirus enhancers.

[0143] Appropriate secretory signals can be incorporated into the expressed polypeptide to secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment. These signals may be endogenous or heterologous to the polypeptide.

[0144] Polypeptides (e.g., engineered IL-21 variants) can be expressed in modified forms such as fusion proteins (e.g., HSA or GST fusions) or with histidine tags, and may include not only secretory signals but also additional heterologous functional regions. For example, additional amino acids, particularly charged amino acid regions, can be added to the N-terminus of the polypeptide to improve stability and persistence within host cells, during purification, and during subsequent handling and storage. Peptide moieties can also be added to polypeptides to facilitate purification. Such regions can be removed before the final preparation of the polypeptide. Adding peptide moieties to polypeptides, particularly to induce secretion or efflux, to improve stability, and to facilitate purification, is a well-known and common technique in the art.

[0145] Treatment method The manipulated IL-21 variants and their protein constructs disclosed herein can be used for a variety of therapeutic purposes.

[0146] In one embodiment, the disclosure provides a method for treating cancer in a subject, a method for reducing the rate of tumor volume growth in a subject over time, a method for reducing the risk of developing metastasis, or a method for reducing the risk of developing additional metastasis in a subject. In some embodiments, the treatment can stop, slow, delay, or inhibit the progression of cancer. In some embodiments, the treatment may result in a reduction in the number, severity, and / or duration of one or more symptoms of cancer in a subject.

[0147] In one embodiment, the Disclosure features a method comprising administering a therapeutically effective dose of an engineered IL-21 variant or protein construct disclosed herein to a subject in need (e.g., a subject having cancer or identified or diagnosed with cancer) (e.g., breast cancer (e.g., triple-negative breast cancer), carcinoid cancer, 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 hematological malignancy). In some embodiments, the cancer is unresectable or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory 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 carcinoma. In some embodiments, the subject has Hodgkin lymphoma. In some embodiments, the subject has triple-negative breast cancer (TNBC), gastric cancer, urothelial carcinoma, Merkel cell carcinoma, or head and neck cancer.

[0148] In some embodiments, the cancer described herein is esophageal adenocarcinoma, lung cancer, hepatocellular carcinoma, bile duct cancer, gallbladder cancer, stomach 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.

[0149] In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of cancer. Cancer patients can be identified in various ways known in the art.

[0150] In one embodiment, the Disclosure provides a method for treating, preventing, or reducing the risk of developing disorders associated with abnormal or undesirable immune responses (e.g., autoimmune disorders) by administering, for example, a therapeutically effective dose of the engineered IL-21 variants and protein constructs disclosed herein to a subject in need. These autoimmune disorders 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 disorders include allergies, asthma, and / or atopic dermatitis. Thus, the engineered IL-21 variants and protein constructs disclosed herein can be used to inhibit immune responses. In some embodiments, the immune disorders described herein include allergies, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes mellitus, pain, or neuropathy.

[0151] As used herein, “effective dose” means an amount or dosage sufficient to produce a beneficial or desired outcome, including stopping, slowing, delaying, or inhibiting the progression of a disease, such as cancer. The effective dose varies depending on the age and weight of the subject to whom the engineered IL-21 variant and protein construct, the vector containing polynucleotides encoding the engineered IL-21 variant and protein construct, and / or their compositions are administered, the severity of the symptoms, and the route of administration, and therefore the dosage can be determined on an individual basis.

[0152] An effective dose may be administered in one or more doses. For example, an effective dose of the engineered IL-21 variant and / or protein construct may be sufficient to improve, halt, stabilize, reverse, inhibit, slow, and / or delay cancer progression in a patient, or sufficient to improve, halt, stabilize, reverse, slow, and / or delay the proliferation of cells (e.g., biopsy cells, any of the cancer cells described herein, or cell lines (e.g., cancer cell lines)) in vitro. As is understood in the art, the effective dose may vary depending, among other factors, such as the patient's medical history and the type (and / or dosage) of the engineered IL-21 variant and protein construct used.

[0153] The effective doses and schedules for administering the engineered IL-21 variants or their protein constructs, polynucleotides encoding the engineered IL-21 variants or protein constructs, and / or compositions disclosed herein may be determined empirically, and making such determinations is within the scope of the art. Those skilled in the art will understand that the dosage to be administered will vary, for example, depending on the mammal to receive the engineered IL-21 variants or their protein constructs, polynucleotides, and / or compositions disclosed herein, the route of administration, the specific type of polynucleotide and / or composition disclosed herein used, and other drugs administered to the mammal.

[0154] A 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 per kg of patient body weight). In some embodiments, the dose may be 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 less than 0.1 mg / kg. In some embodiments, the dosage can be 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 greater than 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 approximately 1-150 mg / kg, approximately 1-100 mg / kg, approximately 1-80 mg / kg, approximately 1-50 mg / kg, approximately 1-30 mg / kg, approximately 1-20 mg / kg, approximately 1-10 mg / kg, approximately 1-5 mg / kg, approximately 5-150 mg / kg, approximately 5-100 mg / kg, approximately 5-80 mg / kg, approximately 5-50 mg / kg, approximately 5-30 mg / kg, approximately 5-20 mg / kg, approximately 5-10 mg / kg, approximately 10-150 mg / kg, approximately 10-100 mg / kg, approximately 10-80 mg / kg, approximately 10- The dosages are 50 mg / kg, approximately 10-30 mg / kg, approximately 10-20 mg / kg, approximately 20-150 mg / kg, approximately 20-100 mg / kg, approximately 20-80 mg / kg, approximately 20-50 mg / kg, approximately 20-30 mg / kg, approximately 30-150 mg / kg, approximately 30-100 mg / kg, approximately 30-80 mg / kg, approximately 30-50 mg / kg, approximately 50-150 mg / kg, approximately 50-100 mg / kg, approximately 50-80 mg / kg, approximately 80-150 mg / kg, approximately 80-100 mg / kg, or approximately 100-150 mg / kg.

[0155] In any of the methods described herein, the manipulated IL-21 variant or its protein construct may be administered to a subject at least once a week (for example, 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).

[0156] 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, one or more additional therapeutic agents are administered to the subject such that there is an overlap in the physiological activity periods of the one or more additional therapeutic agents and the engineered IL-21 variant or its protein construct in the subject.

[0157] In some embodiments, one or more additional therapeutic agents may be administered to the subject. The 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, dual PI3K / mTOR 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).

[0158] 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 the activated Hedgehog signaling pathway, and agents that selectively degrade estrogen receptors.

[0159] In some embodiments, additional therapeutic agents include trabectedin, nab-paclitaxel, trevananib, pazopanib, sediranib, palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, reolysin, alimta, zykadia, sutent, temsirolimus, axitinib, everolimus, sorafenib, votrient, pazopanib, IMA-901, AGS-003, and cabozan. The treatment may include one or more therapeutic agents selected from the group consisting of tinib, vinflunin, Hsp90 inhibitors, Ad-GM-CSF, temazolomide, IL-2, IFNa, vinblastine, thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacitidine, bortezomid, amrubicin, carfilzomib, pralatrexate, and enzastaurin.

[0160] In some embodiments, additional therapeutic agents may include one or more agents selected from the group consisting of adjuvants, TLR agonists, tumor necrosis factor (TNF) alpha, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, CX3CL1-targeted therapies, CXCL9-targeted therapies, CXCL10-targeted therapies, CCL5-targeted therapies, LFA-1 agonists, ICAM1 agonists, and selectin agonists.

[0161] In some embodiments, carboplatin, nab-paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI are administered to the subject.

[0162] In some embodiments, additional therapeutic agents are anti-OX40 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-SIRPα antibody, anti-CD47 antibody, anti-LAG3 antibody, anti-TIGIT antibody, anti-BTLA antibody, anti-CTLA-4 antibody, or anti-GITR antibody. In some embodiments, additional therapeutic agents are anti-CD20 antibody (e.g., rituximab) or anti-EGF receptor antibody (e.g., cetuximab).

[0163] Pharmaceutical composition and route of administration Pharmaceutical compositions containing the manipulated IL-21 variant or its protein construct described herein are also provided herein. The pharmaceutical compositions can be formulated in any way known in the art.

[0164] Pharmaceutical compositions are formulated to be compatible with their intended route of administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The compositions may include sterile diluents (e.g., sterile water or saline), fixative oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents, antibacterial or antifungal agents such as benzyl alcohol or methylparaben, chlorobutanol, phenol, ascorbic acid, or thimerosal, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates, or phosphates, and isotonic agents such as sugars (e.g., dextrose), polyalcohols (e.g., mannitol or sorbitol), or salts (e.g., sodium chloride), or any combination thereof. Liposome suspensions can also be used as pharmaceutically acceptable carriers. Preparations of these compositions can be formulated and encapsulated in ampoules, disposable syringes, or multi-dose vials. If necessary (e.g., in injectable formulations), appropriate fluidity can be maintained by the use of coating agents such as lecithin or surfactants. Drug absorption can be prolonged by including absorption-delaying agents (e.g., aluminum monostearate and gelatin). Alternatively, controlled release can be achieved by implants and microencapsulated delivery systems that may contain biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid).

[0165] Compositions containing the manipulated IL-21 variant or its protein construct described herein can be formulated in dosing unit form (i.e., physically separate units containing a predetermined amount of the active compound for ease of administration and uniformity of dosage) for parenteral administration (e.g., intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, or intraperitoneal).

