Il-2 fusion protein comprising il-2 receptor variant and use thereof

Abstract

Provided in the present invention is a truncated variant of interleukin-2 receptor α (IL-2Rα), the truncated variant comprising 166 to 190 or 167 to 190 consecutive amino acids from the N-terminus of the extracellular domain of wild-type IL-2Rα. Further provided in the present invention are a fusion protein comprising the truncated variant of IL-2Rα and the use thereof. By means of the unique molecular design of the fusion protein, a novel IL-2 therapy with improved therapeutic effect and safety is provided in the present invention.

Description

IL-2 fusion proteins containing IL-2 receptor variants and their uses Technical Field

[0001] This application relates to the field of biomedicine, specifically to an interleukin-2 (IL-2) fusion protein. Background Technology

[0002] Interleukin-2 (IL-2) can stimulate the proliferation and differentiation of T cells, induce the production of cytotoxic T lymphocytes (CTLs) and the differentiation of peripheral blood lymphocytes into cytotoxic killer cells activated by lymphokines, promote the expression of cytokines and cytolytic molecules by T cells, promote the proliferation and differentiation of B cells and the synthesis of immunoglobulins by B cells, and stimulate the proliferation and activation of natural killer (NK) cells. However, the use of high-dose IL-2 to treat cancer can cause serious non-specific toxicity, which may lead to serious complications such as hypotension and acute renal failure, as well as common side effects such as fever, chills, fatigue, nausea and vomiting, placing a significant burden on the patient's body and potentially endangering their life.

[0003] The main serious side effect of IL-2 therapy lies in the fact that systemic administration can activate immune cells throughout the body. If IL-2 could be concentrated in the tumor site rather than distributed evenly throughout the body, it might be possible to reduce side effects while maintaining efficacy against the tumor. Interleukin-2 (IL-2) fusion proteins are genetically engineered to contain both IL-2 and interleukin-2 receptor α (IL-2Rα). IL-2Rα and IL-2 are linked by a cleavable linker. Upon reaching tumor tissue, the linker is cleaved by tumor-associated proteases, releasing IL-2 to exert its function.

[0004] Various IL-2 variants and prodrugs have been developed with the aim of reducing the toxicity of IL-2 in cancer treatment. However, they have encountered several problems, such as diminished efficacy and increased immunogenicity. Fusion proteins containing IL-2 or its variants, such as IL-2-Fc fusion proteins, may be structurally fragile, unstable, and highly toxic. Fusion proteins containing the full length of IL-2Rα suffer from protein aggregation, difficulty in drug formation, and significant weight loss in mice during in vivo experiments, indicating high toxicity. These characteristics severely limit their clinical prospects.

[0005] Therefore, there is a need in the field for novel IL-2 therapies with improved therapeutic efficacy and safety. Summary of the Invention

[0006] This application provides a fusion protein containing an IL-2Rα truncated form, specifically, the fusion protein has reduced toxicity and high stability.

[0007] Specifically, the first aspect of this application relates to the following technical solution:

[0008] 1. A fusion protein comprising:

[0009] Interleukin-2 (IL-2) wild-type or mutant protein;

[0010] A cuttable connector connected to the IL-2;

[0011] Interleukin-2 receptor α (IL-2Rα) truncated bodies connected to the said cleavable connector; and

[0012] Target-binding peptides,

[0013] The IL-2Rα truncated form comprises an amino acid sequence selected from the C-terminus of the IL-2Rα extracellular domain, truncated to a length of 29 to 52 amino acids.

[0014] In this application, the IL-2Rα truncated form comprises an amino acid sequence truncated by 29 to 52 amino acids from the C-terminus of the IL-2Rα extracellular domain, which is equivalent to "the IL-2Rα truncated form comprises 167 to 190 amino acids continuously starting from the N-terminus of the wild-type IL-2Rα extracellular domain".

[0015] 2. The fusion protein according to claim 1, wherein the truncated IL-2Rα comprises: C-terminal amino acids 168-219aa, 169-219aa, 170-219aa, 171-219aa, 172-219aa, 173-219aa, 174-219aa, 175-219aa, 176-219aa, 177-219aa, 1... Amino acid sequences missing from 78-219aa, 179-219aa, 180-219aa, 181-219aa, 182-219aa, 183-219aa, 184-219aa, 185-219aa, 186-219aa, 187-219aa, 188-219aa, 189-219aa, 190-219aa, or 191-219aa.

[0016] 3. The fusion protein according to claim 1, wherein the IL-2Rα truncated form comprises an amino acid sequence in which the C-terminal amino acids 173-219aa, 174-219aa, 175-219aa, 176-219aa, 177-219aa, 178-219aa, 179-219aa, 180-219aa, 181-219aa, 182-219aa, or 183-219aa are missing from the IL-2Rα extracellular domain.

[0017] 4. The fusion protein according to item 1, wherein the IL-2Rα truncated form comprises an amino acid sequence in which amino acids 173-219aa of the C-terminal domain of the IL-2Rα extracellular domain are missing.

[0018] 5. The fusion protein according to any one of items 1-4, wherein the IL-2Rα truncated form comprises any amino acid sequence of SEQ ID NO:5-28.

[0019] 6. The fusion protein according to any one of items 1-5, wherein the target-binding polypeptide is linked to the IL-2Rα truncated form via an uncuttable linker.

[0020] 7. The fusion protein according to any one of items 1-5, wherein the target-binding polypeptide is linked to the wild-type or mutant IL-2 protein via an uncleavable linker.

[0021] 8. A fusion protein comprising, in sequence:

[0022] Target-binding peptides, IL-2Rα truncated forms, and wild-type or mutant IL-2 proteins.

[0023] The IL-2Rα truncated form comprises an amino acid sequence selected from the C-terminus of the IL-2Rα extracellular domain, truncated to a length of 29 to 52 amino acids.

[0024] 9. A fusion protein comprising, in sequence:

[0025] Target-binding peptides, wild-type or mutant IL-2 proteins, and IL-2Rα truncated forms.

[0026] The IL-2Rα truncated form comprises an amino acid sequence selected from the C-terminus of the IL-2Rα extracellular domain, truncated to a length of 28 to 52 amino acids.

[0027] 10. The fusion protein according to item 8, wherein the truncated IL-2Rα comprises: C-terminal amino acids 168-219aa, 169-219aa, 170-219aa, 171-219aa, 172-219aa, 173-219aa, 174-219aa, 175-219aa, 176-219aa, 177-219aa from the IL-2Rα extracellular domain. Amino acid sequences missing from 178-219aa, 179-219aa, 180-219aa, 181-219aa, 182-219aa, 183-219aa, 184-219aa, 185-219aa, 186-219aa, 187-219aa, 188-219aa, 189-219aa, 190-219aa, or 191-219aa.

[0028] 11. The fusion protein according to item 9, wherein the truncated IL-2Rα comprises: IL-2Rα extracellular domain C-terminal amino acids 168-219aa, 169-219aa, 170-219aa, 171-219aa, 172-219aa, 173-219aa, 174-219aa, 175-219aa, 176-219aa, 177-219aa, 178-219aa, 17 ...9-219aa, 170-219aa, 179-219aa, 170-219aa, 179-219aa, 170-219aa, 179-219aa, 170-219aa, 179-219aa, 170-219aa, 179-219aa, 170-219aa, 179-219aa, 179-219aa, 179-219aa, 179-219aa, 179-219aa, 179-219aa, 179-219aa, 179-2 Amino acid sequences missing from 19aa, 179-219aa, 180-219aa, 181-219aa, 182-219aa, 183-219aa, 184-219aa, 185-219aa, 186-219aa, 187-219aa, 188-219aa, 189-219aa, 190-219aa, 191-219aa, or 192-219aa.

[0029] 12. The fusion protein according to any one of items 8 or 9, wherein the IL-2Rα truncated form comprises an amino acid sequence in which the C-terminal amino acids 173-219aa, 174-219aa, 175-219aa, 176-219aa, 177-219aa, 178-219aa, 179-219aa, 180-219aa, 181-219aa, 182-219aa, or 183-219aa are missing.

[0030] 13. The fusion protein according to any one of items 8 or 9, wherein the IL-2Rα truncated form comprises an amino acid sequence in which amino acids 173-219aa of the C-terminal amino acid domain of the IL-2Rα are missing.

[0031] 14. The fusion protein according to any one of items 8-13, wherein the IL-2Rα truncated form comprises any amino acid sequence of SEQ ID NO:4-28.

[0032] 15. The fusion protein according to any one of items 8, 10 or 12-13, wherein the target-binding polypeptide is linked to the IL-2Rα truncated variant via an uncuttable linker.

[0033] 16. The fusion protein according to any one of items 9, 11 or 12-13, wherein the target-binding polypeptide is linked to the wild-type or mutant IL-2 protein via an incleavable linker.

[0034] 17. The fusion protein according to any one of items 8-16, wherein the IL-2 of the fusion protein is connected to the truncated IL-2Rα form by a cleavable linker.

[0035] 18. A fusion protein comprising:

[0036] Half-life extenders;

[0037] Interleukin-2 (IL-2) wild-type or mutant protein;

[0038] A cuttable connector connected to the IL-2; and

[0039] The IL-2Rα truncated section connected to the cuttable connector,

[0040] The IL-2Rα truncated form comprises an amino acid sequence selected from the C-terminus of the IL-2Rα extracellular domain, truncated to a length of 29 to 52 amino acids.

[0041] 19. The fusion protein according to claim 18, wherein the truncated IL-2Rα comprises: C-terminal amino acids 168-219aa, 169-219aa, 170-219aa, 171-219aa, 172-219aa, 173-219aa, 174-219aa, 175-219aa, 176-219aa, 177-219aa from the IL-2Rα extracellular domain. Amino acid sequences missing from 178-219aa, 179-219aa, 180-219aa, 181-219aa, 182-219aa, 183-219aa, 184-219aa, 185-219aa, 186-219aa, 187-219aa, 188-219aa, 189-219aa, 190-219aa, or 191-219aa.

[0042] 20. The fusion protein according to claim 18, wherein the IL-2Rα truncated form comprises an amino acid sequence in which the C-terminal amino acids 173-219aa, 174-219aa, 175-219aa, 176-219aa, 177-219aa, 178-219aa, 179-219aa, 180-219aa, 181-219aa, 182-219aa, or 183-219aa are missing from the IL-2Rα extracellular domain.

[0043] 21. The fusion protein according to item 18, wherein the IL-2Rα truncated form comprises an amino acid sequence in which amino acids 173-219aa of the C-terminal domain of the IL-2Rα extracellular domain are missing.

[0044] 22. The fusion protein according to any one of items 18-21, wherein the IL-2Rα truncated form comprises any amino acid sequence of SEQ ID NO:5-28.

[0045] 23. The fusion protein according to any one of items 18-22, wherein the half-life extender is linked to the IL-2Rα truncated body via an incuttable linker.

[0046] 24. The fusion protein according to any one of items 18-22, wherein the half-life extender is linked to the wild-type or mutant IL-2 protein via an incleavable linker.

[0047] 25. The fusion protein according to any one of items 18-24, wherein the half-life extender is selected from: the Fc region of an antibody, or human serum albumin.

[0048] 26. The fusion protein according to any one of items 18-25, wherein the half-life extender is an antibody Fc region selected from: wild-type Fc region, mutant Fc region, monomeric wild-type Fc region, monomeric mutant Fc region, dimeric wild-type Fc region, or dimeric mutant Fc region.

[0049] 27. The fusion protein according to any one of items 18-26, wherein the half-life extender comprises SEQ ID NO:36.

[0050] 28. The fusion protein according to any one of items 1-27, wherein the uncleavable linker is not cleaved by tumor-associated proteases.

[0051] 29. The fusion protein according to any one of items 1-28, wherein the uncuttable linker is a glycine / serine linker.

[0052] 30. The fusion protein according to any one of items 1-29, wherein the uncuttable linker comprises SEQ ID NO:39.

[0053] 31. The fusion protein according to any one of items 1-30, wherein the cleavable linker is cleavable by a tumor-associated protease.

[0054] 32. The fusion protein according to any one of items 1-31, wherein the cleavable linker is cleaved by a matrix metalloproteinase.

[0055] 33. The fusion protein according to any one of items 1-32, wherein the cleavable linker is cleaved by matrix metalloproteinase 14 (MMP14).

[0056] 34. The fusion protein according to any one of claims 1-33, wherein the cleavable linker is cleaved by caspase, the caspase being selected from: caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11 or caspase 12.

[0057] 35. The fusion protein according to any one of items 1-34, wherein the cleavable linker comprises any amino acid sequence of SEQ ID NO:32-35.

[0058] 36. The fusion protein according to any one of items 1-35, wherein the target-binding polypeptide is an antibody or a functional fragment thereof.

[0059] 37. The fusion protein according to item 36, wherein the antibody or a functional fragment thereof is selected from: Fab, scFv or nanobody.

[0060] 38. The fusion protein according to any one of items 1-37, wherein the target-binding polypeptide is selected from T cell target-binding polypeptides and tumor cell target-binding polypeptides;

[0061] Preferably, the target-binding polypeptide, the first target-binding polypeptide, and / or the second target-binding polypeptide can bind to one or more targets;

[0062] Preferably, the target-binding polypeptide, the first target-binding polypeptide, and / or the second target-binding polypeptide can bind to one or more targets;

[0063] Preferably, the target-binding peptide is capable of binding to one of the following targets: CLDN18.2, PD-1, PD-L1, VEGFR, HER2;

[0064] Preferably, the target-binding peptide can bind to one of the following target combinations: PD-1 / VEGF combination, or CLDN18.2 / CD3 combination;

[0065] Preferably, the target of the T cell target-binding peptide is selected from CD3 and / or PD-1, and the target of the tumor cell target-binding peptide is selected from one or more of CLDN18.2, PD-L1, VEGFR, and HER2.

[0066] 39. The fusion protein according to any one of items 1-38, wherein the target-binding polypeptide comprises any amino acid sequence of SEQ ID NO:36-38.

[0067] 40. The fusion protein according to any one of items 1-39, wherein the wild-type IL-2 comprises at least 95% or 100% the same amino acid sequence as SEQ ID NO:1.

[0068] 41. The fusion protein according to any one of items 1-40, wherein the IL-2 mutant protein comprises one or more amino acid mutations.

[0069] 42. The fusion protein according to any one of items 1-41, wherein the IL-2 mutant protein comprises one or more of the following mutations: R81E, L85T or L85I.

[0070] 43. The fusion protein according to any one of items 1-42, wherein the IL-2 wild-type or mutant protein comprises any amino acid sequence in SEQ ID NO:1-2.

[0071] 44. The fusion protein according to any one of claims 1-43, wherein the fusion protein comprises a first chain and a second chain, the first chain and the second chain comprising a sequence having at least 95% sequence identity with respect to the following amino acid sequence, wherein:

[0072] The amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:40; or

[0073] The amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:41; or

[0074] The amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:42; or

[0075] The amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:43.

[0076] 45. The fusion protein according to any one of items 1-44, wherein the fusion protein is a dimer.

[0077] 46. ​​The fusion protein according to item 45, wherein the fusion protein is a homodimer.

[0078] 47. The fusion protein according to item 45, wherein the fusion protein is a heterodimer.

[0079] 48. The fusion protein according to any one of claims 1-47, wherein the fusion protein containing the truncated IL-2Rα has reduced toxicity compared to the fusion protein containing the untruncated IL-2Rα.

[0080] 49. A polynucleotide encoding a fusion protein described in any one of terms 1-48.

[0081] 50. An expression vector comprising the polynucleotide described in item 49.

[0082] 51. A host cell comprising the polynucleotide of claim 49 or the expression vector of claim 50.

[0083] 52. A method for preparing a fusion protein, comprising culturing a host cell according to item 51.