[0166] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under Good Manufacturing Practice (GMP) conditions. Pharmaceutical compositions can be provided in unit dosage forms (i.e., doses for single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the chosen route of administration. For injection, the engineered IL-21 variant or its protein construct can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, the engineered IL-21 variant or its protein construct may be in a lyophilized form for formulation in a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0167] The toxicity and therapeutic efficacy of a composition can be determined by standard pharmaceutical procedures in cell cultures or experimental animals (e.g., monkeys). For example, one can determine the LD50 (lethal dose for 50% of the population) and ED50 (therapeutably effective dose for 50% of the population): the therapeutic index is the ratio of LD50:ED50. Drugs exhibiting a high therapeutic index are preferred. If a drug exhibits undesirable side effects, care must be taken to minimize potential damage (i.e., reduce the undesirable side effects). Toxicity and therapeutic efficacy can also be determined by other standard pharmaceutical procedures.

[0168] Exemplary doses include milligrams or micrograms of any of the manipulated IL-21 variants or their protein constructs described herein per kilogram of body weight of the subject (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 wide range, those skilled in the art will understand that therapeutic agents can vary their potency and that effective doses can be determined by methods known in the art. Typically, a relatively low dose is administered first, and the attending physician or veterinary expert (in the case of therapeutic use) or researcher (if still working in the development stage) may then gradually increase the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular subject depends on various factors, including the activity of the specific compound used, the subject's age, weight, general health status, sex, and diet, administration time, route of administration, excretion rate, and the in vivo half-life of the engineered IL-21 variant or its protein construct.

[0169] The pharmaceutical composition may be included in a container, pack, or dispenser along with instructions for administration. This disclosure also provides methods for producing engineered IL-21 variants or their protein constructs for the various uses described herein.

[0170] How to manipulate cytokines Methods for improving the properties (e.g., stability, activity, or affinity) of a protein (e.g., cytokine) are provided herein, comprising one or more of the following steps: (a) providing a 3D structure of the protein (e.g., cytokine); (b) modifying one or more amino acid residues in the 3D structure. アルファ(c) measuring the distance between atoms, and (c) selecting two amino acid residues from one or more amino acid residues, wherein the C of the two selected amino acid residues アルファ Select atoms that are within 3-7 Å. In some embodiments, the two selected amino acid residues are C アルファ The atoms are within 4.5 to 6.5 Å, for example, within approximately 6.5 Å, 6.4 Å, 6.3 Å, 6.2 Å, 6.1 Å, 6 Å, 5.9 Å, 5.8 Å, 5.7 Å, 5.6 Å, 5.5 Å, 5.4 Å, 5.3 Å, 5.2 Å, 5.1 Å, 5 Å, 4.9 Å, 4.8 Å, 4.7 Å, 4.6 Å, or 4.5 Å. In some embodiments, the C of two selected amino acid residues is アルファ From C ベータ The directionality to atoms is favorable for the formation of unnatural disulfide bonds. For example, C ベータ The atoms are in a direction that allows them to form disulfide bonds (C アルファ From C ベータ Suitable for. In some cases, the first selected amino acid residue C ベータ Atomic orientation (C of the first selected amino acid residue) アルファ From C ベータ (to) and the second selected amino acid residue C ベータ Atomic orientation (C of the second selected amino acid residue) アルファ From C ベータ The angle between (f) and the angel is less than 120 degrees. In some cases, C ベータ The atoms are not oriented away from each other. In some embodiments, the 3D structure of a protein (e.g., cytokine) is derived from the PDB structure of the protein (e.g., cytokine), its fragments, or the complex of the protein (e.g., cytokine) bound to its binding partner(s). In some embodiments, the spatial coordinates of all atoms in the 3D structure can be loaded into software (e.g., modeling and simulation software) and the distance between any two atoms in the 3D structure can be determined.

[0171] In some embodiments, the first selected amino acid residue C ベータ Atomic orientation and the C of the second selected amino acid residueベータ The angle between the atomic orientation and the surrounding elements is less than 120 degrees, for example, 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 6 8, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 1 degree.

[0172] In some embodiments, the C of two selected amino acid residues described herein アルファ The atomic distances are approximately 3 to 7 Å, 3 to 6.5 Å, 3 to 6 Å, 3 to 5.5 Å, 3 to 5 Å, 3 to 4.5 Å, 3 to 4 Å, 3 to 3.5 Å, 3.5 to 7 Å, 3.5 to 6.5 Å, 3.5 to 6 Å, 3.5 to 5.5 Å, 3.5 to 5 Å, 3.5 to 4.5 Å, 3.5 to 4 Å, 4 to 7 Å, 4 to 6.5 Å, 4 to 6 Å, and The angulations are approximately 4 to 5.5 Å, approximately 4 to 5 Å, approximately 4 to 4.5 Å, approximately 4.5 to 7 Å, approximately 4.5 to 6.5 Å, approximately 4.5 to 6 Å, approximately 4.5 to 5.5 Å, approximately 4.5 to 5 Å, approximately 5 to 7 Å, approximately 5 to 6.5 Å, approximately 5 to 6 Å, approximately 5 to 5.5 Å, approximately 5.5 to 7 Å, approximately 5.5 to 6.5 Å, approximately 5.5 to 6 Å, approximately 6 to 7 Å, approximately 6 to 6.5 Å, or approximately 6.5 to 7 Å.

[0173] In some embodiments, the C of two selected amino acid residues described herein アルファThe atomic distances are approximately 4.5 to 6.5 Å, 4.5 to 6.4 Å, 4.5 to 6.3 Å, 4.5 to 6.2 Å, 4.5 to 6.1 Å, 4.5 to 6.0 Å, 4.5 to 5.9 Å, 4.5 to 5.8 Å, 4.5 to 5.7 Å, 4.5 to 5.6 Å, and 4.5 to 5. 5 Å, approximately 4.5~5.4 Å, approximately 4.5~5.3 Å, approximately 4.5~5.2 Å, approximately 4.5~5.1 Å, approximately 4.5~5.0 Å, approximately 4.5~4.9 Å, approximately 4.5~4.8 Å, approximately 4.5~4.7 Å, approximately 4.5~4.6 Å, approximately 4.6~6.5 Å, approximately 4.6~6.4 Å, approximately 4.6~approx. 6.3 Å, approx. 4.6~approx. 6.2 Å, approx. 4.6~approx. 6.1 Å, approx. 4.6~approx. 6.0 Å, approx. 4.6~approx. 5.9 Å, approx. 4.6~approx. 5.8 Å, approx. 4.6~approx. 5.7 Å, approx. 4.6~approx. 5.6 Å, approx. 4.6~approx. 5.5 Å, approx. 4.6~approx. 5.4 Å, approx. 4.6~approx. 5.3 Å, approx. 4.6~ Approximately 5.2 Å, approximately 4.6-5.1 Å, approximately 4.6-5.0 Å, approximately 4.6-4.9 Å, approximately 4.6-4.8 Å, approximately 4.6-4.7 Å, approximately 4.7-6.5 Å, approximately 4.7-6.4 Å, approximately 4.7-6.3 Å, approximately 4.7-6.2 Å, approximately 4.7-6.1 Å, approximately 4.7-6.0 Å, approximately 4.7~5.9 Å, approximately 4.7~5.8 Å, approximately 4.7~5.7 Å, approximately 4.7~5.6 Å, approximately 4.7~5.5 Å, approximately 4.7~5.4 Å, approximately 4.7~5.3 Å, approximately 4.7~5.2 Å, approximately 4.7~5.1 Å, approximately 4.7~5.0 Å, approximately 4.7~4.9 Å, approximately 4 0.7~approx. 4.8 Å, approx. 4.8~approx. 6.5 Å, approx. 4.8~approx. 6.4 Å, approx. 4.8~approx. 6.3 Å, approx. 4.8~approx. 6.2 Å, approx. 4.8~approx. 6.1 Å, approx. 4.8~approx. 6.0 Å, approx. 4.8~approx. 5.9 Å, approx. 4.8~approx. 5.8 Å, approx. 4.8~approx. 5.7 Å, approx. 4.8~approx. 5.6 Å, approx. 4.8~approx. 5.5 Å, approximately 4.8 to 5.4 Å, approximately 4.8 to 5.3 Å, approximately 4.8 to 5.2 Å, approximately 4.8 to 5.1 Å, approximately 4.8 to 5.0 Å, approximately 4.8 to 4.9 Å, approximately 4.9 to 6.5 Å, approximately 4.9 to 6.4 Å, approximately 4.9 to 6.3 Å, approximately 4.9 to 6.2 Å, approximately 4.9 to 6.1 Å Approximately 4.9-6.0 Å, approximately 4.9-5.9 Å, approximately 4.9-5.8 Å, approximately 4.9-5.7 Å, approximately 4.9-5.6 Å, approximately 4.9-5.5 Å, approximately 4.9-5.4 Å, approximately 4.9-5.3 Å, approximately 4.9-5.2 Å, approximately 4.9-5.1 Å, approximately 4.9-5.0 Å, approximately 5.0 to approximately 6.5 Å, approximately 5.0 to approximately 6.4 Å, approximately 5.0 to approximately 6.3 Å, approximately 5.0 to approximately 6.2 Å, approximately 5.0 to approximately 6.1 Å, approximately 5.0 to approximately 6.0 Å, approximately 5.0 to approximately 5.9 Å, approximately 5.0 to approximately 5.8 Å, approximately 5.0 to approximately 5.7 Å, approximately 5.0 to approximately 5.6 Å, approximately 5.0 to approximately 5.5 Å, approximately 5.0 to approximately 5 0.4 Å, approximately 5.0~5.3 Å, approximately 5.0~5.2 Å, approximately 5.0~5.1 Å, approximately 5.1~6.5 Å, approximately 5.1~6.4 Å, approximately 5.1~6.3 Å, approximately 5.1~6.2 Å, approximately 5.1~6.1 Å, approximately 5.1~6.0 Å, approximately 5.1~5.9 Å, approximately 5.1~5.8 Å, Approximately 5.1-5.7 Å, approximately 5.1-5.6 Å, approximately 5.1-5.5 Å, approximately 5.1-5.4 Å, approximately 5.1-5.3 Å, approximately 5.1-5.2 Å, approximately 5.2-6.5 Å, approximately 5.2-6.4 Å, approximately 5.2-6.3 Å, approximately 5.2-6.2 Å, approximately 5.2-6.1 Å, approximately 5.2 ~6.0 Å, approx. 5.2~5.9 Å, approx. 5.2~5.8 Å, approx. 5.2~5.7 Å, approx. 5.2~5.6 Å, approx. 5.2~5.5 Å, approx. 5.2~5.4 Å, approx. 5.2~5.3 Å, approx. 5.3~6.5 Å, approx. 5.3~6.4 Å, approx. 5.3~6.3 Å, approx. 5.3~6. 2 Å, approximately 5.3 to approximately 6.1 Å, approximately 5.3 to approximately 6.0 Å, approximately 5.3 to approximately 5.9 Å, approximately 5.3 to approximately 5.8 Å, approximately 5.3 to approximately 5.7 Å, approximately 5.3 to approximately 5.6 Å, approximately 5.3 to approximately 5.5 Å, approximately 5.3 to approximately 5.4 Å, approximately 5.4 to approximately 6.5 Å, approximately 5.4 to approximately 6.4 Å, approximately 5.4 to approximately 6.3 Å, approximately 5.4~approx. 6.2 Å, approx. 5.4~approx. 6.1 Å, approx. 5.4~approx. 6.0 Å, approx. 5.4~approx. 5.9 Å, approx. 5.4~approx. 5.8 Å, approx. 5.4~approx. 5.7 Å, approx. 5.4~approx. 5.6 Å, approx. 5.4~approx. 5.5 Å, approx. 5.5~approx. 6.5 Å, approx. 5.5~approx. 6.4 Å, approx. 5.5~approx. 6.3 Å, approx. 5.5~ Approximately 6.2 Å, approximately 5.5 to 6.1 Å, approximately 5.5 to 6.0 Å, approximately 5.5 to 5.9 Å, approximately 5.5 to 5.8 Å, approximately 5.5 to 5.7 Å, approximately 5.5 to 5.6 Å, approximately 5.6 to 6.5 Å, approximately 5.6 to 6.4 Å, approximately 5.6 to 6.3 Å, approximately 5.6 to 6.2 Å, approximately 5.6 to 6.1 Å Å, approximately 5.6~6.0 Å, approximately 5.6~5.9 Å, approximately 5.6~5.8 Å, approximately 5.6~5.7 Å, approximately 5.7~6.5 Å, approximately 5.7~6.4 Å, approximately 5.7~6.3 Å, approximately 5.7~6.2 Å, approximately 5.7~6.1 Å, approximately 5.7~6.0 Å, approximately 5.7~5.9 Å, approximately 5.7 to approximately 5.8 Å, approximately 5.8 to approximately 6.5 Å, approximately 5.8 to approximately 6.4 Å, approximately 5.8 to approximately 6.3 Å, approximately 5.8 to approximately 6.2 Å, approximately 5.8 to approximately 6.1 Å, approximately 5.8 to approximately 6.0 Å, approximately 5.8 to approximately 5.9 Å, approximately 5.9 to approximately 6.5 Å, approximately 5.9 to approximately 6.4 Å, approximately 5.9 to approximately 6.3 Å, approximately 5.9 to approximately 6.2 Å, approximately 5.9 to approximately 6.1 Å, approximately 5.9 to approximately 6.0 Å, approximately 6.0 to approximately 6.5 Å These ranges are approximately 6.0–6.4 Å, 6.0–6.3 Å, 6.0–6.2 Å, 6.0–6.1 Å, 6.1–6.5 Å, 6.1–6.4 Å, 6.1–6.3 Å, 6.1–6.2 Å, 6.2–6.5 Å, 6.2–6.4 Å, 6.2–6.3 Å, 6.3–6.5 Å, 6.3–6.4 Å, or 6.4–6.5 Å. In some embodiments, the ranges described herein are inclusive, meaning that values ​​at the boundaries of the ranges are included within the ranges.

[0174] In some embodiments, the C of two selected amino acid residues described herein ベータ The distances between atoms are approximately 3 to 8 Å, 3 to 7.5 Å, 3 to 7 Å, 3 to 6.5 Å, 3 to 6 Å, 3 to 5.5 Å, 3 to 5 Å, 3 to 4.5 Å, 3 to 4 Å, 3 to 3.5 Å, 3.5 to 8 Å, 3.5 to 7.5 Å, 3.5 to 7 Å, and 3.5 to 6 Å. 5 Å, approximately 3.5-6 Å, approximately 3.5-5.5 Å, approximately 3.5-5 Å, approximately 3.5-4.5 Å, approximately 3.5-4 Å, approximately 4-8 Å, approximately 4-7.5 Å, approximately 4-7 Å, approximately 4-6.5 Å, approximately 4-6 Å, approximately 4-5.5 Å, approximately 4-5 Å, approximately 4-4.5 Å, approximately 4.5-8 Å , approximately 4.5~7.5 Å, approximately 4.5~7 Å, approximately 4.5~6.5 Å, approximately 4.5~6 Å, approximately 4.5~5.5 Å, approximately 4.5~5 Å, approximately 5~8 Å, approximately 5~7.5 Å, approximately 5~7 Å, approximately 5~6.5 Å, approximately 5~6 Å, approximately 5~5.5 Å, approximately 5.5~8 Å, approximately 5.5~ The values ​​are 7.5 Å, approximately 5.5 to 7 Å, approximately 5.5 to 6.5 Å, approximately 5.5 to 6 Å, approximately 6 to 8 Å, approximately 6 to 7.5 Å, approximately 6 to 7 Å, approximately 6 to 6.5 Å, approximately 6.5 to 8 Å, approximately 6.5 to 7.5 Å, approximately 6.5 to 7 Å, approximately 7 to 8 Å, approximately 7 to 7.5 Å, or approximately 7.5 to 8 Å.