[0084] 53. The method for preparing a fusion protein according to item 52, the method further comprising purifying or isolating the produced fusion protein.

[0085] 54. A pharmaceutical composition comprising the fusion protein described in any one of claims 1-48 and a pharmaceutically acceptable excipient, carrier, or diluent.

[0086] 55. A method for treating or alleviating a disease or symptom, comprising:

[0087] Administer to a patient in need a therapeutically effective amount of any of the fusion proteins described in items 1-48, the polynucleotide described in item 49, the expression vector described in item 50, the host cell described in item 51, or the pharmaceutical composition described in item 53.

[0088] 56. The method according to item 55, wherein the patient in need is a human.

[0089] 57. The method according to any one of items 55 or 56, wherein the disease or symptom is cancer.

[0090] 58. The method according to any one of items 55-57, wherein the disease or condition is selected from: acute myeloid leukemia, adrenocortical carcinoma, B-cell lymphoma, urothelial carcinoma of the bladder, ductal carcinoma of the breast, lobular carcinoma of the breast, esophageal cancer, castration-resistant prostate cancer (CRPC), cervical cancer, cholangiocarcinoma, chronic myeloid leukemia, colorectal adenocarcinoma, colorectal cancer (CRC), esophageal cancer, gastric adenocarcinoma, glioblastoma multiforme, squamous cell carcinoma of the head and neck, Hodgkin lymphoma / primary mediastinal B-cell lymphoma, liver cell carcinoma. HCC, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, low-grade glioma, lung adenocarcinoma, lung squamous cell carcinoma, melanoma (MEL), mesothelioma, non-squamous NSCLC, ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma, paraganglioma and pheochromocytoma, prostate cancer, renal cell carcinoma (RCC), sarcoma, cutaneous melanoma, head and neck squamous cell carcinoma, T-cell lymphoma, thymoma, papillary thyroid carcinoma, uterine carcinosarcoma, endometrioid carcinoma of the uterine body or uveal melanoma.

[0091] 59. Use of the fusion protein of any one of items 1-48, the polynucleotide of item 49, the expression vector of item 50, the host cell of item 51, or the pharmaceutical composition of item 53 for the treatment or relief of a disease or condition.

[0092] 60. Use of the fusion protein of any one of items 1-48, the polynucleotide of item 49, the expression vector of item 50, the host cell of item 51, or the pharmaceutical composition of item 53 in the preparation of a medicament for treating or alleviating a disease or condition.

[0093] The second aspect of this application relates to the following technical solution:

[0094] 1. A fusion protein comprising:

[0095] Interleukin-2 (IL-2) wild-type or mutant protein;

[0096] A cuttable connector connected to the IL-2;

[0097] The interleukin-2 receptor α (IL-2Rα) truncated variant linked to the cleavable linker; and

[0098] Target-binding peptides,

[0099] The IL-2Rα truncated form comprises an amino acid sequence selected from the C-terminus of the IL-2Rα extracellular domain, truncated to a length of 29 to 52 amino acids.

[0100] In this application, the IL-2Rα truncated form comprises an amino acid sequence truncated by 29 to 52 amino acids from the C-terminus of the IL-2Rα extracellular domain, which is equivalent to "the IL-2Rα truncated form comprises 167 to 190 amino acids continuously starting from the N-terminus of the wild-type IL-2Rα extracellular domain".

[0101] 2. The fusion protein according to claim 1, wherein the truncated IL-2Rα comprises: C-terminal amino acids 168-219aa, 169-219aa, 170-219aa, 171-219aa, 172-219aa, 173-219aa, 174-219aa, 175-219aa, 176-219aa, 177-219aa, 1... Amino acid sequences missing from 78-219aa, 179-219aa, 180-219aa, 181-219aa, 182-219aa, 183-219aa, 184-219aa, 185-219aa, 186-219aa, 187-219aa, 188-219aa, 189-219aa, 190-219aa, or 191-219aa.

[0102] 3. The fusion protein according to claim 1, wherein the IL-2Rα truncated form comprises an amino acid sequence in which the C-terminal amino acids 173-219aa, 174-219aa, 175-219aa, 176-219aa, 177-219aa, 178-219aa, 179-219aa, 180-219aa, 181-219aa, 182-219aa, or 183-219aa are missing from the IL-2Rα extracellular domain.

[0103] 4. The fusion protein according to item 1, wherein the IL-2Rα truncated form comprises an amino acid sequence in which amino acids 173-219aa of the C-terminal domain of the IL-2Rα extracellular domain are missing.

[0104] 5. The fusion protein according to any one of items 1-4, wherein the IL-2Rα truncated form comprises any amino acid sequence of SEQ ID NO:5-28.

[0105] 6. The fusion protein according to any one of items 1-5, wherein the target-binding polypeptide is linked to the IL-2Rα truncated form via an incleavable linker.

[0106] 7. The fusion protein according to any one of items 1-5, wherein the target-binding polypeptide is linked to the wild-type or mutant IL-2 protein via an incleavable linker.

[0107] 8. A fusion protein selected from any combination of the following polypeptide chains:

[0108] (1) Two first polypeptide chains, wherein the first polypeptide chain comprises, sequentially, a target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is linked to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is linked to the single-chain Fc via a non-cleavable linker.

[0109] (2) The first polypeptide chain: sequentially comprises a target-binding polypeptide, a single-chain Fc, and an IL-2Rα truncated variant; the second polypeptide chain: sequentially comprises a target-binding polypeptide, a single-chain Fc, and IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is linked to the single-chain Fc via a cleavable linker, and the IL-2Rα truncated variant is linked to the single-chain Fc via a non-cleavable linker.

[0110] (3) The first polypeptide chain: sequentially contains a target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated form, and IL-2 wild-type or mutant protein; the second polypeptide chain: sequentially contains a target-binding polypeptide and a single-chain Fc, wherein the IL-2 wild-type or mutant protein is linked to the IL-2Rα truncated form via a cleavable linker.

[0111] (4) First polypeptide chain: sequentially comprising a first target-binding polypeptide with an scFv (single-chain variable fragment) structure, a single-chain Fc, and an IL-2Rα truncated form, wherein the IL-2Rα truncated form is linked to the single-chain Fc via a non-cleavable linker; Second polypeptide chain: comprising the heavy chain variable region (VH) of the first target-binding polypeptide, the heavy chain Fd segment of the second target-binding polypeptide, a single-chain Fc, and wild-type or mutant IL-2 protein, wherein the wild-type or mutant IL-2 protein is linked to the single-chain Fc via a cleavable linker; Third polypeptide chain: comprising the light chain variable region (VL) of the first target-binding polypeptide and one light chain of the second target-binding polypeptide.

[0112] (5) First polypeptide chain: sequentially comprising a first target-binding polypeptide with an scFv (single-chain variable fragment) structure, a single-chain Fc, and wild-type or mutant IL-2 protein, wherein the wild-type or mutant IL-2 protein is linked to the single-chain Fc via a cleavable linker; Second polypeptide chain: comprising the heavy chain variable region (VH) of the first target-binding polypeptide, the heavy chain Fd segment of the second target-binding polypeptide, the single-chain Fc, and an IL-2Rα truncated form, wherein the IL-2Rα truncated form is linked to the single-chain Fc via an incleavable linker; Third polypeptide chain: comprising the light chain variable region (VL) of the first target-binding polypeptide and a light chain of the second target-binding polypeptide.

[0113] (6) Two first polypeptide chains, wherein the first polypeptide chain comprises a light chain containing a first target-binding polypeptide; and two second polypeptide chains, wherein the second polypeptide chain comprises, sequentially, a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is linked to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is linked to the single-chain Fc via a non-cleavable linker.

[0114] (7) Two first polypeptide chains, wherein the first polypeptide chain comprises a light chain of a first target-binding polypeptide; the second polypeptide chain comprises, in sequence, a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a single-chain Fc, and IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is linked to the single-chain Fc via a cleavable linker; and the third polypeptide chain comprises, in sequence, a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a single-chain Fc, and an IL-2Rα truncated form, wherein the IL-2Rα truncated form is linked to the single-chain Fc via an uncleavable linker.

[0115] (8) Two first polypeptide chains, wherein the first polypeptide chain comprises, sequentially, a light chain of the first target-binding polypeptide, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is linked to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is linked to a light chain of the first target-binding polypeptide via a non-cleavable linker; and two second polypeptide chains, wherein the second polypeptide chain comprises, sequentially, a heavy chain Fd segment of the first target-binding polypeptide, a single-chain Fc segment, and a second target-binding polypeptide with a scFv (single-chain variable fragment) structure.

[0116] (9) Two first polypeptide chains, wherein the first polypeptide chain comprises a light chain of a first target-binding polypeptide; and two second polypeptide chains, wherein the second polypeptide chain comprises, in sequence, a wild-type or mutant IL-2 protein, an IL-2Rα truncated form, a heavy chain Fd segment of the first target-binding polypeptide, a single-chain Fc segment, and a scFv (single-chain variable fragment) structure of the second target-binding polypeptide, wherein the wild-type or mutant IL-2 protein is linked to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is linked to the heavy chain Fd segment of the first target-binding polypeptide via a non-cleavable linker.

[0117] (10) Two first polypeptide chains, wherein the first polypeptide chain comprises a light chain containing a first target-binding polypeptide; and two second polypeptide chains, wherein the second polypeptide chain comprises, in sequence, a second target-binding polypeptide with a scFv (single-chain variable fragment) structure, a heavy chain Fd segment of the first target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is linked to the IL-2Rα truncated form via a cleavable linker.

[0118] (11) Two first polypeptide chains, wherein the first polypeptide chain comprises, sequentially, a light chain variable region (VL) of a first target-binding polypeptide and a light chain of a second target-binding polypeptide; and two second polypeptide chains, wherein the second polypeptide chain comprises, sequentially, a heavy chain variable region (VH) of the first target-binding polypeptide, a heavy chain Fd segment of the second target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, the IL-2Rα truncated form is connected to the single-chain Fc via a non-cleavable linker, the light chain variable region (VL) of the first target-binding polypeptide is connected to a light chain of the second target-binding polypeptide via a flexible linker 1, and the heavy chain variable region (VH) of the first target-binding polypeptide is connected to the heavy chain Fd segment of the second target-binding polypeptide via a flexible linker 2.

[0119] (12) Two first polypeptide chains, wherein the first polypeptide chain comprises, sequentially, a light chain variable region (VL) of a first target-binding polypeptide and a light chain of a second target-binding polypeptide; the second polypeptide chain comprises, sequentially, a heavy chain variable region (VH) of the first target-binding polypeptide, a heavy chain Fd segment of the second target-binding polypeptide, a single-chain Fc, and an IL-2Rα truncated form, wherein the IL-2Rα truncated form is connected to the single-chain Fc via a non-cleavable linker; and the third polypeptide chain comprises, sequentially, a heavy chain variable region (VH) of the first target-binding polypeptide, a heavy chain Fd segment of the second target-binding polypeptide, a single-chain Fc, and IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the single-chain Fc via a cleavable linker, the light chain variable region (VL) of the first target-binding polypeptide is connected to a light chain of the second target-binding polypeptide via a flexible linker 1, and the heavy chain variable region (VH) containing the first target-binding polypeptide is connected to the heavy chain Fd segment of the second target-binding polypeptide via a flexible linker 2.

[0120] Among them, the IL-2Rα truncated forms described in (1)-(12) contain amino acid sequences selected from the C-terminus of the extracellular domain of IL-2Rα truncated to a length of 29 to 52 amino acids.

[0121] 9. A fusion protein selected from any combination of the following polypeptide chains:

[0122] (1) Two first polypeptide chains, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus, a target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is linked to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is linked to the single-chain Fc via a non-cleavable linker.

[0123] (2) The first polypeptide chain: from the N-terminus to the C-terminus, it contains a target-binding polypeptide, a single-chain Fc, and an IL-2Rα truncated variant; the second polypeptide chain: from the N-terminus to the C-terminus, it contains a target-binding polypeptide, a single-chain Fc, and IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is linked to the single-chain Fc via a cleavable linker, and the IL-2Rα truncated variant is linked to the single-chain Fc via a non-cleavable linker.

[0124] (3) First polypeptide chain: from N-terminus to C-terminus, it contains a target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated variant, and IL-2 wild-type or mutant protein in sequence; Second polypeptide chain: from N-terminus to C-terminus, it contains a target-binding polypeptide and a single-chain Fc in sequence, wherein the IL-2 wild-type or mutant protein is linked to the IL-2Rα truncated variant via a cleavable linker.

[0125] (4) First polypeptide chain: From N-terminus to C-terminus, it sequentially comprises a first target-binding polypeptide with a scFv (single-chain variable fragment) structure, a single-chain Fc, and an IL-2Rα truncated form, wherein the IL-2Rα truncated form is linked to the single-chain Fc via a non-cleavable linker; Second polypeptide chain: From N-terminus to C-terminus, it comprises the heavy chain variable region (VH) of the first target-binding polypeptide, the heavy chain Fd segment of the second target-binding polypeptide, a single-chain Fc, and wild-type or mutant IL-2 protein, wherein the wild-type or mutant IL-2 protein is linked to the single-chain Fc via a cleavable linker; Third polypeptide chain: From N-terminus to C-terminus, it comprises the light chain variable region (VL) of the first target-binding polypeptide and a light chain of the second target-binding polypeptide.

[0126] (5) First polypeptide chain: From N-terminus to C-terminus, it sequentially comprises a first target-binding polypeptide with a scFv (single-chain variable fragment) structure, a single-chain Fc, and wild-type or mutant IL-2 protein, wherein the wild-type or mutant IL-2 protein is linked to the single-chain Fc via a cleavable linker; Second polypeptide chain: From N-terminus to C-terminus, it comprises the heavy chain variable region (VH) of the first target-binding polypeptide, the heavy chain Fd segment of the second target-binding polypeptide, the single-chain Fc, and an IL-2Rα truncated form, wherein the IL-2Rα truncated form is linked to the single-chain Fc via an incleavable linker; Third polypeptide chain: From N-terminus to C-terminus, it comprises the light chain variable region (VL) of the first target-binding polypeptide and a light chain of the second target-binding polypeptide.

[0127] (6) Two first polypeptide chains, wherein the first polypeptide chain comprises a light chain containing a first target-binding polypeptide; and two second polypeptide chains, wherein the second polypeptide chain comprises, from the N-terminus to the C-terminus, a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is linked to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is linked to the single-chain Fc via a non-cleavable linker.

[0128] (7) Two first polypeptide chains, wherein the first polypeptide chain comprises a light chain containing a first target-binding polypeptide; the second polypeptide chain comprises, from the N-terminus to the C-terminus, a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a single-chain Fc, and IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is linked to the single-chain Fc via a cleavable linker; and the third polypeptide chain comprises, from the N-terminus to the C-terminus, a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a single-chain Fc, and an IL-2Rα truncated form, wherein the IL-2Rα truncated form is linked to the single-chain Fc via an uncleavable linker.

[0129] (8) Two first polypeptide chains, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus, a light chain of the first target-binding polypeptide, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is linked to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is linked to a light chain of the first target-binding polypeptide via a non-cleavable linker; and two second polypeptide chains, wherein the second polypeptide chain comprises, from the N-terminus to the C-terminus, a heavy chain Fd segment of the first target-binding polypeptide, a single-chain Fc segment, and a second target-binding polypeptide with a scFv (single-chain variable fragment) structure.

[0130] (9) Two first polypeptide chains, wherein the first polypeptide chain comprises a light chain containing a first target-binding polypeptide; and two second polypeptide chains, wherein the second polypeptide chain comprises, from the N-terminus to the C-terminus, a wild-type or mutant IL-2 protein, an IL-2Rα truncated form, a heavy chain Fd segment of the first target-binding polypeptide, a single-chain Fc segment, and a second target-binding polypeptide with a single-chain variable fragment (scFv) structure, wherein the wild-type or mutant IL-2 protein is linked to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is linked to the heavy chain Fd segment of the first target-binding polypeptide via a non-cleavable linker.