[0175] In some embodiments, the C of two selected amino acid residues described herein ベータThe atomic distances are approximately 4.5 to 6.5 Å, 4.5 to 6.4 Å, 4.5 to 6.3 Å, 4.5 to 6.2 Å, 4.5 to 6.1 Å, 4.5 to 6.0 Å, 4.5 to 5.9 Å, 4.5 to 5.8 Å, 4.5 to 5.7 Å, 4.5 to 5.6 Å, and 4.5 to 5. 5 Å, approximately 4.5~5.4 Å, approximately 4.5~5.3 Å, approximately 4.5~5.2 Å, approximately 4.5~5.1 Å, approximately 4.5~5.0 Å, approximately 4.5~4.9 Å, approximately 4.5~4.8 Å, approximately 4.5~4.7 Å, approximately 4.5~4.6 Å, approximately 4.6~6.5 Å, approximately 4.6~6.4 Å, approximately 4.6~approx. 6.3 Å, approx. 4.6~approx. 6.2 Å, approx. 4.6~approx. 6.1 Å, approx. 4.6~approx. 6.0 Å, approx. 4.6~approx. 5.9 Å, approx. 4.6~approx. 5.8 Å, approx. 4.6~approx. 5.7 Å, approx. 4.6~approx. 5.6 Å, approx. 4.6~approx. 5.5 Å, approx. 4.6~approx. 5.4 Å, approx. 4.6~approx. 5.3 Å, approx. 4.6~ Approximately 5.2 Å, approximately 4.6-5.1 Å, approximately 4.6-5.0 Å, approximately 4.6-4.9 Å, approximately 4.6-4.8 Å, approximately 4.6-4.7 Å, approximately 4.7-6.5 Å, approximately 4.7-6.4 Å, approximately 4.7-6.3 Å, approximately 4.7-6.2 Å, approximately 4.7-6.1 Å, approximately 4.7-6.0 Å, approximately 4.7~5.9 Å, approximately 4.7~5.8 Å, approximately 4.7~5.7 Å, approximately 4.7~5.6 Å, approximately 4.7~5.5 Å, approximately 4.7~5.4 Å, approximately 4.7~5.3 Å, approximately 4.7~5.2 Å, approximately 4.7~5.1 Å, approximately 4.7~5.0 Å, approximately 4.7~4.9 Å, approximately 4 0.7~approx. 4.8 Å, approx. 4.8~approx. 6.5 Å, approx. 4.8~approx. 6.4 Å, approx. 4.8~approx. 6.3 Å, approx. 4.8~approx. 6.2 Å, approx. 4.8~approx. 6.1 Å, approx. 4.8~approx. 6.0 Å, approx. 4.8~approx. 5.9 Å, approx. 4.8~approx. 5.8 Å, approx. 4.8~approx. 5.7 Å, approx. 4.8~approx. 5.6 Å, approx. 4.8~approx. 5.5 Å, approximately 4.8 to 5.4 Å, approximately 4.8 to 5.3 Å, approximately 4.8 to 5.2 Å, approximately 4.8 to 5.1 Å, approximately 4.8 to 5.0 Å, approximately 4.8 to 4.9 Å, approximately 4.9 to 6.5 Å, approximately 4.9 to 6.4 Å, approximately 4.9 to 6.3 Å, approximately 4.9 to 6.2 Å, approximately 4.9 to 6.1 Å Approximately 4.9-6.0 Å, approximately 4.9-5.9 Å, approximately 4.9-5.8 Å, approximately 4.9-5.7 Å, approximately 4.9-5.6 Å, approximately 4.9-5.5 Å, approximately 4.9-5.4 Å, approximately 4.9-5.3 Å, approximately 4.9-5.2 Å, approximately 4.9-5.1 Å, approximately 4.9-5.0 Å, approximately 5.0 to approximately 6.5 Å, approximately 5.0 to approximately 6.4 Å, approximately 5.0 to approximately 6.3 Å, approximately 5.0 to approximately 6.2 Å, approximately 5.0 to approximately 6.1 Å, approximately 5.0 to approximately 6.0 Å, approximately 5.0 to approximately 5.9 Å, approximately 5.0 to approximately 5.8 Å, approximately 5.0 to approximately 5.7 Å, approximately 5.0 to approximately 5.6 Å, approximately 5.0 to approximately 5.5 Å, approximately 5.0 to approximately 5 0.4 Å, approximately 5.0~5.3 Å, approximately 5.0~5.2 Å, approximately 5.0~5.1 Å, approximately 5.1~6.5 Å, approximately 5.1~6.4 Å, approximately 5.1~6.3 Å, approximately 5.1~6.2 Å, approximately 5.1~6.1 Å, approximately 5.1~6.0 Å, approximately 5.1~5.9 Å, approximately 5.1~5.8 Å, Approximately 5.1-5.7 Å, approximately 5.1-5.6 Å, approximately 5.1-5.5 Å, approximately 5.1-5.4 Å, approximately 5.1-5.3 Å, approximately 5.1-5.2 Å, approximately 5.2-6.5 Å, approximately 5.2-6.4 Å, approximately 5.2-6.3 Å, approximately 5.2-6.2 Å, approximately 5.2-6.1 Å, approximately 5.2 ~6.0 Å, approx. 5.2~5.9 Å, approx. 5.2~5.8 Å, approx. 5.2~5.7 Å, approx. 5.2~5.6 Å, approx. 5.2~5.5 Å, approx. 5.2~5.4 Å, approx. 5.2~5.3 Å, approx. 5.3~6.5 Å, approx. 5.3~6.4 Å, approx. 5.3~6.3 Å, approx. 5.3~6. 2 Å, approximately 5.3 to approximately 6.1 Å, approximately 5.3 to approximately 6.0 Å, approximately 5.3 to approximately 5.9 Å, approximately 5.3 to approximately 5.8 Å, approximately 5.3 to approximately 5.7 Å, approximately 5.3 to approximately 5.6 Å, approximately 5.3 to approximately 5.5 Å, approximately 5.3 to approximately 5.4 Å, approximately 5.4 to approximately 6.5 Å, approximately 5.4 to approximately 6.4 Å, approximately 5.4 to approximately 6.3 Å, approximately 5.4~approx. 6.2 Å, approx. 5.4~approx. 6.1 Å, approx. 5.4~approx. 6.0 Å, approx. 5.4~approx. 5.9 Å, approx. 5.4~approx. 5.8 Å, approx. 5.4~approx. 5.7 Å, approx. 5.4~approx. 5.6 Å, approx. 5.4~approx. 5.5 Å, approx. 5.5~approx. 6.5 Å, approx. 5.5~approx. 6.4 Å, approx. 5.5~approx. 6.3 Å, approx. 5.5~ Approximately 6.2 Å, approximately 5.5 to 6.1 Å, approximately 5.5 to 6.0 Å, approximately 5.5 to 5.9 Å, approximately 5.5 to 5.8 Å, approximately 5.5 to 5.7 Å, approximately 5.5 to 5.6 Å, approximately 5.6 to 6.5 Å, approximately 5.6 to 6.4 Å, approximately 5.6 to 6.3 Å, approximately 5.6 to 6.2 Å, approximately 5.6 to 6.1 Å Å, approximately 5.6~6.0 Å, approximately 5.6~5.9 Å, approximately 5.6~5.8 Å, approximately 5.6~5.7 Å, approximately 5.7~6.5 Å, approximately 5.7~6.4 Å, approximately 5.7~6.3 Å, approximately 5.7~6.2 Å, approximately 5.7~6.1 Å, approximately 5.7~6.0 Å, approximately 5.7~5.9 Å, approximately 5.7 to approximately 5.8 Å, approximately 5.8 to approximately 6.5 Å, approximately 5.8 to approximately 6.4 Å, approximately 5.8 to approximately 6.3 Å, approximately 5.8 to approximately 6.2 Å, approximately 5.8 to approximately 6.1 Å, approximately 5.8 to approximately 6.0 Å, approximately 5.8 to approximately 5.9 Å, approximately 5.9 to approximately 6.5 Å, approximately 5.9 to approximately 6.4 Å, approximately 5.9 to approximately 6.3 Å, approximately 5.9 to approximately 6.2 Å, approximately 5.9 to approximately 6.1 Å, approximately 5.9 to approximately 6.0 Å, approximately 6.0 to approximately 6.5 Å These ranges are approximately 6.0–6.4 Å, 6.0–6.3 Å, 6.0–6.2 Å, 6.0–6.1 Å, 6.1–6.5 Å, 6.1–6.4 Å, 6.1–6.3 Å, 6.1–6.2 Å, 6.2–6.5 Å, 6.2–6.4 Å, 6.2–6.3 Å, 6.3–6.5 Å, 6.3–6.4 Å, or 6.4–6.5 Å. In some embodiments, the ranges described herein are inclusive, meaning that values ​​at the boundaries of the ranges are included within the ranges.

[0176] In some embodiments, the C of two selected amino acid residues アルファThe distance between the atoms is close enough to form a non-natural disulfide bond. In some embodiments, the two selected amino acid residues do not participate in the interaction of the protein (e.g., cytokine) with its binding partner (e.g., a receptor that binds to the protein (e.g., cytokine)). In some embodiments, the method further comprises expressing a protein variant (e.g., a cytokine variant), the 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 change the 3D structure of the protein (e.g., cytokine). For example, the cysteine mutation does not substantially interfere with the overall structure of the protein (e.g., cytokine). In some cases, the mutated residue does not show a spatial collision with the non-mutated residues. In some cases, one skilled in the art may perform a simulation of the mutated protein (e.g., mutated cytokine) structure in silico and determine the associated conformational changes caused by the cysteine mutation. In some embodiments, the associated conformational changes are minimal, e.g., the RMSD value is considered to be small by one skilled in the art.

[0177] Also provided herein is a method of screening for cytokine variants having improved anti-tumor efficacy, the method comprising: (a) providing the 3D structure of a cytokine; and (b) measuring the distance of the C アルファ atoms of one or more amino acid residues in the 3D structure; and (c) selecting two amino acid residues from the one or more amino acid residues, wherein the C アルファThe atoms are within the range of 7.0 Å, 6.9 Å, 6.8 Å, 6.7 Å, 6.6 Å, 6.5 Å, 6.4 Å, 6.3 Å, 6.2 Å, 6.1 Å, 6 Å, 5.9 Å, 5.8 Å, 5.7 Å, 5.6 Å, 5.5 Å, 5.4 Å, 5.3 Å, 5.2 Å, 5.1 Å, 5 Å, 4.9 Å, 4.8 Å, 4.7 Å, 4.6 Å, or 4.5 Å, 4.4 Å, 4.3 Å, 4.2 Å, 4.1 Å, 4.0 Å, 3.9 Å, 3.8 Å, 3.7 Å, 3.6 Å, 3.5 Å, 3.4 Å, 3.3 Å, 3.2 Å, 3.1 Å, 3.0 Å, or any of the ranges described herein.

[0178] In some embodiments, the protein or cytokine has 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 or fewer amino acid residues.

[0179] In some embodiments, the method further comprises (d) expressing a cytokine variant, wherein the variant includes a non-natural disulfide bond formed by mutating 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 body weight of an animal administered with the cytokine variant does not decrease significantly compared to the body weight of a reference animal (e.g., an animal administered with a vehicle).

[0180] In some applications, the cytokine described herein is IL-21 (e.g., human IL-21). In some applications, 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 applications, the cytokine is IL-2, IL-7, IL-10, IL-15, IL-21, IFNα, GM-CSF, or FLT-3.

[0181] In some embodiments, functional assays are performed to compare one or more expressed protein variants (e.g., cytokine variants). [Examples]

[0182] The present invention will be further illustrated by the following examples, but these examples will not limit the scope of the present invention as described in the claims.

[0183] Example 1. Design of an IL-21 variant by introducing an artificial disulfide bond. Interleukin-21 (IL-21) is a pleomorphic cytokine composed of four α-helical bundles, primarily produced by natural killer T (NKT) cells, T follicular helper (TFH) cells, and TH17 cells. IL-21 transmits 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 1 to 24 residues constitute the signal peptide.

[0184] To screen for IL-21 variants with enhanced stability and different functional potencies (e.g., T cell proliferation and STAT-3 signaling), a variant of wild-type IL-21 (SEQ ID NO: 2; without signal peptide) was identified by replacing two residues in the 3D structure of IL-21 (e.g., PDB ID: 2OQP) with C2. アルファ Atomic distance and C ベータ The design was achieved by selectively mutating two spatially adjacent residues to cysteine ​​according to the orientation of the atoms. The two newly introduced cysteine ​​residues can form an artificial disulfide bond, thereby intended to stabilize IL-21 and / or alter its functional potency.

[0185] The sequences of wild-type IL-21 and its variants are shown in the table below.

[0186] [Table 1] TIFF2026509566000002.tif194160

[0187] An IL-21 variant fused with a His tag (SEQ ID NO: 22) was expressed in Expi293 and CHO-S cells. The expressed proteins were purified by affinity chromatography using a Ni-HisTrap® column (Cytiva, catalog number: 17371206) and by size-exclusive chromatography (SEC) using a Superdex® 75 column (Cytiva, catalog number: 29148721). The purified proteins were also analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0188] As shown in Figure 1, the results showed that H8-IL21-C04, H8-IL21-C06, and H8-IL21-C13 can be expressed in either Expi293 or CHO-S cells, while H8-IL21-C02, H8-IL21-C05, H8-IL21-C09, H8-IL21-C10, and H8-IL21-C12 are 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 can be expressed in both Expi293 and CHO-S cells.

[0189] Example 2. Determination of efficacy in inducing STAT-3 phosphorylation and T cell proliferation. The efficacy of IL-21 variants on T cell responses was determined by STAT-3 phosphorylation and T cell proliferation. Specifically, in the STAT-3 phosphorylation assay, CD3+ T cells were isolated from hPBMC (human peripheral blood mononuclear cell) donors according to the manufacturing protocol. CD3+ T cells were activated with CD3 / CD28 Dynabeads® at a cell-to-bead ratio of 1:4 for 3 days. The activated T cells were rested for 24 hours and then subjected to 5 × 10⁻¹⁶ ray growth. 4 CD3+ T cells were incubated with His-tagged IL-21 variant at the indicated concentration (2nM, 5-fold serial dilution, 6 spots) at 37°C for 30 minutes. After incubation, CD3+ 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 the T cell proliferation assay, CD3+ T cells were isolated from hPBMC donors according to the manufacturing protocol. CD3+ T cells were activated with CD3 / CD28 Dynabeads® at a cell-to-bead ratio of 1:4 for 3 days. Activated T cells were rested for 24 hours and then subjected to 5 × 10⁻⁶ densitometry. 4CD3+ T cells were incubated for 3 days with the indicated concentrations of His-tagged IL-21 variant in the presence of pre-coated 1 μg / ml anti-CD3 antibody. Post-incubation, T cell proliferation was determined by the CellTiter-Glo® bioluminescent cell viability assay (Promega, catalog number: G7573).

[0190] As shown in Figure 2A, all IL-21 variants showed relatively low efficacy in inducing STAT-3 phosphorylation, with slight differences. As shown in Figure 2B, H8-IL21-C11 showed the highest efficacy in inducing T cell proliferation. H8-IL21-C03 showed relatively weak ability to induce T cell proliferation. H8-IL21-C01, H8-IL21-C07, and H8-IL21-C08 showed similar efficacy in inducing T cell proliferation compared to the wild-type control (H8-IL21-WT). His-tagged mesothelin extracellular domain (MSLN-His) was used as a negative control.

[0191] Based on the results of protein expression and T cell responses of IL-21 variants, 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.

[0192] Example 3. Expression and purification of HSA-fused IL-21 variant To enhance the stability of the IL-21 variants, plasmids (UCOE® single-expression puromycin vector CET1019 HS-puro-SceI; Merck, catalog number: UC0E01) with the human promoter uHS or HS encoding wild-type IL-21 (SEQ ID NO: 2) and selected IL-21 variants (IL21-C01, IL21-C03, IL21-C07, and IL21-C08) fused with HSA (human serum albumin) were constructed. Expi293 cells were transfected to express 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.

[0193] First, the purity of the HSA-fused IL-21 variants was analyzed by SDS-PAGE and HPLC-SEC (high-performance liquid chromatography size exclusion chromatography), respectively. As shown in Figure 3A, all of the selected HSA-fused IL-21 variants showed no aggregation or only slight fragmentation by non-reducing (NR) or reducing (R) SDS-PAGE. As shown in Figure 3B, the HPLC-SEC analysis results demonstrated that the purity (indicated by "Main %") of all of the selected HSA-fused IL-21 variants exceeded 95%.

[0194] In addition, the aggregation (T agg ) and onset (T onset ) temperatures of the HSA-fused IL-21 variants were analyzed by dynamic light scattering and static light scattering (DLS / SLS). As shown in Figure 3C, compared with HSA-IL21-WT, all of the selected HSA-fused IL-21 variants showed significantly increased T agg and T onset temperatures. The results indicated that the selected IL-21 variants had better stability than wild-type IL-21.

[0195] Example 4. Determination of the efficacy of HSA-fused IL-21 variant in inducing STAT-3 phosphorylation and T cell proliferation. The efficacy of the HSA-fused IL-21 variant on T cell response 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. CD8+ T cells were activated with CD3 / CD28 Dynabeads® at a cell-to-bead ratio of 1:5 for 3 days. The activated T cells were rested for 24 hours and then subjected to 5 × 10⁻¹⁶ ray growth. 4 CD8+ T cells were incubated with His-tagged HSA-fused IL-21 variants at the indicated concentration (1 nM, 20-fold serial dilution, 4 spots) at 37°C for 30 minutes. 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 the T cell proliferation assay, CD8+ T cells were isolated from hPBMC donors according to the manufacturing protocol. CD8+ T cells were activated with CD3 / CD28 Dynabeads® at a cell-to-bead ratio of 1:8 for 3 days. Activated T cells were rested for 24 hours and then subjected to 5 × 10⁻⁶ saturation. 4 CD8+ T cells were incubated for 6 days with His-tagged HSA-fused IL-21 variants at the indicated concentrations (10 nM, 5-fold serial dilutions, 6 spots) in the presence of pre-coated 1 μg / ml anti-CD3 antibody. Post-incubation, T cell proliferation was determined by the CellTiter-Glo® bioluminescent cell viability assay (Promega, catalog number: G7573).

[0196] As shown in Figure 4A, HSA-IL21-C03, HSA-IL21-C07, and HSA-IL21-C08 showed relatively weaker efficacy in inducing STAT-3 phosphorylation than HSA-IL21-WT. HSA-IL21-C01 showed similar efficacy in inducing STAT-3 phosphorylation compared to HSA-IL21-WT. As shown in Figure 4B, HSA-IL21-C03 showed the highest efficacy in inducing T cell proliferation compared to HSA-IL21-WT and other HSA-fusion IL-21 variants. However, HSA-IL21-C08 showed relatively weaker efficacy in inducing T cell proliferation than HSA-IL21-WT, although the difference was slight. As negative controls, His-tagged HSA (HSA-His), MSLN-His, and SIRPα-G4Fc (Trillium) (or SIRPα-G4Fc-WT (Trillium); Sequence ID No. 23) were used.

[0197] The results also showed that IgG4 Fc-fused wild-type IL-21 (G4Fc-IL21-WT; SEQ ID NO: 16) and H8-IL21-WT (H8-IL21-WT_CHOS) expressed in CHO-S cells had a relatively weaker effect on inducing STAT-3 phosphorylation and T cell proliferation compared to HSA-fused wild-type IL-21 and its variants expressed in Expi293 cells.

[0198] Example 5. Determination of the efficacy of HSA-fused IL-21 variant in inducing NK-92 cell proliferation. The efficacy of an HSA-fused IL-21 variant in inducing NK-92 cell proliferation was determined by the CellTiter-Glo® bioluminescent cell viability assay (Promega, catalog number: G7573). Specifically, NK-92 cells were starved for 24 hours in a medium without IL-2 supplementation. After starvation, 5 × 10⁶ cells were observed. 4NK-92 cells were incubated for a further 24 hours in complete medium (containing 75% MEM-α, ribonucleoside and deoxyribonucleoside (Gibco, catalog number: 12571-048) + 12.5% ​​horse serum + 12.5% ​​FBS + 150 U / mL IL-2) with the HSA-IL-21 variant at the indicated concentration (100 nM, 10-fold serial dilution, 8 spots). Growth was determined by the CellTiter-Glo® bioluminescent cell viability assay.

[0199] The EC50 values ​​for each HSA-fused IL-21 variant are shown in the table below.

[0200] [Table 2]

[0201] As shown in Figure 5 and the table above, HSA-IL21-C03 showed better efficacy than HSA-IL21-WT in inducing NK-92 cell proliferation. HSA-IL21-C08 showed the lowest efficacy among the HSA-fused IL-21 variants in inducing NK-92 cell proliferation. HSA-His protein (Acro) was used as a negative control.

[0202] Example 6. Determination of the efficacy of HSA-fused IL-21 variant in alleviating Treg-mediated T cell suppression. The efficacy of HSA-fused IL-21 variants on Treg function was determined by a Treg-mediated T cell suppression assay.