[0131] (10) Two first polypeptide chains, wherein the first polypeptide chain comprises a light chain containing a first target-binding polypeptide; and two second polypeptide chains, wherein the second polypeptide chain comprises, from the N-terminus to the C-terminus, a second target-binding polypeptide with a scFv (single-chain variable fragment) structure, a heavy chain Fd segment of the first target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is linked to the IL-2Rα truncated form via a cleavable linker.

[0132] (11) Two first polypeptide chains, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus, a light chain variable region (VL) of a first target-binding polypeptide and a light chain of a second target-binding polypeptide; and two second polypeptide chains, wherein, from the N-terminus to the C-terminus, the second polypeptide chain comprises, in sequence, a heavy chain variable region (VH) of the first target-binding polypeptide, a heavy chain Fd segment of the second target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, the IL-2Rα truncated form is connected to the single-chain Fc via a non-cleavable linker, the light chain variable region (VL) of the first target-binding polypeptide is connected to a light chain of the second target-binding polypeptide via a flexible linker 1, and the heavy chain variable region (VH) of the first target-binding polypeptide is connected to the heavy chain Fd segment of the second target-binding polypeptide via a flexible linker 2.

[0133] (12) Two first polypeptide chains, wherein the first polypeptide chain comprises, from the N-terminus to the C-terminus, a light chain variable region (VL) of the first target-binding polypeptide and a light chain of the second target-binding polypeptide; the second polypeptide chain comprises, from the N-terminus to the C-terminus, a heavy chain variable region (VH) of the first target-binding polypeptide, a heavy chain Fd segment of the second target-binding polypeptide, a single-chain Fc, and an IL-2Rα truncated form, wherein the IL-2Rα truncated form is connected to the single-chain Fc via an inclevable linker; and the third polypeptide chain comprises, from the N-terminus to the C-terminus, a light chain variable region (VL) of the first target-binding polypeptide and a light chain of the second target-binding polypeptide; The first target-binding polypeptide comprises a heavy chain variable region (VH), a heavy chain Fd segment of a second target-binding polypeptide, a single-chain Fc, and wild-type or mutant IL-2 protein, wherein the wild-type or mutant IL-2 protein is linked to the single-chain Fc via a cleavable linker; the light chain variable region (VL) of the first target-binding polypeptide is linked to one light chain of the second target-binding polypeptide via a flexible linker 1; and the heavy chain variable region (VH) containing the first target-binding polypeptide is linked to the heavy chain Fd segment of the second target-binding polypeptide via a flexible linker 2.

[0134] Among them, the IL-2Rα truncated forms described in (1)-(12) contain amino acid sequences selected from the C-terminus of the extracellular domain of IL-2Rα truncated to a length of 29 to 52 amino acids.

[0135] 10. The fusion protein according to item 8 or 9, wherein the truncated IL-2Rα comprises: C-terminal amino acids 168-219aa, 169-219aa, 170-219aa, 171-219aa, 172-219aa, 173-219aa, 174-219aa, 175-219aa, 176-219aa, and 177-219aa of the IL-2Rα extracellular domain. Amino acid sequences missing from 178-219aa, 179-219aa, 180-219aa, 181-219aa, 182-219aa, 183-219aa, 184-219aa, 185-219aa, 186-219aa, 187-219aa, 188-219aa, 189-219aa, 190-219aa, or 191-219aa.

[0136] 11. The fusion protein according to item 8 or 9, wherein the IL-2Rα truncated form comprises an amino acid sequence in which the C-terminal amino acids 173-219aa, 174-219aa, 175-219aa, 176-219aa, 177-219aa, 178-219aa, 179-219aa, 180-219aa, 181-219aa, 182-219aa, or 183-219aa are missing.

[0137] 12. The fusion protein according to item 8 or 9, wherein the IL-2Rα truncated form comprises an amino acid sequence in which amino acids 173-219aa of the C-terminal amino acid domain of the IL-2Rα extracellular domain are missing.

[0138] 13. The fusion protein according to any one of items 8-12, wherein the IL-2Rα truncated form comprises any amino acid sequence of SEQ ID NO:4-28.

[0139] 14. A fusion protein comprising:

[0140] Half-life extenders;

[0141] Interleukin-2 (IL-2) wild-type or mutant protein;

[0142] A cuttable connector connected to the IL-2; and

[0143] The IL-2Rα truncated body connected to the cuttable connector,

[0144] The IL-2Rα truncated form comprises an amino acid sequence selected from the C-terminus of the IL-2Rα extracellular domain, truncated to a length of 29 to 52 amino acids.

[0145] 15. The fusion protein according to claim 14, wherein the truncated IL-2Rα comprises: C-terminal amino acids 168-219aa, 169-219aa, 170-219aa, 171-219aa, 172-219aa, 173-219aa, 174-219aa, 175-219aa, 176-219aa, 177-219aa from the IL-2Rα extracellular domain. Amino acid sequences missing from 178-219aa, 179-219aa, 180-219aa, 181-219aa, 182-219aa, 183-219aa, 184-219aa, 185-219aa, 186-219aa, 187-219aa, 188-219aa, 189-219aa, 190-219aa, or 191-219aa.

[0146] 16. The fusion protein according to claim 14, wherein the IL-2Rα truncated form comprises an amino acid sequence in which the C-terminal amino acids 173-219aa, 174-219aa, 175-219aa, 176-219aa, 177-219aa, 178-219aa, 179-219aa, 180-219aa, 181-219aa, 182-219aa, or 183-219aa are missing.

[0147] 17. The fusion protein according to item 14, wherein the IL-2Rα truncated form comprises an amino acid sequence in which amino acids 173-219aa of the C-terminal domain of the IL-2Rα extracellular domain are missing.

[0148] 18. The fusion protein according to any one of items 14-17, wherein the IL-2Rα truncated form comprises any amino acid sequence of SEQ ID NO:5-28.

[0149] 19. The fusion protein according to any one of items 14-18, wherein the half-life extender is linked to the IL-2Rα truncated form via an incuttable linker.

[0150] 20. The fusion protein according to any one of items 14-18, wherein the half-life extender is linked to the wild-type or mutant IL-2 protein via an incuttable linker.

[0151] 21. The fusion protein according to any one of items 14-20, wherein the half-life extender is selected from: the Fc region of an antibody, or human serum albumin.

[0152] 22. The fusion protein according to any one of items 14-21, wherein the half-life extender is an antibody Fc region selected from: wild-type Fc region, mutant Fc region, monomeric wild-type Fc region, monomeric mutant Fc region, dimeric wild-type Fc region, or dimeric mutant Fc region.

[0153] 23. The fusion protein according to any one of items 14-22, wherein the half-life extender comprises SEQ ID NO:36.

[0154] 24. The fusion protein according to any one of items 14-23, wherein the wild-type or mutant IL-2 protein is connected to the truncated IL-2Rα protein by a cleavable linker.

[0155] 25. The fusion protein according to any one of items 1-24, wherein the cleavable linker is not cleaved by tumor-associated proteases.

[0156] 26. The fusion protein according to any one of items 1-25, wherein the uncleavable linker comprises glycine / serine.

[0157] 27. The fusion protein according to any one of items 1-26, wherein the uncleavable linker comprises SEQ ID NO:39.

[0158] 28. The fusion protein according to any one of items 1-27, wherein the cleavable linker is cleavable by a tumor-associated protease.

[0159] 29. The fusion protein according to any one of items 1-28, wherein the cleavable linker is cleaved by a matrix metalloproteinase.

[0160] 30. The fusion protein according to any one of items 1-29, wherein the cleavable linker is cleaved by matrix metalloproteinase 14 (MMP14).

[0161] 31. The fusion protein according to any one of claims 1-30, wherein the cleavable linker is cleaved by caspase, the caspase being selected from: caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11 or caspase 12.

[0162] 32. The fusion protein according to any one of items 1-31, wherein the cleavable linker comprises any amino acid sequence of SEQ ID NO:32-35.

[0163] 33. The fusion protein according to any one of items 1-32, wherein the target-binding polypeptide is an antibody or a functional fragment thereof.

[0164] 34. The fusion protein according to any one of items 1-33, wherein the target-binding polypeptide is selected from T cell target-binding polypeptides and tumor cell target-binding polypeptides;

[0165] Preferably, the target-binding polypeptide can bind to one or more targets;

[0166] Preferably, the target-binding peptide can bind to one of the following targets: CLDN18.2, PD-1, PD-L1, VEGFR, HER2;

[0167] Preferably, the target-binding peptide can bind to one of the following target combinations: PD-1 / VEGF combination, or CLDN18.2 / CD3 combination;

[0168] Preferably, the target of the T cell target-binding peptide is selected from CD3 and / or PD-1, and the target of the tumor cell target-binding peptide is selected from one or more of CLDN18.2, PD-L1, VEGFR, and HER2.

[0169] 35. The fusion protein according to any one of items 1-34, wherein the target-binding polypeptide comprises any amino acid sequence of SEQ ID NO:36-38.

[0170] 36. The fusion protein according to any one of items 1-35, wherein the wild-type IL-2 comprises at least 95% or 100% the same amino acid sequence as SEQ ID NO:1.

[0171] 37. The fusion protein according to any one of items 1-36, wherein the IL-2 mutant protein contains one or more of the following mutations compared to the wild-type IL-2 protein: R81E, N88D, L85T or L85I.

[0172] 38. The fusion protein according to items 8-36, wherein flexible linker 1 and flexible linker 2 are selected from the antibody flexible linker region or contain an amino acid sequence composed of glycine and serine, preferably the antibody flexible linker region is an elbow region or a hinge region, and preferably flexible linker 1 and flexible linker 2 are each 5-15 amino acids.

[0173] In this application, the elbow region, located inside the Fab segment, is the bend between the heavy chain variable region (VH) and the heavy chain constant region 1 (CH1), and between the light chain variable region (VL) and the light chain constant region (CL). Preferably, the flexible linker 1 and the flexible linker 2 can be selected from amino acid sequences of different lengths in the heavy chain elbow region and the light chain elbow region, respectively, such as ASTKGP (HC-γS), ASTKGPSVFPLAP (HC-γL), TVAAP (LC-κS), TVAAPSVFIFPP (LC-κL), QPKAAP (LC-λS), or QPKAAPSVTLFPP (LC-λL).

[0174] In this application, the hinge region, located between the Fab and Fc segments, is the amino acid sequence between the heavy chain constant region 1 (CH1) and constant region 2 (CH2). Preferably, the flexible linker 1 and flexible linker 2 can be selected from amino acid sequences of different lengths in the hinge region, such as PNLLGGP (HC-hS), PAPNLLGGP (HC-hL), PAPNLLGGP (LC-hS), or PTISPAPNLLGGP (LC-hL).

[0175] 39. The fusion protein according to any one of items 1-38, wherein the IL-2 wild-type or mutant protein comprises any amino acid sequence of SEQ ID NO:1-2.

[0176] 40. The fusion protein according to any one of claims 1-39, wherein the fusion protein comprises a first chain and a second chain, the first chain and the second chain comprising a sequence having at least 95% sequence identity with respect to the following amino acid sequence, wherein:

[0177] The amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:40; or

[0178] The amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:41; or

[0179] The amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:42; or

[0180] The amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:43.

[0181] 41. The fusion protein according to any one of items 1-40, wherein the fusion protein is a homodimer or a heterodimer.

[0182] 42. The fusion protein according to item 41, wherein the homodimer or heterodimer is formed by a covalent bond between two single-chain Fcs, preferably by a covalent bond between CH and CL and between two single-chain Fcs.

[0183] 43. The fusion protein according to item 42, wherein the single-chain Fc includes an amino acid mutation, preferably a Knob, Hole, or LALA mutation.

[0184] 44. The fusion protein according to any one of claims 1-43, wherein the fusion protein containing the truncated IL-2Rα has reduced toxicity compared to the fusion protein containing the untruncated IL-2Rα.

[0185] 45. A polynucleotide encoding a fusion protein described in any one of terms 1-44.

[0186] 46. ​​An expression vector comprising the polynucleotide described in item 45.

[0187] 47. A host cell comprising the polynucleotide according to claim 45 or the expression vector according to claim 46.

[0188] 48. A method for preparing a fusion protein, comprising culturing host cells according to item 47.

[0189] 49. The method for preparing a fusion protein according to item 48, the method further comprising purifying or isolating the produced fusion protein.

[0190] 50. A pharmaceutical composition comprising the fusion protein described in any one of claims 1-44 and a pharmaceutically acceptable excipient, carrier, or diluent.

[0191] 51. A method for treating or alleviating a disease or symptom, comprising:

[0192] Administer to a patient in need a therapeutically effective amount of the fusion protein of any one of claims 1-44, the polynucleotide of claim 45, the expression vector of claim 46, the host cell of claim 47, or the pharmaceutical composition of claim 50.

[0193] 52. The method according to item 51, wherein the patient in need is a human.

[0194] 53. The method according to item 51 or 52, wherein the disease or symptom is cancer.

[0195] 54. The method according to any one of items 51-53, wherein the disease or condition is selected from: acute myeloid leukemia, adrenocortical carcinoma, B-cell lymphoma, urothelial carcinoma of the bladder, ductal carcinoma of the breast, lobular carcinoma of the breast, esophageal cancer, castration-resistant prostate cancer (CRPC), cervical cancer, cholangiocarcinoma, chronic myeloid leukemia, colorectal adenocarcinoma, colorectal cancer (CRC), esophageal cancer, gastric adenocarcinoma, glioblastoma multiforme, squamous cell carcinoma of the head and neck, Hodgkin lymphoma / primary mediastinal B-cell lymphoma, liver cell carcinoma. HCC, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, low-grade glioma, lung adenocarcinoma, lung squamous cell carcinoma, melanoma (MEL), mesothelioma, non-squamous NSCLC, ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma, paraganglioma and pheochromocytoma, prostate cancer, renal cell carcinoma (RCC), sarcoma, cutaneous melanoma, head and neck squamous cell carcinoma, T-cell lymphoma, thymoma, papillary thyroid carcinoma, uterine carcinosarcoma, endometrioid carcinoma of the uterine body or uveal melanoma.

[0196] 55. Use of the fusion protein of any one of claims 1-44, the polynucleotide of claim 45, the expression vector of claim 46, the host cell of claim 47, or the pharmaceutical composition of claim 50 in the treatment or relief of a disease or condition.

[0197] 56. Use of the fusion protein of any one of claims 1-44, the polynucleotide of claim 45, the expression vector of claim 46, the host cell of claim 47, or the pharmaceutical composition of claim 50 in the preparation of a medicament for treating or alleviating a disease or condition.

[0198] The third aspect of this application relates to the following technical solution:

[0199] 1. A truncated form of interleukin-2 receptor α (IL-2Rα) comprising 166 to 190 or 167 to 190 amino acids continuously from the N-terminus of the extracellular domain of wild-type IL-2Rα.

[0200] 2. The IL-2Rα truncated form according to claim 1, wherein the IL-2Rα truncated form comprises 166 to 182 or 167 to 182 amino acids continuously from the N-terminus of the extracellular domain of wild-type IL-2Rα;

[0201] Preferably, the IL-2Rα truncated form comprises 166 to 172 or 167 to 172 amino acids consecutively starting from the N-terminus of the extracellular domain of wild-type IL-2Rα.

[0202] 3. The IL-2Rα truncated form according to item 1 or 2, wherein the IL-2Rα truncated form comprises an amino acid sequence as shown in any one of SEQ ID NO: 5-29, or the amino acid sequence of the IL-2Rα truncated form is as shown in any one of SEQ ID NO: 5-29;

[0203] Preferably, the IL-2Rα truncated form comprises an amino acid sequence as shown in any one of SEQ ID NO:13-29, or the amino acid sequence of the IL-2Rα truncated form is as shown in any one of SEQ ID NO:13-29;

[0204] More preferably, the IL-2Rα truncated form comprises an amino acid sequence as shown in any one of SEQ ID NO:23-29, or the amino acid sequence of the IL-2Rα truncated form is as shown in any one of SEQ ID NO:23-29.