[0203] Specifically, CD4 + CD25 + CD127 低Treg cells were isolated from hPBMC donors according to the manufacturing protocol. The Treg cells were activated with CD3 / CD28 Dynabeads® at a cell-to-bead ratio of 1:10 for 4 days. After activation, CD8+ T cells were isolated from another hPBMC donor according to the manufacturing protocol and then stained with 1 μM CFSE (carboxyfluorescein succinimimidyl ester) dye. Treg and CFSE-labeled CD8+ T cells were incubated at a cell-to-cell ratio of 1:4 and then treated with HSA-fused IL-21 and its variants at the indicated concentrations for 3 days. The cells were then harvested and stained with PE-anti-CD8 antibody. T cell proliferation was controlled by CD8 + CFSE 低 It was determined by the proportion of cells.

[0204] As shown in Figure 6, all selected HSA-fusion IL-21 variants (HSA-IL21-C01, HSA-IL21-C03, HSA-IL21-C07, and HSA-IL21-C08) showed a significant enhancement (e.g., at approximately 0.0586 nM) in mitigating Treg-mediated T cell suppression compared to HSA-IL21-WT. In particular, HSA-IL21-C08 showed lower efficacy in mitigating Treg-mediated T cell suppression than the other HSA-fusion IL-21 variants. HSA-IL21-C01, HSA-IL21-C03, and HSA-IL21-C07 showed similar efficacy against Treg function. SIRPα-G4Fc-WT (Trillium) was used as a negative control.

[0205] The results also showed that HSA-IL21-WT was more effective than H8-IL21-WT (SEQ ID NO: 24) in alleviating Treg-mediated T cell suppression.

[0206] Example 7. Determination of the efficacy of HSA-fused IL-21 variant in inducing cytotoxicity in primary NK cells. The potency of the HSA-fused IL-21 variants against the induction of primary NK cell cytotoxicity was determined by the DELFIA® EuTDA cytotoxicity reagent (PerkinElmer). Specifically, primary NK cells were isolated from hPBMC donors according to the production protocol and then incubated for 24 hours with or without the indicated concentrations of HSA-fused IL-21 and its variants. Before incubation with primary NK cells, K-562 tumor cells were treated with 20 ng / ml of IFN-γ for 24 hours and then labeled with the fluorescence-enhancing ligand BATDA (bis(acetoxymethyl) 2,2’:6’,2’’-terpyridine-6,6’’-dicarboxylate) according to the production protocol of the DELFIA® EuTDA cytotoxicity reagent (PerkinElmer). Next, effector cells (primary NK cells) and target cells (BATDA-labeled K-562) were co-incubated at a cell-to-cell ratio of 4:1 in a 96-well U-bottom cell culture plate at 37 °C for 4 hours in the presence of 1 - 100 nM of the HSA-fused IL-21 variant. The cell supernatant was carefully collected from the assay plate without disturbing the cells. 20 μl of the clarified supernatant sample was mixed with 200 μl of europium solution in the attached DELFIA® strip plate. The signal was detected using a compatible plate reader (excitation at 340 nm and emission at 615 nm) with the DELFIA® time-resolved fluorescence (TRF) setting. The percentage of specific release was determined by the following formula: (Experimental release - Spontaneous release) / (Maximum release - Spontaneous release) × 100 as determined by

[0207] As shown in Figure 7, HSA-IL21-C01 and HSA-IL21-C08 were shown to be less potent than HSA-IL21-WT against the induction of primary NK cell cytotoxicity. The results indicate that the selected IL-21 variants can induce specific lysis of K-562 tumor cells mediated by primary NK cells.

[0208] Based on the above feature determination and the results of the in vitro assay, HSA-IL21-C01 and HSA-IL21-C08 were lead candidates and were selected for subsequent experiments.

[0209] Example 8. Evaluation of the Antitumor Efficacy and In Vivo Toxicity of HSA-Fusion IL-21 Variants The antitumor efficacy of HSA-fusion IL-21 and its variants was evaluated in a CT26 tumor-bearing BALB / c mouse model. The treatment plan and dosing schedule are shown in Figure 8A. Specifically, 5- to 6-week-old BALB / c mice were selected and 2 × 10 5 individual mouse colon cancer cells CT26 were inoculated subcutaneously (s.c.). When the tumor volume reached approximately 100-200 mm 3 (4 days later), the mice were randomly assigned to three treatment groups and one control group (10 mice per group). The treatment group mice were intraperitoneally (i.p.) injected with 10 mg / kg of HSA-IL21-WT (G2), HSA-IL21-C01 (G3), or HSA-IL21-C08 (G4). The control group mice (G1) were injected with 10 mL / kg vehicle (isotonic sodium chloride solution). The treatment started on the grouping day (day 0) and injections were performed twice a week (for a total of 3 weeks).

[0210] As shown in Figure 8B, compared with HSA-IL21-WT, HSA-IL21-C01 and HSA-IL21-C08 showed significant tumor growth inhibition. The individual tumor growth curves and the tumor volumes of each mouse on day 21 are shown in Figures 8C-8F and 8G, respectively. The results indicate that in the CT26 syngeneic mouse model, HSA-IL21-C01 and HSA-IL21-C08 induced significant tumor shrinkage. The results of the body weight changes in Figure 8H suggest that the treatment with HSA-fusion IL-21 and its variants did not induce significant toxicity in CT26 syngeneic mice. An overall summary table of the tumor growth inhibition rate (%TGI) is shown in Figure 8I. The results indicate that in the CT26 syngeneic mouse model, the TGI% of HSA-IL21-C01 and HSA-IL21-C08 was 91% and 99%, respectively. The corresponding P-values were determined to be less than 0.01.

[0211] Example 9. Determination of the efficacy of HSA-fused IL-21 variant in alleviating Treg-mediated T cell suppression. CD4 + CD25 + CD127 低 Treg cells were isolated from hPBMC donors according to the manufacturing protocol. The Treg cells were activated with CD3 / CD28 Dynabeads® (Thermo Fisher, catalog number: 11132D) at a cell-to-bead ratio of 1:10 and treated for 4 days with or without the indicated concentrations of HSA-fused IL-21 and its variants. After 4 days of treatment, the CD3 / CD28 Dynabeads® and IL-21-containing medium was discarded. In addition, CD8 + T cells were isolated from another hPBMC donor according to the manufacturing protocol and then stained with 1 μM CFSE (Thermo Fisher, catalog number: C34554) dye. Treg and CFSE-labeled CD8 + T cells were incubated for 6 days at a cell-to-cell ratio of 1:8. The cells were then harvested and stained with PE-anti-CD8 antibody (BioLegend, catalog number: 980902). T cell proliferation was observed using CD8 antibody. + CFSE 低 It was determined by the proportion of cells.

[0212] As shown in Figures 9A-9B, all IL-21 variants are CD8 mediated by Treg cells. + We were able to mitigate the induction of T cell suppression. Compared to HSA-IL21-WT, all selected HSA-fusion IL-21 variants (HSA-IL21-C01, HSA-IL21-C03, HSA-IL21-C07, and HSA-IL21-C08) showed a significant enhancement in mitigating Treg-mediated T cell suppression.

[0213] Example 10. Determination of the efficacy of HSA-fused IL-21 variant in inducing cytotoxicity in primary NK cells. The efficacy of the HSA-fused IL-21 variant in inducing NK cell toxicity against K-562 cells was detected as described in Example 7.

[0214] As shown in Figure 10, IL21-WT and the variants demonstrated efficacy in inducing NK cell cytotoxicity. All of the selected HSA-fused IL-21 variants (HSA-IL21-C01, HSA-IL21-C03, HSA-IL21-C07, and HSA-IL21-C08) showed similar efficacy 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.

[0215] Example 11. Evaluation of the antitumor and memory effects of the HSA-fused IL-21 variant. The antitumor efficacy and memory effect of HSA-fused IL-21 and its variants were evaluated in a CT26 tumor-carrying BALB / c mouse model. The treatment plan and administration schedule are shown in Figure 11A. To determine antitumor efficacy, 5-6 week old BALB / c mice were selected and administered 2 × 10⁶ times. 5 Individual mouse colon cancer cells (CT26) were subcutaneously inoculated (sc) into one side of all mice. The tumor volume was approximately 100-200 mm². 3 When the mice reached the required stage (4 days after tumor inoculation), they were randomly assigned to three treatment groups (16 mice per group) and one control group (including 8 mice). Treatment mice were intraperitoneally (ip) injected with 10 mg / kg of HSA-IL21-WT (G2), HSA-IL21-C01 (G3), or HSA-IL21-C08 (G4). Control mice (G1) were injected with 10 mL / kg vehicle (isotonic sodium chloride solution). Treatment began on the grouping day (day 4) and injections were given twice a week (for a total of 3 weeks).

[0216] After six doses, the mice were allowed to rest (no treatment after 21 days), and tumor growth was observed. 40 days after the first vaccination, the tumor size was 50 mm. 3CT26 tumor-bearing mice from groups G3 and G4 that were below a certain level were selected for tumor rechallenge, and the memory effect of HSA-fused IL-21 and its variants was determined. Ten out of 16 mice from the original G3 group and eleven out of 16 mice from the original G4 group were given 2 × 10⁶ doses. 5 CT26 cells were subcutaneously (sc) inoculated into the opposite side of the mouse. A new control group of mice (G5, 6 mice) was used as a re-challenge control group, with 2 × 10⁶ cells inoculated. 5 Individual CT26 cells were inoculated subcutaneously (sc).