[0205] 4. A fusion protein comprising

[0206] Interleukin-2 (IL-2) wild-type or mutant protein;

[0207] A cleavable linker that connects to the wild-type or mutant IL-2 protein;

[0208] Interleukin-2 receptor α (IL-2Rα) truncated bodies connected to the said cleavable connector; and

[0209] Target-binding peptides;

[0210] The IL-2Rα truncated body is any one of the IL-2Rα truncated bodies described in items 1-3.

[0211] 5. The fusion protein according to item 4, wherein the target-binding polypeptide is linked to the IL-2Rα truncated form via an uncuttable linker, or the target-binding polypeptide is linked to the wild-type or mutant IL-2 protein via an uncuttable linker.

[0212] 6. The fusion protein according to item 4 or 5, wherein the fusion protein comprises, in sequence:

[0213] (i) a target-binding peptide, an IL-2Rα truncated form, and a wild-type or mutant IL-2 protein, wherein the target-binding peptide is linked to the IL-2Rα truncated form via an uncleavable linker; or

[0214] (ii) a target-binding polypeptide, wild-type or mutant IL-2 protein and a truncated IL-2Rα protein, wherein the target-binding polypeptide is linked to the wild-type or mutant IL-2 protein via an uncuttable linker.

[0215] 7. A fusion protein comprising:

[0216] Half-life extenders;

[0217] Interleukin-2 (IL-2) wild-type or mutant protein;

[0218] Cleavable linkers to the wild-type or mutant IL-2 protein; and

[0219] A truncated form of interleukin-2 receptor α (IL-2Rα) connected to the cuttable connector;

[0220] The IL-2Rα truncated body is any one of the IL-2Rα truncated bodies described in items 1-3.

[0221] 8. The fusion protein according to item 7, wherein the half-life extender is connected to the IL-2Rα truncated form via an uncuttable linker, or the half-life extender is connected to the wild-type or mutant IL-2 protein via an uncuttable linker.

[0222] 9. The fusion protein according to item 7 or 8, wherein the fusion protein is selected from any of the following:

[0223] (i) a half-life extender, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the half-life extender is connected to the IL-2Rα truncated form via an incuttable linker;

[0224] (ii) a half-life extender, wild-type or mutant IL-2 protein and IL-2Rα truncated form, wherein the half-life extender is connected to the wild-type or mutant IL-2 protein via an incuttable linker;

[0225] (iii) A target-binding peptide, a half-life extender, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the half-life extender is linked to the IL-2Rα truncated form via an incleavable linker; or

[0226] (iv) A target-binding peptide, a half-life extender, a wild-type or mutant IL-2 protein, and an IL-2Rα truncated form, wherein the half-life extender is linked to the wild-type or mutant IL-2 protein via an uncuttable linker.

[0227] 10. The fusion protein according to any one of items 7-9, wherein the fusion protein is selected from any one of the following:

[0228] (1) Two first polypeptide chains, wherein the first polypeptide chains sequentially comprise: a first target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated form and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the single-chain Fc via an uncleavable linker.

[0229] (2) Two first polypeptide chains, each first polypeptide chain comprising, in sequence: a light chain of a first target-binding polypeptide, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the light chain of the first target-binding polypeptide via an uncleavable linker; and two second polypeptide chains, each second polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a single-chain Fc segment, and a second target-binding polypeptide with an scFv structure;

[0230] (3) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; and two second polypeptide chains, each second polypeptide chain comprising, in sequence: wild-type or mutant IL-2 protein, a truncated IL-2Rα protein, a heavy chain Fd segment of the first target-binding polypeptide, a single chain Fc segment, and a second target-binding polypeptide with an scFv structure, wherein the wild-type or mutant IL-2 protein is connected to the truncated IL-2Rα protein via a cleavable linker, and the truncated IL-2Rα protein is connected to the heavy chain of the first target-binding polypeptide via an uncleavable linker.

[0231] (4) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; two second polypeptide chains, each second polypeptide chain comprising, in sequence: a second target-binding polypeptide with an scFv structure, a heavy chain Fd segment of the first target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the single-chain Fc via an uncleavable linker;

[0232] (5) Two first polypeptide chains, each first polypeptide chain comprising: a light chain variable region of a second target-binding polypeptide and a light chain of the first target-binding polypeptide; two second polypeptide chains, each second polypeptide chain comprising: a heavy chain variable region of a second target-binding polypeptide, a heavy chain Fd segment of the first target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated body, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated body via a cleavable linker, and the IL-2Rα truncated body is connected to the single-chain Fc via an uncleavable linker;

[0233] (6) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; and two second polypeptide chains, each second polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the single-chain Fc via an uncleavable linker.

[0234] (7) Two first polypeptide chains, each first polypeptide chain comprising, in sequence: a light chain of a first target-binding polypeptide, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the light chain of the first target-binding polypeptide via an uncleavable linker; and two second polypeptide chains, each second polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), and a single-chain Fc.

[0235] (8) Two first polypeptide chains, each first polypeptide chain comprising, in sequence: wild-type or mutant IL-2 protein, IL-2Rα truncated form, and light chain of first target-binding polypeptide, wherein the wild-type or mutant IL-2 protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the light chain of first target-binding polypeptide via an uncleavable linker; two second polypeptide chains, each second polypeptide chain comprising, in sequence: heavy chain Fd segment of first target-binding polypeptide, second target-binding polypeptide in the form of nanobody (VHH or V-NAR), and single chain Fc;

[0236] (9) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; two second polypeptide chains, each second polypeptide chain comprising, in sequence: wild-type or mutant IL-2 protein, an IL-2Rα truncated form, a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), and a single chain Fc, wherein the wild-type or mutant IL-2 protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the heavy chain Fd segment of the first target-binding polypeptide via an uncleavable linker;

[0237] (10) A first polypeptide chain, comprising in sequence: a first target-binding polypeptide with an scFv structure, a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the single-chain Fc via an uncleavable linker; a second polypeptide chain, comprising in sequence: a heavy chain variable region of the first target-binding polypeptide, a heavy chain Fd segment of the second target-binding polypeptide, and a single-chain Fc; a third polypeptide chain, comprising in sequence: a light chain variable region of the first target-binding polypeptide and a light chain of the second target-binding polypeptide;

[0238] (11) Two first polypeptide chains, comprising: a light chain of a first target-binding polypeptide; a second polypeptide chain, comprising in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide of scFv structure, a first single chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the first single chain Fc via an uncleavable linker; a third polypeptide chain, comprising in sequence: a heavy chain Fd segment of the first target-binding polypeptide and a second single chain Fc.

[0239] 11. A fusion protein selected from any of the following:

[0240] (1) Two first polypeptide chains, wherein the first polypeptide chains sequentially comprise: a first target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated form and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the single-chain Fc via an uncleavable linker.

[0241] (2) Two first polypeptide chains, each first polypeptide chain comprising, in sequence: a light chain of a first target-binding polypeptide, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the light chain of the first target-binding polypeptide via an uncleavable linker; and two second polypeptide chains, each second polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a single-chain Fc segment, and a second target-binding polypeptide with an scFv structure;

[0242] (3) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; and two second polypeptide chains, each second polypeptide chain comprising, in sequence: wild-type or mutant IL-2 protein, a truncated IL-2Rα protein, a heavy chain Fd segment of the first target-binding polypeptide, a single chain Fc segment, and a second target-binding polypeptide with an scFv structure, wherein the wild-type or mutant IL-2 protein is connected to the truncated IL-2Rα protein via a cleavable linker, and the truncated IL-2Rα protein is connected to the heavy chain of the first target-binding polypeptide via an uncleavable linker.

[0243] (4) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; two second polypeptide chains, each second polypeptide chain comprising, in sequence: a second target-binding polypeptide with an scFv structure, a heavy chain Fd segment of the first target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the single-chain Fc via an uncleavable linker;

[0244] (5) Two first polypeptide chains, each first polypeptide chain comprising: a light chain variable region of a second target-binding polypeptide and a light chain of the first target-binding polypeptide; two second polypeptide chains, each second polypeptide chain comprising: a heavy chain variable region of a second target-binding polypeptide, a heavy chain Fd segment of the first target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated body, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated body via a cleavable linker, and the IL-2Rα truncated body is connected to the single-chain Fc via an uncleavable linker;

[0245] (6) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; and two second polypeptide chains, each second polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the single-chain Fc via an uncleavable linker.

[0246] (7) Two first polypeptide chains, each first polypeptide chain comprising, in sequence: a light chain of a first target-binding polypeptide, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the light chain of the first target-binding polypeptide via an uncleavable linker; and two second polypeptide chains, each second polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), and a single-chain Fc.

[0247] (8) Two first polypeptide chains, each first polypeptide chain comprising, in sequence: wild-type or mutant IL-2 protein, IL-2Rα truncated form, and light chain of first target-binding polypeptide, wherein the wild-type or mutant IL-2 protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the light chain of first target-binding polypeptide via an uncleavable linker; two second polypeptide chains, each second polypeptide chain comprising, in sequence: heavy chain Fd segment of first target-binding polypeptide, second target-binding polypeptide in the form of nanobody (VHH or V-NAR), and single chain Fc;

[0248] (9) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; two second polypeptide chains, each second polypeptide chain comprising, in sequence: wild-type or mutant IL-2 protein, an IL-2Rα truncated form, a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), and a single chain Fc, wherein the wild-type or mutant IL-2 protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the heavy chain Fd segment of the first target-binding polypeptide via an uncleavable linker;

[0249] (10) A first polypeptide chain, comprising in sequence: a first target-binding polypeptide with an scFv structure, a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the single-chain Fc via an uncleavable linker; a second polypeptide chain, comprising in sequence: a heavy chain variable region of the first target-binding polypeptide, a heavy chain Fd segment of the second target-binding polypeptide, and a single-chain Fc; a third polypeptide chain, comprising in sequence: a light chain variable region of the first target-binding polypeptide and a light chain of the second target-binding polypeptide;

[0250] (11) Two first polypeptide chains, comprising: a light chain of a first target-binding polypeptide; a second polypeptide chain, comprising in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide of scFv structure, a first single chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the first single chain Fc via an uncleavable linker; a third polypeptide chain, comprising in sequence: a heavy chain Fd segment of the first target-binding polypeptide and a second single chain Fc;

[0251] The IL-2Rα truncated body is any one of the IL-2Rα truncated bodies described in items 1-3.

[0252] 12. A fusion protein comprising:

[0253] Half-life extenders;

[0254] Interleukin-2 (IL-2) wild-type or mutant protein;

[0255] Interleukin-2 receptor α (IL-2Rα) truncated variant; and

[0256] Cuttable connectors;

[0257] The IL-2Rα truncated body is any one of the IL-2Rα truncated bodies described in items 1-3.

[0258] 13. The fusion protein according to claim 12, wherein the fusion protein comprises:

[0259] (i) a first polypeptide chain, the first polypeptide chain comprising a first single-chain Fc and an IL-2Rα truncated form; and

[0260] (ii) A second polypeptide chain comprising a second single-chain Fc and wild-type or mutant IL-2 protein;

[0261] In this embodiment, the wild-type or mutant IL-2 protein is connected to the second single-chain Fc via a cleavable linker, and the truncated IL-2Rα protein is connected to the first single-chain Fc via an uncleavable linker; or, the wild-type or mutant IL-2 protein is connected to the second single-chain Fc via an uncleavable linker, and the truncated IL-2Rα protein is connected to the first single-chain Fc via a cleavable linker.

[0262] 14. The fusion protein according to item 12 or 13, wherein the fusion protein is selected from any of the following:

[0263] (1) A first polypeptide chain, the first polypeptide chain comprising, in sequence: a Fab segment of a first target-binding polypeptide, a first single-chain Fc, and an IL-2Rα truncated form; a second polypeptide chain, the second polypeptide chain comprising, in sequence: a Fab segment of the first target-binding polypeptide, a second single-chain Fc, and an IL-2 wild-type or mutant protein, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker;

[0264] (2) A first polypeptide chain, comprising in sequence: a first target-binding polypeptide with an scFv structure, a first single-chain Fc, and an IL-2Rα truncated form; a second polypeptide chain, comprising in sequence: a heavy chain variable region of the first target-binding polypeptide, a heavy chain Fd segment of the second target-binding polypeptide, a second single-chain Fc, and an IL-2 wild-type or mutant protein; a third polypeptide chain, comprising in sequence: a light chain variable region of the first target-binding polypeptide and a light chain of the second target-binding polypeptide, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker;

[0265] (3) Two first polypeptide chains, comprising: a light chain of a first target-binding polypeptide; a second polypeptide chain, comprising in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide with an scFv structure, a first single-chain Fc, and an IL-2Rα truncated form; and a third polypeptide chain, comprising in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second single-chain Fc, and an IL-2 wild-type or mutant protein, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker.

[0266] (4) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; a second polypeptide chain, the second polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a first single-chain Fc, and an IL-2Rα truncated form; and a third polypeptide chain, the third polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a second single-chain Fc, and an IL-2 wild-type or mutant protein, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker.

[0267] 15. A fusion protein selected from any of the following:

[0268] (1) A first polypeptide chain, the first polypeptide chain comprising, in sequence: a Fab segment of a first target-binding polypeptide, a first single-chain Fc, and an IL-2Rα truncated form; a second polypeptide chain, the second polypeptide chain comprising, in sequence: a Fab segment of the first target-binding polypeptide, a second single-chain Fc, and an IL-2 wild-type or mutant protein, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker;

[0269] (2) A first polypeptide chain, comprising in sequence: a first target-binding polypeptide with an scFv structure, a first single-chain Fc, and an IL-2Rα truncated form; a second polypeptide chain, comprising in sequence: a heavy chain variable region of the first target-binding polypeptide, a heavy chain Fd segment of the second target-binding polypeptide, a second single-chain Fc, and an IL-2 wild-type or mutant protein; a third polypeptide chain, comprising in sequence: a light chain variable region of the first target-binding polypeptide and a light chain of the second target-binding polypeptide, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker;

[0270] (3) Two first polypeptide chains, comprising: a light chain of a first target-binding polypeptide; a second polypeptide chain, comprising in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide with an scFv structure, a first single-chain Fc, and an IL-2Rα truncated form; and a third polypeptide chain, comprising in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second single-chain Fc, and an IL-2 wild-type or mutant protein, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker.

[0271] (4) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; a second polypeptide chain, the second polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a first single-chain Fc, and an IL-2Rα truncated form; and a third polypeptide chain, the third polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a second single-chain Fc, and an IL-2 wild-type or mutant protein, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker.

[0272] The IL-2Rα truncated body is any one of the IL-2Rα truncated bodies described in items 1-3.

[0273] 16. The fusion protein according to any one of items 7-15, wherein the half-life extender is selected from:

[0274] (i) Antibody Fc region or human serum albumin

[0275] (ii) wild-type Fc region, mutant Fc region, wild-type Fc monomer, mutant Fc monomer, wild-type Fc dimer or mutant Fc dimer, wild-type single-stranded Fc, or mutant single-stranded Fc, preferably, the mutant single-stranded Fc, mutant Fc monomer or mutant Fc dimer is a mutant single-stranded Fc, mutant Fc monomer or mutant Fc dimer modified by site-directed mutagenesis;

[0276] (iii) A single-chain Fc containing an amino acid mutation, a first single-chain Fc and / or a second single-chain Fc;

[0277] (iv) Single-stranded Fc, first single-stranded Fc and / or second single-stranded Fc containing Knob, Hole or LALA mutations;

[0278] (v) Contains an amino acid sequence as shown in SEQ ID NO:36.

[0279] 17. The fusion protein according to any one of items 7-9 and 12-13, wherein the fusion protein further comprises a first target-binding polypeptide and / or a second target-binding polypeptide.

[0280] 18. The fusion protein according to claim 17, wherein the target-binding polypeptide, the first target-binding polypeptide, and / or the second target-binding polypeptide are selected from:

[0281] (i) an antibody or a functional fragment thereof; or

[0282] (ii) Fab, scFv, or nanobody.

[0283] 19. The fusion protein according to any one of items 4-6, 10-11, 14-15 and 17-18, wherein the target-binding polypeptide, the first target-binding polypeptide and / or the second target-binding polypeptide are selected from T cell target-binding polypeptides and tumor cell target-binding polypeptides;

[0284] Preferably, the target-binding polypeptide, the first target-binding polypeptide, and / or the second target-binding polypeptide can bind to one or more targets;

[0285] Preferably, the target-binding peptide, the first target-binding peptide, and / or the second target-binding peptide are capable of binding to one of the following targets: CLDN18.2, PD-1, PD-L1, VEGFR, HER2;

[0286] Preferably, the target-binding peptide, the first target-binding peptide, and / or the second target-binding peptide can bind to one of the following target combinations: PD-1 / VEGF combination, or CLDN18.2 / CD3 combination;

[0287] Preferably, the target of the T cell target-binding peptide is selected from CD3 and / or PD-1, and the target of the tumor cell target-binding peptide is selected from one or more of CLDN18.2, PD-L1, VEGFR, and HER2.

[0288] 20. The fusion protein according to any one of items 5-6, 8-11, and 13-15, wherein the uncleavable linker is selected from at least one of the following:

[0289] (i) Not cleaved by tumor-associated proteases;

[0290] (ii) is a glycine / serine linker;

[0291] (iii) When the IL2 and the IL2Rα truncated form are sequentially connected, the non-cleavable linker used to connect the IL2Rα truncated form and the half-life extender or the target-binding peptide is 4 G4S;

[0292] (iv) When the IL2 is connected to the first / second target-binding polypeptide or the first / second single-chain Fc, the non-cleavable linker for connecting the IL2Rα truncated form and the second / first target-binding polypeptide or the second / first single-chain Fc is 3 G4S;

[0293] (v) Contains or is an amino acid sequence as shown in SEQ ID NO:39.

[0294] 21. The fusion protein according to any one of items 4-20, wherein the cleavable linker is selected from at least one of the following:

[0295] (i) It can be cleaved by tumor-associated proteases;

[0296] (ii) It can be cleaved by matrix metalloproteinases;

[0297] (iii) It can be cleaved by matrix metalloproteinase 14 (MMP14);

[0298] (iv) It can be cleaved by caspase;

[0299] (v) Can be cleaved by caspase, wherein the caspase is selected from one or more of the following: caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11 or caspase 12;

[0300] (vi) Contains an amino acid sequence as shown in any one of SEQ ID NO:32-35

[0301] (vii) contains glycine / serine;

[0302] (viii) When the IL2 and the IL2Rα truncated form are sequentially connected, the cleavable linker for connecting the IL2 and the half-life extender or the target-binding peptide contains 4 G4S;

[0303] (ix) When the IL2 is linked to the first / second target-binding peptide or the first / second single-chain Fc, the cleavable linker for linking the IL2 and the second / first target-binding peptide or the second / first single-chain Fc contains two G4S.

[0304] 22. The fusion protein according to any one of items 4-21, wherein the wild-type or mutant IL-2 protein is selected from at least one of the following:

[0305] (i) The wild-type IL-2 protein contains or is an amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO:1;

[0306] (ii) Compared with the wild-type IL-2 protein, the mutant IL-2 protein contains one or more amino acid mutations;

[0307] (iii) Compared with the wild-type IL-2 protein, the mutant IL-2 protein contains one or more of the N88D, R81E, and L85T / I mutations;

[0308] (iv) Compared with the wild-type I-2 protein, the mutant IL-2 protein contains R81E and L85T / I mutations;

[0309] (v) Compared with the wild-type IL-2 protein, the mutant IL-2 protein contains R81E, L85T / I and N88D mutations;

[0310] (vi) The IL-2 mutant protein contains or has an amino acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:65.

[0311] 23. The fusion protein according to any one of items 4-22, wherein the fusion protein is selected from (i) or (ii):

[0312] (i) The fusion protein comprises a first chain and a second chain, wherein the first chain and the second chain comprise the following amino acid sequences or sequences having at least 95% identity with the following amino acid sequences, wherein:

[0313] The amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:40; or, the amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:41; or, the amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:42; or, the amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:43; or, the amino acid sequence of the first chain is shown in SEQ ID NO:49, and the amino acid sequence of the second chain is shown in SEQ ID NO:50; or, the amino acid sequence of the first chain is shown in SEQ ID NO:53, and the amino acid sequence of the second chain is shown in SEQ ID NO:54; or, the amino acid sequence of the first chain is shown in SEQ ID NO:57, and the amino acid sequence of the second chain is shown in SEQ ID NO:58; or, the amino acid sequence of the first chain is shown in SEQ ID NO:59, and the amino acid sequence of the second chain is shown in SEQ ID NO:50. The amino acid sequence of the first chain is shown in SEQ ID NO:60; or, the amino acid sequence of the second chain is shown in SEQ ID NO:61, and the amino acid sequence of the second chain is shown in SEQ ID NO:62; or, the amino acid sequence of the first chain is shown in SEQ ID NO:63, and the amino acid sequence of the second chain is shown in SEQ ID NO:64; or, the amino acid sequence of the first chain is shown in SEQ ID NO:93, and the amino acid sequence of the second chain is shown in SEQ ID NO:94; or, the amino acid sequence of the first chain is shown in SEQ ID NO:95, and the amino acid sequence of the second chain is shown in SEQ ID NO:96; or, the amino acid sequence of the first chain is shown in SEQ ID NO:97, and the amino acid sequence of the second chain is shown in SEQ ID NO:98; or, the amino acid sequence of the first chain is shown in SEQ ID NO:99, and the amino acid sequence of the second chain is shown in SEQ ID NO:100; or, the amino acid sequence of the first chain is shown in SEQ ID NO:101, and the amino acid sequence of the second chain is shown in SEQ ID NO:102; or, the amino acid sequence of the first chain is shown in SEQ ID NO:103, and the amino acid sequence of the second chain is shown in SEQ ID NO:104. As shown in NO:104; or, the amino acid sequence of the first chain is shown in SEQ ID NO:105, and the amino acid sequence of the second chain is shown in SEQ ID NO:106;Alternatively, the amino acid sequence of the first chain is as shown in SEQ ID NO:107, and the amino acid sequence of the second chain is as shown in SEQ ID NO:108; or, the amino acid sequence of the first chain is as shown in SEQ ID NO:109, and the amino acid sequence of the second chain is as shown in SEQ ID NO:110.

[0314] (ii) The fusion protein comprises a first chain, a second chain, and a third chain, wherein the first chain, the second chain, and the third chain comprise the following amino acid sequences or sequences having at least 95% identity with the following amino acid sequences, wherein:

[0315] The amino acid sequence of the first chain is shown in SEQ ID NO:66, the amino acid sequence of the second chain is shown in SEQ ID NO:67, and the amino acid sequence of the third chain is shown in SEQ ID NO:68; or, the amino acid sequence of the first chain is shown in SEQ ID NO:69, the amino acid sequence of the second chain is shown in SEQ ID NO:70, and the amino acid sequence of the third chain is shown in SEQ ID NO:71; or, the amino acid sequence of the first chain is shown in SEQ ID NO:72, the amino acid sequence of the second chain is shown in SEQ ID NO:73, and the amino acid sequence of the third chain is shown in SEQ ID NO:74; or, the amino acid sequence of the first chain is shown in SEQ ID NO:75, the amino acid sequence of the second chain is shown in SEQ ID NO:76, and the amino acid sequence of the third chain is shown in SEQ ID NO:77; or, the amino acid sequence of the first chain is shown in SEQ ID NO:78, the amino acid sequence of the second chain is shown in SEQ ID NO:79, and the amino acid sequence of the third chain is shown in SEQ ID NO:80; or, the amino acid sequence of the first chain is shown in SEQ ID NO:69. As shown in NO:81, the amino acid sequence of the second chain is as shown in SEQ ID NO:82, and the amino acid sequence of the third chain is as shown in SEQ ID NO:83; or, the amino acid sequence of the first chain is as shown in SEQ ID NO:84, the amino acid sequence of the second chain is as shown in SEQ ID NO:85, and the amino acid sequence of the third chain is as shown in SEQ ID NO:86; or, the amino acid sequence of the first chain is as shown in SEQ ID NO:87, the amino acid sequence of the second chain is as shown in SEQ ID NO:88, and the amino acid sequence of the third chain is as shown in SEQ ID NO:89.

[0316] 24. The fusion protein according to any one of items 4-23, wherein the fusion protein is a dimer;

[0317] Preferably, the fusion protein is a homodimer or a heterodimer.

[0318] 25. The fusion protein according to any one of claims 4-24, wherein the fusion protein containing the truncated form of IL-2Rα has reduced toxicity compared to a fusion protein containing untruncated IL-2Rα.

[0319] 26. A polynucleotide encoding an IL-2Rα truncated form as described in any one of items 1-3, or a fusion protein as described in any one of items 4-25.

[0320] 27. An expression vector comprising the polynucleotide as described in item 26.

[0321] 28. A host cell comprising the polynucleotide as described in item 26 or the expression vector as described in item 27.

[0322] 29. A method for preparing an IL-2Rα truncated form or fusion protein, comprising culturing host cells as described in item 28 to prepare an IL-2Rα truncated form as described in any one of items 1-3, or a fusion protein as described in any one of items 4-25;

[0323] Preferably, the method further includes purifying or isolating the produced IL-2Rα truncated form or fusion protein.

[0324] 30. A pharmaceutical composition comprising the fusion protein as described in any one of items 4-25;

[0325] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, carrier, or diluent.

[0326] 31. A method for treating or alleviating a disease or symptom, comprising:

[0327] Administer to a patient in need a therapeutically effective amount of any of the fusion proteins described in items 4-25, the polynucleotide described in item 26, the expression vector described in item 27, the host cell described in item 28, or the pharmaceutical composition described in item 30;

[0328] Preferably, the patient in need is a human being.

[0329] 32. The method according to item 31, wherein the disease or symptom is cancer;

[0330] Preferably, the disease or condition is selected from: acute myeloid leukemia, adrenocortical carcinoma, B-cell lymphoma, urothelial carcinoma of the bladder, ductal carcinoma of the breast, lobular carcinoma of the breast, esophageal cancer, castration-resistant prostate cancer (CRPC), cervical cancer, cholangiocarcinoma, chronic myeloid leukemia, colorectal adenocarcinoma, colorectal cancer (CRC), esophageal cancer, gastric adenocarcinoma, glioblastoma multiforme, squamous cell carcinoma of the head and neck, Hodgkin lymphoma / primary mediastinal B-cell lymphoma, hepatocellular carcinoma (HCC), kidney... Chromophobe carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, low-grade glioma, lung adenocarcinoma, squamous cell lung carcinoma, melanoma (MEL), mesothelioma, non-squamous NSCLC, ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma, paraganglioma and pheochromocytoma, prostate cancer, renal cell carcinoma (RCC), sarcoma, cutaneous melanoma, head and neck squamous cell carcinoma, T-cell lymphoma, thymoma, papillary thyroid carcinoma, uterine carcinosarcoma, endometrioid carcinoma of the uterine body or uveal melanoma.

[0331] 33. Use of the fusion protein as described in any one of items 4-25, the polynucleotide as described in item 26, the expression vector as described in item 27, the host cell as described in item 28, or the pharmaceutical composition as described in item 30 for the treatment or relief of a disease or condition.

[0332] 34. Use of any of the fusion proteins described in items 4-25, the polynucleotides described in item 26, the expression vectors described in item 27, the host cells described in item 28, or the pharmaceutical compositions described in item 30 in the preparation of a medicament for treating or alleviating a disease or condition.

[0333] The fourth aspect of this application relates to the following technical solution:

[0334] This application provides an activatable IL-2 fusion protein, characterized in that it comprises a target-binding polypeptide capable of specifically binding to a target, a truncated form of interleukin-2 receptor α (IL-2Rα), and an IL-2 wild-type polypeptide or mutant.

[0335] In some embodiments, the IL-2Rα truncated section and the IL-2 are connected by a cuttable connector.

[0336] In some embodiments, the cleavable connector is capable of being cleaved by tumor-associated proteases in the tumor microenvironment but not in normal tissue. Upon cleavage, the shielding portion of the cleavable connector loses its function, and IL-2 is released to perform its function. Preferably, the cleavable connector is selected from: SEQ ID NO:32-35.

[0337] In some embodiments, the cleavable linker comprises an amino acid sequence: XXXD, where X represents any single amino acid. For example, the amino acid sequence comprised by the cleavable linker is selected from: DEVD, DEVDG, DQTDG, VEID, VEVD, VEIDN, IEDT, LETD, LQTDG, LEHD, LEHDS, YVAD, WEHD, YVHDA.

[0338] In some embodiments, the IL-2Rα truncated derivative comprises the C-terminal amino acids 168-219aa, 169-219aa, 170-219aa, 171-219aa, 172-219aa, 173-219aa, 174-219aa, 175-219aa, 176-219aa, 177-219aa, and 178-219aa from the IL-2Rα extracellular domain. Amino acid sequences missing from 179-219aa, 180-219aa, 181-219aa, 182-219aa, 183-219aa, 184-219aa, 185-219aa, 186-219aa, 187-219aa, 188-219aa, 189-219aa, 190-219aa, 191-219aa, or 192-219aa. In some embodiments, the sequence of the IL-2Rα truncated form is as shown in SEQ ID NO:4-28.

[0339] In some embodiments, the IL-2 is wild-type or a mutant. Preferably, the IL-2 mutation is R81E, L85T, or L85I. In some embodiments, the sequence of the IL-2 is as shown in SEQ ID NO:1-2. In some embodiments, the mutation of the IL-2 can reduce its affinity for IL-2Rα and increase its affinity for IL-2Rβ / γ.

[0340] In some embodiments, the IL-2Rα truncated form can inhibit the binding of IL-2 to IL-2 receptors in the environment.

[0341] In some embodiments, the target-binding peptide is able to specifically bind to the target.

[0342] In some embodiments, the target-binding polypeptide moiety is selected from: Fab, scFv, nanobody.

[0343] In some embodiments, the target is a cell surface molecule, such as a protein, lipid, or polysaccharide. In some embodiments, the target is on tumor cells, virus-infected cells, bacterial-infected cells, damaged red blood cells, arterial plaque cells, or fibrotic tissue cells.

[0344] In some embodiments, the target-binding polypeptide is selected from T-cell target-binding polypeptides and tumor cell target-binding polypeptides.

[0345] In some embodiments, the target-binding polypeptide can bind to one or more targets.

[0346] In some embodiments, the target-binding peptide can bind to one of the following targets: CLDN18.2, PD-1, PD-L1, VEGFR, HER2.

[0347] In some embodiments, the target-binding peptide can bind to one of the following target combinations: PD-1 / VEGF combination, or CLDN18.2 / CD3 combination.

[0348] In some embodiments, the target of the T cell target-binding peptide is selected from CD3 and / or PD-1, and the target of the tumor cell target-binding peptide is selected from one or more of CLDN18.2, PD-L1, VEGFR, and HER2.

[0349] In some embodiments, the target-binding polypeptide of the fusion protein is linked to the IL-2Rα truncated polypeptide via an uncleavable linker.