[0217] As shown in Figure 11B, HSA-IL21-C01 and HSA-IL21-C08 showed significant inhibition of tumor growth compared to HSA-IL21-WT. Individual tumor growth curves for each mouse in groups G1 to G4 are shown in Figures 11C to 11F, respectively. In these figures, the dashed line represents half of the average tumor volume of the vehicle group (G1) on day 14, i.e., 458 mm². 3 This shows that the ratios after "D14" and "D40" represent the total number of mice in the original group, with tumor sizes of 458 mm at 14 and 40 days after tumor inoculation, respectively. 3 The number of mice is less than 1. The results show that HSA-IL21-C01 and HSA-IL21-C08 induced significant tumor reduction in the CT26 syngeneic mouse model. The results of body weight change in Figure 11G suggest that treatment with HSA-fused IL-21 and its variants did not induce significant toxicity in CT26 syngeneic mice. The mouse survival curves in Figure 11H show that HSA-IL21-C01 and HSA-IL21-C08 significantly extended mouse survival compared to HSA-IL21-WT. As shown in Figures 11I-11K, HSA-IL21-C01 and HSA-IL21-C08 showed a memory effect in the CT26-carrying BALB / c syngeneic mouse model. The results indicate that IL-21 signaling is important for generating a tumor-specific memory response to tumor rechallenge, and that the HSA-fused IL-21 variants tested were able to induce a robust tumor-specific memory response.

[0218] Example 12. Expression and purification of Fc-fused IL-21 variant To test other forms of IL-21 variants, heterodimer Fc-fusion IL-21 variants were designed. Specifically, each heterodimer Fc-fusion IL-21 variant comprises two polypeptide chains: a first polypeptide chain (or "hole chain") containing, from N-terminus to C-terminus, a human IgG4 hinge region (SEQ ID NO: 31), a human IgG4 Fc region with a hole mutation (SEQ ID NO: 33), a linker peptide (SEQ ID NO: 34), and an IL-21 variant (e.g., any one of the above IL-21 variants); and a second polypeptide chain (or "knob chain") containing, from N-terminus to C-terminus, a human IgG4 hinge region (SEQ ID NO: 31) and a human IgG4 Fc region with a knob mutation (SEQ ID NO: 32). Plasmids expressing heterodimer Fc-fusion IL-21 and its variants were constructed. Expi293 cells were transfected to express the following molecules: heterodimer Fc-IL21-WT (hole chain: SEQ ID NO: 35; knob chain: SEQ ID NO: 29), heterodimer Fc-IL21-C01 (hole chain: SEQ ID NO: 36; knob chain: SEQ ID NO: 29), heterodimer Fc-IL21-C03 (hole chain: SEQ ID NO: 37; knob chain: SEQ ID NO: 29), heterodimer Fc-IL21-C07 (hole chain: SEQ ID NO: 38; knob chain: SEQ ID NO: 29), and heterodimer Fc-IL21-C08 (hole chain: SEQ ID NO: 39; knob chain: SEQ ID NO: 29).

[0219] The following reasons were considered in order to construct a heterodimer Fc-fusion IL-21 variant. First, several reports indicated that HSA fusion proteins may have precipitation problems under 37°C and acidic pH conditions. Second, it was intended that the heterodimer Fc fusion protein would be more stable than the HSA fusion protein. Finally, preliminary data showed that heterodimer Fc-IL21-WT exhibits stronger IL-21 signaling induction activity than homodimer Fc-IL21-WT (e.g., G4Fc-IL21-WT containing two identical polypeptide chains with the amino acid sequences of each chain shown in SEQ ID NO: 16).

[0220] The purified heterodimers Fc-IL21-WT, Fc-IL21-C01, Fc-IL21-C03, Fc-IL21-C07, and Fc-IL21-C08 were used in subsequent experiments.

[0221] Example 13. Determination of the efficacy of heterodimer Fc-fused IL-21 variant in inducing IL-21-STAT3 reporter signaling. An IL-21 reporter assay was performed to test the IL-21-phosphorylated STAT3 (pSTAT3) signaling induction activity of heterodimer Fc-fused IL-21 and its variants. Specifically, 2.8 × 10⁻¹⁶ 5 HEK-Blue® IL-21 (InvivoGen, catalog number: hkb-il21) at a concentration of cells / mL was resuspended in pre-warmed assay medium (DMEM (Dulbecco's modified Eagle medium; CORNING, catalog number: 10-013-CV), 10% (v / v) thermally inactivated FBS (Gibco, catalog number: A31606-01), and 100× penicillin-streptomycin solution (CORNING, catalog number: 30-002-CI)). 180 μL of HEK-Blue® IL-21 cells were incubated with 20 μL of serially diluted heterodimerized Fc-fused IL-21 and its variants in a 96-well TC-treated microplate (CORNING, catalog number: 3599) at 37°C for 22 hours. After incubation, 20 μL of the supernatant of induced HEK-Blue® IL-21 cells was incubated with 180 μL of resuspended QUANTI-Blue® solution (InvivoGen, catalog number: rep-qbs2) in a flat-bottomed 96-well plate (Paul Bottger, catalog number: 05-031-0100) at 37°C for 2-3 hours. SEAP (secreted alkaline phosphatase) activity was measured at 630 nm using a spectrophotometer (Varioskan® LUK, Thermo Scientific, model 3020).

[0222] The EC50 values ​​for each heterodimer Fc-fusion IL-21 variant are shown in the table below.

[0223] [Table 3]

[0224] As shown in Figure 12 and the table above, heterodimers Fc-IL21-C01, C03, C07, and C08 showed similar IL-21-STAT3 signaling induction activity compared to heterodimer Fc-IL21-WT.

[0225] Example 14. Determination of the efficacy of heterodimer Fc-fused IL-21 variant in inducing proliferation of T cells and NK-92 cells. In the case of a T cell proliferation assay, CD8 + T cells were isolated from hPBMC donors according to the manufacturing protocol. CD8 + T cells were activated for 2 days with CD3 / CD28 Dynabeads® (Thermo Fisher, catalog number: 11132D) at a cell-to-bead ratio of 4:1. The activated T cells were rested for 24 hours, and then 5 × 10⁶ 4 CD8 in cells / wells + T cells were incubated for 5 days with heterodimer Fc-fused IL-21 and its variants at the indicated concentrations (200 nM, 5-fold serial dilution, 10 spots) in the presence of 1 μg / ml soluble anti-CD3 antibody (BioLegend, catalog number: 317347). After incubation, T cell proliferation was determined by the CellTiter-Glo® luminescence cell viability assay (Promega, catalog number: G7573). For the NK-92 cell proliferation assay, NK-92 cells were starved for 24 hours in a medium without IL-2 supplementation. After starvation, 5 × 10⁶ cells were observed. 4NK-92 cells per cell / well were further incubated for 24 hours in complete medium (75% MEM-α, ribonucleosides and deoxyribonucleosides (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 (BioLegend, catalog number: 589108)) with the indicated concentrations (200 nM, 5-fold serial dilution, 8 spots) of the heterodimeric Fc-fusion IL-21 or its variants. Proliferation was determined by the CellTiter-Glo® Luminescent Cell Viability Assay.

[0226] As shown in Figure 13A and the following table, the heterodimers Fc-IL21-C01, C03, C07, and C08 showed relatively stronger potency than the heterodimer Fc-IL21-WT for the induction of activated CD8 + T cell proliferation.

[0227]

Table 4

[0228] As shown in Figure 13B and the following table, the heterodimers Fc-IL21-C01, C07, and C08 showed better potency than the heterodimers Fc-IL21-WT and C03 for the induction of NK-92 cell proliferation.

[0229]

Table 5

[0230] Example 15. Determination of the potency of heterodimeric Fc-fusion IL-21 variants for the induction of cytotoxicity in primary NK cells The efficacy of heterodimer Fc-fused IL-21 variants in inducing cytotoxicity in primary NK cells 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 stained with 5 μM CellTrace®-Violet dye (ThermoFisher, catalog number: C34557). Effector cells (primary NK cells) and target cells (Violet-labeled K-562) were co-incubated at 8:1 cell-to-cell ratio in 96-well U-type cell culture plates at 37°C for 24 hours in the presence of 0.00001–100 nM heterodimer Fc-fused IL-21 or its variants. Subsequently, the cells were harvested and stained with 7-AAD viability stain (BioLegend, catalog number: 420404). The following formula: (CellTrace-Violet + 7-AAD + Cell) / (CellTrace-Violet + The cytotoxicity of NK cells was determined by the percentage of K-562 tumor-dead cells calculated as (cells) × 100 (%).

[0231] As shown in Figure 14, the heterodimer Fc-IL21-WT and its variants demonstrated efficacy in inducing cytotoxicity in NK cells. Heterodimers Fc-IL21-C07 and C08 showed slightly stronger efficacy in inducing cytotoxicity in primary NK cells than heterodimer Fc-IL21-WT and other variants. The results indicate that selected heterodimer Fc-IL-21 variants were able to induce specific lysis of K-562 tumor cells mediated by primary NK cells.