[0350] In some embodiments, the target-binding peptide of the fusion protein is linked to IL-2 via an incleavable linker.

[0351] In some embodiments, the uncuttable connector is a glycine / serine connector.

[0352] In some embodiments, the incuttable connector comprises: SEQ ID NO:39.

[0353] This application provides an activatable IL-2 fusion protein, characterized in that it comprises a half-life extender capable of prolonging the drug half-life, an IL-2Rα truncated peptide, and an IL-2 wild-type polypeptide or mutant, wherein the IL-2Rα truncated peptide and IL-2 are linked by a cleavable linker.

[0354] In some embodiments, the IL-2Rα truncated derivative comprises the C-terminal amino acids 168-219aa, 169-219aa, 170-219aa, 171-219aa, 172-219aa, 173-219aa, 174-219aa, 175-219aa, 176-219aa, 177-219aa, and 178-219aa from the IL-2Rα extracellular domain. Amino acid sequences missing from 179-219aa, 180-219aa, 181-219aa, 182-219aa, 183-219aa, 184-219aa, 185-219aa, 186-219aa, 187-219aa, 188-219aa, 189-219aa, 190-219aa, 191-219aa, or 192-219aa. In some embodiments, the sequence of the IL-2Rα truncated form is as shown in SEQ ID NO:4-28.

[0355] In some embodiments, the IL-2 is wild-type or a mutant. Preferably, the IL-2 mutation is R81E, L85T, or L85I. In some embodiments, the sequence of the IL-2 is as shown in SEQ ID NO:1-2. In some embodiments, the mutation of the IL-2 can reduce its affinity for IL-2Rα and increase its affinity for IL-2Rβ / γ.

[0356] In some embodiments, the IL-2Rα truncated form can inhibit the binding of IL-2 to IL-2 receptors in the environment.

[0357] In some embodiments, the cutable connector is selected from: SEQ ID NO:32-35.

[0358] In some embodiments, the cleavable linker comprises an amino acid sequence: XXXD, where X represents any single amino acid. For example, the amino acid sequence comprised by the cleavable linker is selected from: DEVD, DEVDG, DQTDG, VEID, VEVD, VEIDN, IEDT, LETD, LQTDG, LEHD, LEHDS, YVAD, WEHD, YVHDA.

[0359] In some embodiments, the half-life extender of the fusion protein is linked to the IL-2Rα truncated via an uncuttable linker.

[0360] In some embodiments, the half-life extender of the fusion protein is linked to IL-2 via an incisional linker.

[0361] In some embodiments, the uncuttable connector is a glycine / serine connector.

[0362] In some embodiments, the incuttable connector comprises: SEQ ID NO:39.

[0363] In some embodiments, the half-life extender is selected from antibody fragments or human serum albumin.

[0364] In some embodiments, the half-life extender is the antibody Fc region.

[0365] In some embodiments, the half-life extender comprises: SEQ ID NO:36.

[0366] This application also includes a method of treating or alleviating a disease or symptom, comprising administering a therapeutically effective amount of a fusion protein, polynucleotide, expression vector, host cell, or pharmaceutical composition to a patient in need.

[0367] On the one hand, the disease or symptom is cancer, selected from: bladder cancer, bone cancer, breast cancer, carcinoid, cervical cancer, colon cancer, endometrial cancer, glioma, head and neck cancer, liver cancer, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, sarcoma, skin cancer, stomach cancer, testicular cancer, thyroid cancer, urogenital cancer, or urothelial carcinoma.

[0368] On the other hand, the diseases or conditions selected are: acute myeloid leukemia, adrenocortical carcinoma, B-cell lymphoma, urothelial carcinoma of the bladder, ductal carcinoma of the breast, lobular carcinoma of the breast, esophageal cancer, castration-resistant prostate cancer (CRPC), cervical cancer, cholangiocarcinoma, chronic myeloid leukemia, colorectal adenocarcinoma, colorectal cancer (CRC), esophageal cancer, gastric adenocarcinoma, glioblastoma multiforme, squamous cell carcinoma of the head and neck, Hodgkin lymphoma / primary mediastinal B-cell lymphoma, hepatocellular carcinoma (HCC), renal stenosis. Chromoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, low-grade glioma, lung adenocarcinoma, squamous cell lung carcinoma, melanoma (MEL), mesothelioma, non-squamous NSCLC, ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma, paraganglioma and pheochromocytoma, prostate cancer, renal cell carcinoma (RCC), sarcoma, cutaneous melanoma, head and neck squamous cell carcinoma, T-cell lymphoma, thymoma, papillary thyroid carcinoma, uterine carcinosarcoma, endometrioid carcinoma of the uterine body or uveal melanoma. Beneficial effects:

[0369] (1) This invention provides a truncated form of interleukin-2 receptor α (IL-2Rα) and a fusion protein containing the same. This invention truncates the IL-2Rα protein, making its binding to IL-2 more tight. This design effectively reduces the non-specific binding of the fusion protein to regulatory T cells (Tregs) and peripheral targets, thereby reducing its peripheral toxicity and enhancing its targeting into the tumor microenvironment.

[0370] (2) Furthermore, the present invention has engineered the IL-2 protein by adding mutations that reduce the affinity of IL-2 for its receptor, thereby further reducing the activity and toxicity of the fusion protein in the periphery and providing additional safety assurance.

[0371] (3) The fusion protein of this invention, through its unique molecular design, effectively combines peripheral attenuation with intratumoral enhancement, enabling it to be efficiently enriched in the tumor microenvironment and effectively activate T cells to kill tumor cells, thereby achieving therapeutic effects while ensuring safety. Therefore, this invention provides a novel IL-2 fusion protein and its therapy with improved therapeutic effects and safety.

[0372] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0373] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0374] Figure 1 shows the in vitro binding activity of the fusion proteins described in this application to their target sites. Specifically, Figure 1A shows the binding activity of fusion protein 4 to the CLDN18.2 target site; Figure 1B shows the binding activity of fusion protein 24-3 to the PD-L1 target site; Figure 1C shows the binding activity of fusion protein 22-6 to the PD-1 target site; Figure 1D shows the binding activity of fusion proteins 33-3 and 33-4 to the VEGFR target site; Figure 1E shows the binding activity of fusion protein 35-2 to the HER2 target site; Figure 1F shows the binding activity of fusion proteins 5-2, 5-4, 5-7, and 5-9 to the PD-1 target site; and Figure 1G shows the binding activity of fusion proteins 5-11, 5-13, and 5-17 to the PD-1 target site.

[0375] Figure 2 shows the in vitro binding activity of the fusion proteins described in this application to the target. Specifically, Figure 2A shows the binding activity of fusion proteins 22-23 and 22-27 to the target PD-1; Figure 2B shows the binding activity of fusion proteins 22-38 and 22-39 to the target PD-L1.

[0376] Figure 3 shows the in vitro binding activity of the fusion proteins described in this application with the IL-2R receptor. Specifically, Figure 3A shows the binding activity of fusion protein 4 with the IL-2R receptor; Figure 3B shows the binding activity of fusion proteins 22-6 with the IL-2R receptor; Figure 3C shows the binding activity of fusion proteins 24-3 with the IL-2R receptor; and Figure 3D shows the binding activity of fusion proteins 3-31 with the IL-2R receptor.

[0377] Figure 4 shows the in vitro binding activity of the fusion proteins described in this application with the IL-2R receptor. Specifically, Figure 4A shows the binding activity of fusion proteins 3-19 and 3-23 with the IL-2R receptor; Figure 4B shows the binding activity of fusion proteins 22-23 and 22-27 with the IL-2R receptor; Figure 4C shows the binding activity of fusion proteins 3-19 and 3-30 with the IL-2R receptor; Figure 4D shows the binding activity of fusion proteins 22-38 and 22-39 with the IL-2R receptor; and Figure 4E shows the binding activity of fusion proteins 3-51 and 3-52 with the IL-2R receptor.

[0378] Figure 5 shows the changes in the in vitro bioactivity of the fusion proteins described in this application. Specifically, Figure 5A shows the changes in the in vitro bioactivity of fusion protein 4; Figure 5B shows the changes in the in vitro bioactivity of fusion proteins 22-6; Figure 5C shows the changes in the in vitro bioactivity of fusion proteins 5-2, 5-4, 5-7, and 5-9; and Figure 5D shows the changes in the in vitro bioactivity of fusion proteins 3-23 and 3-31.

[0379] Figure 6 shows the changes in the in vitro bioactivity of the fusion proteins described in this application. Specifically, Figure 6A shows the changes in the in vitro bioactivity of fusion proteins 3-19 and 3-23; Figure 6B shows the changes in the in vitro bioactivity of fusion proteins 3-19 and 3-30; Figure 6C shows the changes in the in vitro bioactivity of fusion proteins 22-38 and 22-39; Figure 6D shows the changes in the in vitro bioactivity of fusion proteins 3-51 and 3-52; and Figure 6E shows the changes in the in vitro bioactivity of fusion proteins 22-23 and 22-27.

[0380] Figure 7 shows the in vitro toxicity test results of the fusion proteins described in this application. Specifically, Figure 7A shows the in vitro toxicity test results for fusion proteins 22-6, 33-3, and 35-2; Figure 7B shows the in vitro toxicity test results for fusion proteins 3-23 and 3-31; and Figure 7C shows the in vitro toxicity test results for fusion proteins 5-2, 5-4, 5-7, 5-9, 5-11, 5-13, 5-15, and 5-17.

[0381] Figure 8 shows the in vitro toxicity test results of the fusion proteins described in this application. Specifically, Figure 8A shows the in vitro toxicity test results for fusion proteins 22-23 and 22-27; Figure 8B shows the in vitro toxicity test results for fusion proteins 3-19 and 3-30; Figure 8C shows the in vitro toxicity test results for fusion proteins 22-38 and 22-39; and Figure 8D shows the in vitro toxicity test results for fusion proteins 3-51 and 3-52.

[0382] Figure 9 shows the in vivo activity changes and safety assays of the fusion protein (fusion protein 4) described in this application.

[0383] Figure 10 shows the in vivo activity changes and safety assays of the fusion protein (fusion protein 22-6) described in this application.

[0384] Figure 11 shows the in vivo activity changes and safety assays of the fusion proteins (fusion proteins 24-3 and 24-2) described in this application.

[0385] Figure 12 shows the in vivo activity changes and safety assays of the fusion proteins (fusion proteins 33-3 and 33-4) described in this application.

[0386] Figure 13 shows the in vivo activity changes and safety assays of the fusion protein (fusion protein 35-2) described in this application.

[0387] Figure 14 shows the in vivo activity changes and safety assays of the fusion protein (fusion protein 3-52) described in this application.

[0388] Figure 15 shows the in vivo activity changes and safety assays of the fusion proteins (fusion proteins 22-38 and 22-39) described in this application.

[0389] Figure 16 shows a schematic diagram of the enzyme digestion results of the fusion protein described in this application. In the figure, 1 represents fusion protein 3-23+ undigested, 2 represents fusion protein 3-23+ digested with MMP14, 3 represents fusion protein 3-29+ undigested, and 4 represents fusion protein 3-29+ digested with MMP14.

[0390] Figure 17 shows various structural schematic diagrams of the fusion protein described in this application. Detailed Implementation

[0391] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0392] definition

[0393] In this application, the terms “interleukin-2 receptor α”, “interleukin-2 receptor a”, “IL-2Rα”, “IL2Rα”, “IL-2Ra” or “IL2Ra”, also known as CD25, are one of the receptors for IL-2 and have a relatively low affinity for IL-2.

[0394] In this application, the term "wild-type IL-2Rα" refers to the wild-type human interleukin-2 receptor α chain (IL-2Rα, whose amino acid sequence is referenced to UniProt database accession number P01589), and any human natural variant that has at least 85% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) amino acid sequence identity with the reference sequence and retains the function of binding to IL-2.

[0395] In this application, the term "wild-type IL-2Rα extracellular domain" refers to the extracellular functional region of the wild-type human interleukin-2 receptor α chain (IL-2Rα, whose amino acid sequence is referenced to UniProt database accession number P01589), and any corresponding extracellular functional region of a human natural variant that has at least 85% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) amino acid sequence identity with that extracellular functional region and retains the function of specifically binding to IL-2. In some embodiments, the wild-type IL-2Rα extracellular domain refers to an amino acid sequence fragment known in the art and commonly used to consist of positions Glu22 to Gln240 (219 amino acids) or Glu22 to Ser233 (212 amino acids), wherein the amino acid position numbering is defined based on the corresponding amino acid positions indicated in UniProt accession number P01589. In some embodiments, the wild-type IL-2Rα extracellular domain comprises an amino acid sequence as shown in SEQ ID NO:3, or comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:3. In some embodiments, the amino acid sequence of the wild-type IL-2Rα extracellular domain is as shown in SEQ ID NO:3, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:3. In some embodiments, the amino acid sequence of the wild-type IL-2Rα extracellular domain comprises the sequence of SEQ ID NO:3 after removing the last 7 amino acid residues from the C-terminus, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with that sequence. In some embodiments, the amino acid sequence of the wild-type IL-2Rα extracellular domain consists of the sequence of SEQ ID NO:3 after removing the last 7 amino acid residues from the C-terminus, or consists of the sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with that sequence.For a description of the extracellular domain of wild-type IL-2Rα, see, for example: Deborah J et al. Crystal structure of the IL-2 signaling complex: paradigm for a heterotrimeric cytokine receptor.[J]. Proc Natl Acad Sci USA, 2006, 103: 2788-93.

[0396] Those skilled in the art will understand that, in order to facilitate the structural stability of the wild-type full-length IL-2Rα fragment in the fusion protein, the above-mentioned wild-type full-length IL-2Rα sequence can be artificially mutated at a specific site (C192S) (that is, the natural cysteine ​​at position 192 of the mature protein is replaced with serine, C192S). This mutation only eliminates the free thiol group at this site in the wild-type sequence, without changing the natural characteristics of the full-length IL-2Rα fragment or the core function of IL-2 specific binding, and can be used as a functionally equivalent form of the wild-type full-length IL-2Rα fragment in the fusion protein of the present invention.

[0397] In this application, the term "N-terminus of the wild-type IL-2Rα extracellular domain" refers to the N-terminal start site of the mature extracellular domain of the wild-type human interleukin-2 receptor α chain (IL-2Rα, whose amino acid sequence is referenced in UniProt database accession number P01589) after natural post-translational processing and excision of the N-terminal signal peptide. This site corresponds to the Glu22 position in the reference sequence (UniProt database accession number P01589). This N-terminal definition also applies to any mature extracellular domain of a human natural variant that has at least 85% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) amino acid sequence identity with the extracellular domain of the reference sequence (UniProt database accession number P01589) and retains its IL-2-specific binding function.

[0398] In this application, the term "IL-2Rα truncated variant" or "IL-2Ra truncated variant" refers to a molecule obtained by truncating the C-terminus of the IL-2Rα extracellular domain (e.g., the wild-type IL-2Rα extracellular domain) to different lengths of amino acids. In some embodiments, in addition to C-terminal deletion, the "IL-2Rα truncated variant" also contains one or more mutations.

[0399] In this application, the terms “interleukin-2”, “IL-2” or “IL-2” generally refer to a T cell growth factor called interleukin 2 and include wild-type IL-2 and variants containing one or more mutations in the amino acid sequence of wild-type IL-2, which may include, in one embodiment, human and mammalian forms, conserved amino acid substitutions, glycoform modifications or variants.

[0400] In this application, the term "target-binding polypeptide" refers to a polypeptide that binds to a target biomolecule (such as a T cell surface receptor, tumor-associated antigen, immune checkpoint molecule, or its epitope) through specific molecular interactions, and whose functions include, but are not limited to: directing the IL-2 functional domain of a fusion protein to a specific cell type or tissue; regulating downstream signaling pathways by binding to the target (such as enhancing / inhibiting receptor activation); and synergizing with IL-2 to enhance therapeutic effects (such as targeting immune cells in the tumor microenvironment). Further, the target-binding polypeptide is an antibody or a functional fragment thereof. In some embodiments, the target-binding polypeptide comprises an antibody or a functional fragment thereof.

[0401] In this application, the term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with multi-epitope specificity, bispecific antibodies, dimerized antibodies, single-chain molecules, etc. The term "antibody" is used in the broadest sense and encompasses a wide variety of antibody structures, including not only complete (i.e., full-length) antibodies, but also their antigen-binding fragments (e.g., Fab, Fab', F(ab')2, Fv), their variants, fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, biantibodies, linear antibodies, single-chain antibodies (scFV), VHH antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configurations of immunoglobulin molecules containing antigen recognition sites of desired specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Typically, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and first, second, and third constant regions (CH1, CH2, CH3). Each light chain contains a variable region (VL) and a constant region (CL). The constant region includes either κ (Kappa) or λ (Lambda). Full-length antibodies can be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses mentioned above), but the antibody does not need to belong to any specific class. Immunoglobulins can be assigned to different classes based on the antibody amino acid sequence of the constant region of the heavy chain. Typically, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant regions of the heavy chain corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.

[0402] In this application, the term "functional fragment" of an antibody refers to any portion of a full-length antibody, but which at least contains a portion of the variable region of the antibody that binds to an antigen (e.g., one or more CDRs and / or one or more antibody binding sites), and thus retains binding specificity and at least a portion of the specific binding ability of the full-length antibody. Therefore, an antigen-binding fragment refers to an antibody fragment containing an antigen-binding portion that binds to the same antigen as an antibody fragment derived from the antibody. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetically produced derivatives, such as recombinant derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, biantibodies, Fd and Fd' fragments, and other fragments, including modified fragments. The fragments may include multiple chains linked together, for example by disulfide bonds and / or by peptide linkers. An antibody's "functional fragment" generally contains at least or about 50 amino acids, and typically at least or about 200 amino acids. An antigen-binding fragment includes any antibody fragment that, when inserted into an antibody frame, acquires an antibody that immunely and specifically binds to an antigen.

[0403] In some embodiments, the target-binding polypeptide comprises an antibody variable region (e.g., VH, VL, scFv, VHH, etc.) that specifically binds to the target antigen, and may further fuse one or more of an antibody constant region sequence to the N-terminus or C-terminus of the antibody variable region, wherein the constant region sequence is selected from the CL, CH1, Fc region, hinge region, or a combination thereof. Based on this, the target-binding polypeptide may take various forms, including but not limited to: a single-chain fragment consisting only of the antibody variable region, a Fab fragment comprising the antibody variable region and CL / CH1, an F(ab')2 fragment formed by connecting Fab fragments via hinge regions, scFv-Fc or Fab-Fc fusion proteins formed by fusing the antibody variable region and the Fc region, and a complete antibody comprising the complete constant region.

[0404] In this application, the term "uncleanable linker" refers to a peptide whose amino acid sequence lacks the substrate sequence of a protease, making it difficult to be cleaved by endogenous or exogenous proteases, hydrolases, or other biochemical processes under physiological conditions or target application environments. For example, this application uses a glycine / serine linker.

[0405] In this application, the term "cleavable linker" refers to a peptide whose amino acid sequence contains one or more substrates of one or more proteases.

[0406] In this application, the term "tumor-associated protease" refers to an enzyme that catalyzes the hydrolysis of cleavable linkers in the tumor microenvironment. The tumor microenvironment is an acidic environment. The "tumor-associated protease" is expressed at high levels in the tumor microenvironment but is almost not expressed in normal tissues. For example, MMP proteases are highly expressed in tumors and can specifically cleave proteins within the tumor.

[0407] In this application, the term "tumor-associated antigen" or "TAA" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) that is fully expressed or expressed on the surface of cancer cells and can be used to preferentially target pharmaceutical agents to cancer cells. In some embodiments, TAAs are markers expressed by both normal and cancer cells, such as lineage markers. In some embodiments, TAAs are cell surface molecules that are overexpressed in cancer cells compared to normal cells, for example, 1-fold, 2-fold, 3-fold, or more overexpression compared to normal cells. In some embodiments, TAAs are cell surface molecules that are inappropriately synthesized in cancer cells, such as molecules containing deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, TAAs are expressed entirely or as fragments (e.g., MHC / peptides) specifically on the cell surface of cancer cells and are not synthesized or expressed on the surface of normal cells. Therefore, the term "TAA" encompasses cancer cell-specific antigens, sometimes referred to in the art as tumor-specific antigens.

[0408] In this application, the term "T cell surface receptor" refers to a molecule (typically a protein, carbohydrate, lipid, or some combination thereof) expressed on the surface of T lymphocytes and useful for preferentially targeting a specific site with a drug. In some embodiments, this site is cancerous tissue and / or the T cell antigen is a tumor-reactive lymphocyte antigen, a cell surface molecule of tumor or viral lymphocytes, or a checkpoint inhibitor expressed on T lymphocytes, such as PD-1.

[0409] In this application, the term "immune checkpoint molecule" generally refers to a molecule that regulates the immune system. For example, an immune checkpoint molecule can prevent the immune system from attacking cells. For example, an immune checkpoint molecule could be PD-L1.

[0410] In this application, the term "half-life extender" refers to a domain attached to a target component (e.g., a cytokine or a functional fragment thereof, or a masking portion) to prolong the half-life of a target component in serum. The term "half-life extender" encompasses, for example, antibodies, antibody fragments, bispecific antibodies, albumins, binding proteins, polyamino acid sequences, and antibody derivatives. For example, a half-life extender is the Fc region of an antibody.

[0411] In this application, the term "antibody Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, which contains at least a CH2 domain and a CH3 domain, or further contains a portion of a hinge region. The "Fc region" can be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as the segment from the amino acid residue at position Cys226 or Pro230 to its carboxyl terminus. As is known in the art, the Fc region can exist in dimer or monomeric form. In some embodiments, the antibody Fc region is selected from: wild-type Fc regions, mutant Fc regions, wild-type Fc monomers, mutant Fc monomers, wild-type Fc dimers, or mutant Fc dimers, wherein the mutant Fc monomers or mutant Fc dimers are, for example, site-directed mutagenesis-modified mutant Fc monomers or mutant Fc dimers.

[0412] In this application, the terms "single-chain Fc" and "Fc monomer" are used interchangeably, referring to an antibody Fc fragment existing in monomeric form, consisting of a single polypeptide chain, which at least contains the CH2 and CH3 domains of the immunoglobulin heavy chain constant region, and may further contain a portion of the hinge region (e.g., for human IgG, the segment from Cys226 or Pro230 to the carboxyl terminus). This term encompasses both native Fc monomers and mutant Fc monomers obtained through site-directed mutagenesis or other modifications.

[0413] In this application, the term "mutation" generally refers to a change in the nucleotide or amino acid sequence of a wild-type molecule. Mutations in DNA can alter codons, resulting in changes in the amino acid sequence. DNA changes can include substitutions, deletions, insertions, alternative splicing, or truncation. Amino acid changes can include substitutions, deletions, insertions, additions, truncations, or errors in protein processing or cleavage. Alternatively, mutations in the nucleotide sequence can result in silent mutations in the amino acid sequence as well understood in the art.

[0414] In this application, the term "dimer" refers to a molecule having two peptide chains that are covalently or non-covalently linked, with or without a linker.

[0415] In this application, the term "homodimer" refers to a molecule comprising two polypeptide chains of comparable length, wherein the two polypeptide chains have the same amino acid sequence at corresponding positions, wherein the corresponding positions are determined according to the Kabat EU index.

[0416] In this application, the term "heterodimer" refers to a molecule comprising two polypeptide chains (e.g., of comparable length), wherein the two polypeptide chains have an amino acid sequence having at least one different amino acid residue at a corresponding position, the corresponding position being determined according to the Kabat EU index.

[0417] In this application, the term "treatment" generally refers to the application or administration of a therapeutic agent to a patient, or to the application or administration of a therapeutic agent to a tissue or cell line isolated from a patient who has a disease, symptoms of a disease, or a predisposition to a disease, with the aim of treating, curing, alleviating, relieving, altering, remedying, improving, enhancing, or influencing the disease, symptoms of a disease, or a predisposition to a disease. For example, "treatment" for a patient for whom symptoms or clinically relevant manifestations of a disease or disorder have not yet been identified is preventative or preventive treatment, while clinical, curative, or remittent "treatment" for a patient for whom symptoms or clinically relevant manifestations of a disease or disorder have been identified generally does not constitute preventative or preventive treatment.

[0418] In this application, the term "therapeuticly effective amount" generally refers to an amount of substance, compound, material, or composition containing a compound that, when applied to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or condition.

[0419] In this application, the term "host cell" refers to a cell used to receive, maintain, replicate, and amplify the vector. The host cell can also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acids contained in the vector replicate, thereby amplifying the nucleic acids. The host cell can be a eukaryotic or prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells, and CHO (transient and stable transfection) cells.

[0420] In this application, the term "vector" generally refers to a reproducible nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into a suitable host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically through restriction enzyme digestion and ligation. Vectors also include those containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for amplification of nucleic acids or for expression / display of polypeptides encoded by nucleic acids. Vectors are typically kept free but can be designed to integrate genes or portions thereof into the chromosome of the genome.

[0421] In this application, the term "pharmaceuticalally acceptable excipient" includes any material that, when combined with an active ingredient, allows that ingredient to retain its biological activity and does not react with the immune system of a subject. Examples include, but are not limited to, any standard drug carrier, such as phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, and various types of wetting agents.

[0422] In this application, the percentage of "identity," such as 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%, refers to the degree of similarity between amino acid sequences or nucleotide sequences determined by sequence alignment, which is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5%. For example, it is the percentage of positions with identical bases or amino acid residues relative to the total number of positions, determined after introducing vacancies or other methods to make two sequences have as many identical residues as possible. The percentage of "identity" can be determined using software programs known in the art. It is preferred to use default parameters for alignment. A preferred alignment program is BLAST. Preferred programs are BLASTN and BLASTP. Details of these procedures can be found at the following internet address: blast.ncbi.nlm.nih.gov / Blast.cgi.

[0423] In this application, the terms "nucleic acid," "nucleic acid sequence," "nucleotide sequence," "polynucleotide," "polynucleotide sequence," "RNA sequence," or "DNA sequence" refer to oligonucleotides, nucleotides, or polynucleotides and fragments or portions thereof, and refer to DNA or RNA of genomic or synthetic origin, which may be single-stranded or double-stranded and represent sense or antisense strands. Sequences may be non-coding sequences, coding sequences, or mixtures of both. The nucleic acid sequences of this application can be prepared using standard techniques well known to those skilled in the art. Nucleotides are designated by their individual letter names as follows: "A" for adenosine or deoxyadenosine (corresponding to RNA or DNA, respectively), "C" for cytidine or deoxycytidine, "G" for guanosine or deoxyguanosine, "U" for uridine, "T" for deoxythymidine, "R" for purine (A or G), "Y" for pyrimidine (C or T), "K" for G or T, "H" for A, C, or T, "I" for inosine, and "N" for any nucleotide.

[0424] In this application, the term "polynucleotide" refers to a polymeric form of a nucleotide having a length of at least 10 bases, which can be any one of ribonucleotides or deoxyribonucleotides or modified forms of said two nucleotides. The term includes both single-stranded and double-stranded forms of DNA.

[0425] In this application, the term "amino acid" or "amino acid sequence" refers to an oligopeptide, peptide, polypeptide, or protein sequence, or any fragment thereof, and refers to a naturally occurring or synthetic molecule. When "amino acid sequence" is described herein as referring to the amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and similar terms are not intended to limit the amino acid sequence to the complete naturally occurring amino acid sequence associated with the described protein molecule. In this document, "amino acid" may be referred to by its name, its commonly known three-letter symbol, or a single-letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0426] Invention Details

[0427] To address the shortcomings of existing technologies, this application provides a truncated IL-2Rα variant and an IL-2 fusion protein containing the same, wherein the IL-2 fusion protein exhibits reduced toxicity and good anti-tumor effects. In this application, the inventors have found that existing IL-2 fusion proteins suffer from poor stability and high toxicity, severely limiting their clinical application prospects. To solve the above technical problems, this application adopts the following technical solution.

[0428] IL-2Rα truncated body

[0429] In a first aspect, this application provides a truncated form of IL-2Rα comprising 166 to 190 or 167 to 190 amino acids continuously from the N-terminus of the extracellular domain of wild-type IL-2Rα.

[0430] For example, it contains 166 to 190, 167 to 190, 168 to 190, 169 to 190, 170 to 190, 175 to 190, 180 to 190, 181 to 190, 182 to 190, 183 to 190, 184 to 190, 185 to 190, 190, 185, 180, 175, 174, 173, 172, 171, 170, 169, 168, 167, or 166 amino acids consecutively starting from the N-terminus of the wild-type IL-2Rα extracellular domain.

[0431] The amino acid sequence of the wild-type IL-2Rα extracellular domain is as shown in SEQ ID NO:3, or is the sequence of SEQ ID NO:3 truncated by 7 amino acids at its C-terminus. This application is not intended to limit the specific sequence of the wild-type IL-2Rα extracellular domain; SEQ ID NO:3 and its truncated form are merely exemplary reference sequences. In different embodiments, the wild-type IL-2Rα extracellular domain may also be an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:3, as long as it can form a truncated version with corresponding biological activity.

[0432] Furthermore, those skilled in the art will understand that, using SEQ ID NO:3 as a reference sequence, the phrase "comprising 166 to 190 or 167 to 190 amino acids consecutively starting from the N-terminus of the wild-type IL-2Rα extracellular domain" is structurally equivalent to "comprising a sequence obtained by truncating the C-terminus of the IL-2Rα extracellular domain by 29 to 52 or 29 to 53 amino acids." In other words, retaining a specific length of amino acid sequence at the N-terminus means truncating the corresponding length at the C-terminus, and both are equivalent definitions of the truncated structure described in this application.

[0433] In some embodiments, the IL-2Rα truncated form comprises 166 to 182 or 167 to 182 amino acids consecutively starting from the N-terminus of the wild-type IL-2Rα extracellular domain. For example, it comprises 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, or 182 amino acids consecutively starting from the N-terminus of the wild-type IL-2Rα extracellular domain.

[0434] In some embodiments, the IL-2Rα truncated form comprises 166 to 172 or 167 to 172 amino acids consecutively starting from the N-terminus of the wild-type IL-2Rα extracellular domain. For example, it comprises 166, 167, 168, 169, 170, 171, or 172 amino acids consecutively starting from the N-terminus of the wild-type IL-2Rα extracellular domain.

[0435] In some embodiments, the IL-2Rα truncated form comprises an amino acid sequence truncated to 29 to 52 or 29 to 53 amino acids at the C-terminus of the IL-2Rα extracellular domain, compared to the 219aa wild-type IL-2Rα extracellular domain. For example, amino acid sequences containing IL-2Rα extracellular domains truncated to C-terminus lengths of 29 to 53, 29 to 52, 29 to 51, 29 to 50, 29 to 45, 29 to 40, 29 to 35, 30 to 53, 35 to 53, 40 to 53, 45 to 53, 46 to 53, 47 to 53, 48 to 53, 49 to 53, 50 to 53, 29, 30, 35, 40, 45, 46, 47, 48, 49, 50, 51, 52, or 53 amino acid sequences.

[0436] In some embodiments, the IL-2Rα truncated form comprises an amino acid sequence truncated to 37 to 52 or 37 to 53 amino acids at the C-terminus of the IL-2Rα extracellular domain, compared to the 219aa wild-type IL-2Rα extracellular domain. For example, it comprises an amino acid sequence truncated to 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 amino acids at the C-terminus of the IL-2Rα extracellular domain.