[0232] Example 16. Evaluation of the antitumor efficacy of heterodimer Fc-fused IL-21 variant. The antitumor efficacy of heterodimer Fc-fused IL-21 and its variants was evaluated in a CT26 tumor-carrying BALB / c mouse model. The treatment plan and administration schedule are shown in Figure 15A. Specifically, 5-week-old BALB / c mice were selected and administered 2 × 10⁶ times. 5100 mice were subcutaneously inoculated with CT26 colon cancer cells. Treatment with the tested molecules was initiated four days after tumor inoculation. The treatment group mice received intraperitoneal (ip) injections of 10 mg / kg of heterodimer Fc-IL21-WT (G2), heterodimer Fc-IL21-C01 (G3), heterodimer Fc-IL21-C03 (G4), heterodimer Fc-IL21-C07 (G5), and heterodimer Fc-IL21-C08 (G6), respectively. The control group mice (G1) were injected with 10 mL / kg vehicle (isotonic sodium chloride solution). Injections were given twice a week for a total of three weeks.

[0233] The individual tumor growth curves for each mouse in groups G1 to G6 are shown in Figures 15B to 15G, respectively. In these figures, the dashed line represents half of the average tumor volume of the vehicle group (G1) on day 13, i.e., 286 mm². 3 This shows that the ratios after "D13," "D20," and "D41" represent the tumor size at 13, 20, and 41 days after tumor inoculation relative to the total number of surviving mice on the same day, respectively. 3 The number of mice less than 100 is shown. The results showed that heterodimer Fc-IL21-C01 significantly inhibited tumor growth compared to heterodimer Fc-IL21-WT and other variants. In addition, after discontinuation of treatment (after 20 days), heterodimers Fc-IL21-C01, C03, and C08 induced significant tumor reduction in this CT26 syngeneic mouse model. It is possible that heterodimers Fc-IL21-C01, C03, and C08 inhibited tumor growth by inducing a tumor-specific memory response. The results of body weight change in Figure 15H suggest that treatment with heterodimer Fc-fused IL-21 and its variants did not induce significant toxicity in CT26 syngeneic mice.

[0234] Other Embodiments While the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to be illustrative and not to limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A modified IL-21 polypeptide containing unnatural disulfide bonds.

2. The manipulated IL-21 polypeptide according to claim 1, comprising an amino acid sequence that is at least 80% identical to SEQ ID NO:

2.

3. An engineered IL-21 polypeptide according to claim 1 or 2, which can bind to a complex formed by the human IL-21 receptor (IL-21R) and the common cytokine γ chain (γc).

4. below: (a) The amino acid corresponding to position 8 of Sequence ID No. 2 is cysteine ​​(C). (b) The amino acid corresponding to position 19 of sequence number 2 is C. (c) The amino acid corresponding to position 29 of Sequence ID No. 2 is C. (d) The amino acid corresponding to position 31 of sequence number 2 is C. (e) The amino acid corresponding to position 33 of sequence number 2 is C. (f) The amino acid corresponding to position 36 of sequence number 2 is C. (g) The amino acid corresponding to position 39 of Sequence ID No. 2 is C, and (h) The amino acid corresponding to position 56 of Sequence ID No. 2 is C. An operated IL-21 polypeptide according to any one of claims 1 to 3, comprising one or more of the above.

5. below: (a) The amino acid corresponding to position 61 of sequence number 2 is C. (b) The amino acid corresponding to position 62 of sequence number 2 is C. (c) The amino acid corresponding to position 63 of Sequence ID No. 2 is C. (d) The amino acid corresponding to position 80 of Sequence ID No. 2 is C. (e) The amino acid corresponding to position 86 of sequence number 2 is C. (f) The amino acid corresponding to position 105 of sequence number 2 is C. (g) The amino acid corresponding to position 106 of sequence number 2 is C. (h) The amino acid corresponding to position 107 of sequence number 2 is C. (i) The amino acid corresponding to position 110 of sequence number 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 sequence number 2 is C. An operated IL-21 polypeptide according to any one of claims 1 to 4, comprising one or more of the above.

6. below: (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, (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. An operated IL-21 polypeptide according to any one of claims 1 to 5, comprising one or more of the above.

7. below: (a) The amino acid corresponding to position 12 of sequence number 2 is M. (b) The amino acid corresponding to position 16 of Sequence ID No. 2 is R. (c) The amino acid corresponding to position 19 of sequence number 2 is I. (d) The amino acid corresponding to position 23 of Sequence ID No. 2 is D. (e) The amino acid corresponding to position 105 of sequence number 2 is E. (f) The amino acid corresponding to position 114 of sequence number 2 is E. (g) The amino acid corresponding to position 118 of sequence number 2 is S. (h) The amino acid corresponding to position 121 of sequence number 2 is Q. (i) The amino acid corresponding to position 122 of sequence number 2 is K. (j) The amino acid corresponding to position 124 of sequence number 2 is I. (k) The amino acid corresponding to position 125 of Sequence ID No. 2 is H, and (l) The amino acid corresponding to position 128 of sequence number 2 is L. An operated IL-21 polypeptide according to any one of claims 1 to 6, comprising one or more of the above.

8. An engineered IL-21 polypeptide according to any one of claims 1 to 7, comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

9. The manipulated IL-21 polypeptide according to claim 8, comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

3.

10. The manipulated IL-21 polypeptide according to claim 8, comprising an amino acid sequence that is at least 90% identical to that of SEQ ID NO:

5.

11. The manipulated IL-21 polypeptide according to claim 8, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:

9.

12. The manipulated IL-21 polypeptide according to claim 8, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:

10.

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

14. A modified IL-21 polypeptide according to any one of claims 1 to 13, which can induce phosphorylation of STAT-3.

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

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

17. The fusion protein according to claim 16, wherein the HSA comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

26.

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

19. The fusion protein according to claim 18, wherein the linker peptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

25.

20. A fusion protein according to any one of claims 15 to 19, further comprising a His tag optionally at its N-terminus.

21. A fusion protein according to any one of claims 15 to 20, comprising an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17, 18, 19, 20, or 21.

22. Selectively, from the N-terminus towards the C-terminus, (a) Optionally, the His tag and (b) HSA and, (c) Linker peptide and (d) Manipulated IL-21 polypeptide and A fusion protein containing [the specified ingredient].

23. The His tag contains an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22, the HSA contains an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 26, and the linker peptide contains an amino acid sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 25 The fusion protein according to claim 22, comprising an amino acid sequence that is 96%, 97%, 98%, 99%, or 100% identical, and / or the manipulated IL-21 polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

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

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

26. A fusion protein according to any one of claims 15 to 25, which can induce cytotoxicity in primary NK cells.

27. (a) A first polypeptide comprising, from the N-terminus to the C-terminus, an optionally first hinge region, a first Fc region, an optionally linker peptide, and an operated IL-21 polypeptide according to any one of claims 1 to 14, (b) A second polypeptide comprising an optional second hinge region and a second Fc region extending from the N-terminus toward the C-terminus A protein complex containing [the specified ingredient].

28. The protein complex according to 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 according to claim 27 or 28, wherein the first Fc region and / or the second Fc region comprises one or more knob-into-hole (KIH) mutations.

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

31.

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

34.

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

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

35. A protein complex according to any one of claims 27 to 34, which can induce proliferation of immune cells (e.g., T cells or NK cells) and / or induce cytotoxicity of primary NK cells.

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 described in claim 37.

39. A cell comprising the nucleic acid described in claim 37 or the vector described in claim 38.

40. The cell according to claim 39, which is an Expi293 cell or a CHO-S cell.

41. A method for producing a manipulated IL-21 polypeptide or a fusion protein comprising the manipulated IL-21 polypeptide, (a) Culturing the cells according to claim 39 or 40 under conditions sufficient to produce the manipulated IL-21 polypeptide or the fusion protein, (b) Recovering the manipulated IL-21 polypeptide, the fusion protein, or the protein complex produced by the cells. The method, including the method described above.

42. A method for treating a subject having cancer, 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 according to claim 42, wherein the subject has a solid tumor or hematological cancer.

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

45. A method for reducing the rate of tumor growth, comprising contacting tumor cells with an 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.

46. A method for killing tumor cells, comprising contacting the tumor cells with an 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.

47. A method for improving the stability of proteins (e.g., cytokines), (a) To provide the 3D structure of the protein (e.g., cytokine), (b) One or more C amino acid residues in the 3D structure アルファ Measuring the distance between atoms, (c) Selecting two amino acid residues from one or more of the aforementioned amino acid residues, wherein the C of the two selected amino acid residues アルファ The atom is within 3 to 7 angstroms, and the selection is as described above. The method, including the method described above.

48. The method according to 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 according to 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 200 or fewer amino acid residues.

51. A method for screening cytokine variants that have improved antitumor efficacy, (a) To provide the 3D structure of cytokines, (b) One or more C amino acid residues in the 3D structure アルファ Measuring the distance between atoms, (c) Selecting two amino acid residues from one or more of the aforementioned amino acid residues, wherein the C of the two selected amino acid residues アルファ The atom is within 3 to 7 angstroms, and the selection is as described above. The method, including the method described above.

52. (d) Expressing a cytokine variant, wherein the variant includes a non-natural disulfide bond formed by mutating the two selected amino acid residues to cysteine, (e) Administering the cytokine variant to tumor-bearing animals, (f) Determining tumor growth in the tumor-carrying animal (for example, by measuring tumor volume) The method according to claim 51, further comprising:

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

Images