[0437] In some embodiments, the IL-2Rα truncated derivative comprises the C-terminal amino acids 167-219aa, 168-219aa, 169-219aa, 170-219aa, 171-219aa, 172-219aa, 173-219aa, 174-219aa, 175-219aa, 176-219aa, 177-219aa, 178-219aa, 17 ...9-219aa, 170-219aa, 179-219aa, 170-219aa, 179-219aa, 170-219aa, 179-219aa, 170-219aa, 179-219aa, 170-219aa, 17 Amino acid sequences missing from 19aa, 179-219aa, 180-219aa, 181-219aa, 182-219aa, 183-219aa, 184-219aa, 185-219aa, 186-219aa, 187-219aa, 188-219aa, 189-219aa, 190-219aa, 191-219aa, or 192-219aa.

[0438] In some embodiments, the IL-2Rα truncated derivative comprises the C-terminal amino acids 167-219aa, 168-219aa, 169-219aa, 170-219aa, 171-219aa, 172-219aa, 173-219aa, 174-219aa, 175-219aa, 176-219aa, 177-219aa, 178-219aa, 17 ...9-219aa, 170-219aa, 179-219aa, 170-219aa, 179-219aa, 170-219aa, 179-219aa, 170-219aa, 179-219aa, 170-219aa, 17 Amino acid sequences missing from 19aa, 179-219aa, 180-219aa, 181-219aa, 182-219aa, 183-219aa, 184-219aa, 185-219aa, 186-219aa, 187-219aa, 188-219aa, 189-219aa, 190-219aa, 191-219aa, or 192-219aa.

[0439] In some embodiments, the IL-2Rα truncated form comprises an amino acid sequence as shown in any one of SEQ ID NO: 5-29, or the amino acid sequence of the IL-2Rα truncated form is as shown in any one of SEQ ID NO: 5-29. In some embodiments, the IL-2Rα truncated form comprises an amino acid sequence as shown in any one of SEQ ID NO: 13-29, or the amino acid sequence of the IL-2Rα truncated form is as shown in any one of SEQ ID NO: 13-29. In some embodiments, the IL-2Rα truncated form comprises an amino acid sequence as shown in any one of SEQ ID NO: 23-29, or the amino acid sequence of the IL-2Rα truncated form is as shown in any one of SEQ ID NO: 23-29.

[0440] Those skilled in the art will understand that the IL-2Rα truncated variant is not limited to the specific sequences listed above. The IL-2Rα truncated variant should encompass sequences that, compared to any of the sequences shown in SEQ ID NO:5-29, contain one, two, or three or more amino acid mutations, but are substantially functionally identical to them; it also includes sequences that, compared to any of the sequences shown in SEQ ID NO:5-29, have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0441] In some embodiments, the IL-2Rα truncated variant also includes one or more mutations in addition to truncation.

[0442] This application does not impose any restrictions on the method for preparing IL-2Rα truncated variants. The method can be modified according to conventional methods in the art, such as directed mutagenesis, random mutagenesis, or construction of synthetic oligonucleotides, and then the modified DNA sequence can be expressed in host cells to obtain IL-2Rα truncated variants with amino acid sequence substitution, insertion, deletion and / or truncation.

[0443] Secondly, this application provides a polynucleotide that encodes the truncated IL-2Rα variant as described in the first aspect of this application.

[0444] Thirdly, this application provides an expression vector comprising the polynucleotides described in the second aspect of this application.

[0445] In some embodiments, the expression vector is a plasmid, granule, bacteriophage, or viral vector, preferably a plasmid. For example, a polynucleotide encoding the truncated IL-2Rα variant can be cloned into a suitable expression vector, which can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and amplification, or for expression, or both, such as plasmids and viruses.

[0446] Fourthly, this application provides a host cell containing the polynucleotide described in the second aspect of this application or the expression vector described in the third aspect of this application.

[0447] In some implementations, the expression vector is transformed into a host cell for further expression or cloning within the host cell.

[0448] The host cell refers to a cell in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include primary transformed cells and their derived progeny, regardless of passage number. Progeny cells may not be entirely identical to parent cells in terms of nucleic acid content and may contain mutations.

[0449] In some embodiments, the host cell is a eukaryotic cell, a prokaryotic cell, or a bacterial cell.

[0450] Fifthly, this application provides a method for preparing an IL-2Rα truncated form, which includes culturing the host cells described in the fourth aspect of this application to prepare the IL-2Rα truncated form described in the first aspect of this application.

[0451] In some embodiments, the method further includes purifying or separating the produced IL-2Rα truncated form. The purification or separation methods are conventional in the art.

[0452] IL-2 fusion protein

[0453] Sixthly, this application provides a fusion protein comprising:

[0454] Interleukin-2 (IL-2) wild-type or mutant protein;

[0455] A cleavable linker that connects to the wild-type or mutant IL-2 protein;

[0456] Interleukin-2 receptor α (IL-2Rα) truncated bodies connected to the said cleavable connector; and

[0457] Target-binding peptides;

[0458] The IL-2Rα truncated body is the IL-2Rα truncated body described in the first aspect of this application.

[0459] In some embodiments, the target-binding polypeptide is linked to the IL-2Rα truncated variant via an uncuttable linker. In some embodiments, the target-binding polypeptide is linked to the wild-type or mutant IL-2 protein via an uncuttable linker.

[0460] In some embodiments, the fusion protein sequentially comprises: a target-binding polypeptide, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein; wherein the target-binding polypeptide is linked to the IL-2Rα truncated form via an uncuttable linker.

[0461] In some embodiments, the fusion protein sequentially comprises: a target-binding polypeptide, a wild-type or mutant IL-2 protein, and an IL-2Rα truncated form; wherein the target-binding polypeptide is linked to the wild-type or mutant IL-2 protein via an uncuttable linker.

[0462] Seventhly, this application provides a fusion protein comprising:

[0463] Half-life extenders;

[0464] Interleukin-2 (IL-2) wild-type or mutant protein;

[0465] Cleavable linkers to the wild-type or mutant IL-2 protein; and

[0466] A truncated form of interleukin-2 receptor α (IL-2Rα) connected to the cuttable connector;

[0467] The IL-2Rα truncated body is the IL-2Rα truncated body described in the first aspect of this application.

[0468] In some embodiments, the half-life extender is linked to the IL-2Rα truncated variant via an uncuttable linker. In some embodiments, the half-life extender is linked to the wild-type or mutant IL-2 protein via an uncuttable linker.

[0469] In some embodiments, the half-life extender is selected from...

[0470] (i) Antibody Fc region or human serum albumin

[0471] (ii) wild-type Fc region, mutant Fc region, wild-type Fc monomer, mutant Fc monomer, wild-type Fc dimer or mutant Fc dimer, wild-type single-stranded Fc, or mutant single-stranded Fc, preferably, the mutant single-stranded Fc, mutant Fc monomer or mutant Fc dimer is a mutant single-stranded Fc, mutant Fc monomer or mutant Fc dimer modified by site-directed mutagenesis;

[0472] (iii) A single-chain Fc containing an amino acid mutation, a first single-chain Fc and / or a second single-chain Fc;

[0473] (iv) Single-stranded Fc, first single-stranded Fc and / or second single-stranded Fc containing Knob, Hole or LALA mutations;

[0474] (v) Contains an amino acid sequence as shown in SEQ ID NO:36.

[0475] In some embodiments, the half-life extender is an antibody Fc region selected from: wild-type Fc region, mutant Fc region, wild-type Fc monomer, mutant Fc monomer, wild-type Fc dimer, or mutant Fc dimer. In some embodiments, the mutant Fc monomer or mutant Fc dimer is a site-directed mutagenesis-modified mutant Fc monomer or mutant Fc dimer. In some embodiments, the half-life extender comprises the amino acid sequence shown in SEQ ID NO:36.

[0476] In some embodiments, the fusion protein further comprises a first target-binding polypeptide and / or a second target-binding polypeptide.

[0477] In some embodiments, the fusion protein is selected from a combination of any of the following polypeptide chains:

[0478] (1) Two first polypeptide chains, wherein the first polypeptide chains sequentially comprise (preferably from N-terminus to C-terminus): a first target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated form and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the single-chain Fc via an uncleavable linker;

[0479] (2) Two first polypeptide chains, each first polypeptide chain sequentially comprising (preferably from N-terminus to C-terminus): a light chain of a first target-binding polypeptide, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the light chain of the first target-binding polypeptide via an uncleavable linker; two second polypeptide chains, each second polypeptide chain sequentially comprising (preferably from N-terminus to C-terminus): a heavy chain Fd segment of the first target-binding polypeptide, a single-chain Fc, and a second target-binding polypeptide with an scFv structure, wherein preferably, the single-chain Fc is connected to the second target-binding polypeptide with an scFv structure via a flexible linker 1, and preferably, in the second target-binding polypeptide with an scFv structure, VH and VL are connected via a flexible linker 2;

[0480] (3) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; two second polypeptide chains, each second polypeptide chain comprising (preferably from N-terminus to C-terminus): wild-type or mutant IL-2 protein, IL-2Rα truncated form, heavy chain Fd segment of the first target-binding polypeptide, single chain Fc, and a second target-binding polypeptide of scFv structure, wherein the wild-type or mutant IL-2 protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the heavy chain of the first target-binding polypeptide via an uncleavable linker. Preferably, the single chain Fc is connected to the second target-binding polypeptide of scFv structure via a flexible linker 1. Preferably, in the second target-binding polypeptide of scFv structure, VH and VL are connected via a flexible linker 2.

[0481] (4) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; two second polypeptide chains, each second polypeptide chain comprising (preferably from N-terminus to C-terminus): a second target-binding polypeptide of scFv structure, a heavy chain Fd segment of the first target-binding polypeptide, a single chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the single chain Fc via an uncleavable linker. Preferably, the heavy chain Fd segment of the first target-binding polypeptide is connected to the second target-binding polypeptide of scFv structure via a flexible linker 1. Preferably, in the second target-binding polypeptide of scFv structure, VH and VL are connected via a flexible linker 2.

[0482] (5) Two first polypeptide chains, each first polypeptide chain comprising: a light chain variable region of a second target-binding polypeptide and a light chain of the first target-binding polypeptide; two second polypeptide chains, each second polypeptide chain comprising (preferably from N-terminus to C-terminus) in sequence: a heavy chain variable region of the second target-binding polypeptide, a heavy chain Fd segment of the first target-binding polypeptide, a single chain Fc, an IL-2Rα truncated body, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated body via a cleavable linker, and the IL-2Rα truncated body is connected to the single chain Fc via an uncleavable linker; preferably, the heavy chain variable region of the second target-binding polypeptide is connected to the heavy chain Fd segment of the first target-binding polypeptide via a flexible linker 1; and preferably, the light chain variable region of the second target-binding polypeptide is connected to the light chain of the first target-binding polypeptide via a flexible linker 2.

[0483] (6) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; two second polypeptide chains, each second polypeptide chain comprising (preferably from N-terminus to C-terminus): a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the single-chain Fc via an uncleavable linker;

[0484] (7) Two first polypeptide chains, each first polypeptide chain sequentially comprising (preferably from N-terminus to C-terminus): a light chain of a first target-binding polypeptide, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the light chain of the first target-binding polypeptide via an uncleavable linker; two second polypeptide chains, each second polypeptide chain sequentially comprising (preferably from N-terminus to C-terminus): a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), and a single-chain Fc;

[0485] (8) Two first polypeptide chains, each first polypeptide chain sequentially comprising (preferably from N-terminus to C-terminus): wild-type or mutant IL-2 protein, IL-2Rα truncated form, and a light chain of a first target-binding polypeptide, wherein the wild-type or mutant IL-2 protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the light chain of the first target-binding polypeptide via an uncleavable linker; two second polypeptide chains, each second polypeptide chain sequentially comprising (preferably from N-terminus to C-terminus): a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), and a single-chain Fc;

[0486] (9) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; two second polypeptide chains, each second polypeptide chain comprising (preferably from N-terminus to C-terminus): wild-type or mutant IL-2 protein, an IL-2Rα truncated body, a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), and a single chain Fc, wherein the wild-type or mutant IL-2 protein is connected to the IL-2Rα truncated body via a cleavable linker, and the IL-2Rα truncated body is connected to the heavy chain Fd segment of the first target-binding polypeptide via an uncleavable linker;

[0487] (10) A first polypeptide chain, comprising (preferably from N-terminus to C-terminus) the following: a first target-binding polypeptide of the scFv structure, a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is linked to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is linked to the single-chain Fc via an uncleavable linker; a second polypeptide chain, comprising (preferably from N-terminus to C-terminus) the following: a first target-binding polypeptide The first target-binding polypeptide has a heavy chain variable region, a heavy chain Fd segment of the second target-binding polypeptide, and a single chain Fc; the third polypeptide chain comprises (preferably from the N-terminus to the C-terminus) the following in sequence: a light chain variable region of the first target-binding polypeptide and a light chain of the second target-binding polypeptide. Preferably, the heavy chain variable region of the first target-binding polypeptide is connected to the heavy chain Fd segment of the second target-binding polypeptide via a flexible linker 1, and preferably, the light chain variable region of the first target-binding polypeptide is connected to the light chain of the second target-binding polypeptide via a flexible linker 2.

[0488] (11) Two first polypeptide chains, comprising: a light chain of a first target-binding polypeptide; a second polypeptide chain, comprising (preferably from N-terminus to C-terminus) the following in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide of scFv structure, a first single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker; a third polypeptide chain, comprising (preferably from N-terminus to C-terminus) the following in sequence: a heavy chain Fd segment of the first target-binding polypeptide and a second single-chain Fc.

[0489] In the polypeptide chains described in (1)-(11) above, VH and VL in the target-binding polypeptide with the scFv structure can be connected by a flexible linker; and the first target-binding polypeptide and the second target-binding polypeptide can also be connected by a flexible linker. The flexible linkers between VH and VL and between the first and second target-binding polypeptides may be the same or different.

[0490] The flexible linker, flexible linker 1, and flexible linker 2 are selected from the antibody flexible linker region or contain an amino acid sequence composed of glycine and serine. The antibody flexible linker region is preferably an elbow region or a hinge region. The flexible linker 1 and flexible linker 2 are preferably 5-15 amino acids each.

[0491] In this application, the elbow region, located inside the Fab segment, is the bend between the heavy chain variable region (VH) and the heavy chain constant region 1 (CH1), and between the light chain variable region (VL) and the light chain constant region (CL). Preferably, the flexible linker 1 and the flexible linker 2 can be selected from amino acid sequences of different lengths in the heavy chain elbow region and the light chain elbow region, respectively, such as ASTKGP (HC-γS), ASTKGPSVFPLAP (HC-γL), TVAAP (LC-κS), TVAAPSVFIFPP (LC-κL), QPKAAP (LC-λS), or QPKAAPSVTLFPP (LC-λL).

[0492] In this application, the hinge region, located between the Fab and Fc segments, is the amino acid sequence between the heavy chain constant region 1 (CH1) and constant region 2 (CH2). Preferably, the flexible linker 1 and flexible linker 2 can be selected from amino acid sequences of different lengths in the hinge region, such as PNLLGGP (HC-hS), PAPNLLGGP (HC-hL), PAPNLLGGP (LC-hS), or PTISPAPNLLGGP (LC-hL).

[0493] In some embodiments, the single-chain Fc, the first single-chain Fc, and / or the second single-chain Fc contain amino acid mutations; preferably, the amino acid mutations are Knob, Hole, or LALA mutations.

[0494] Eighthly, this application provides a fusion protein comprising:

[0495] Half-life extenders;

[0496] Interleukin-2 (IL-2) wild-type or mutant protein;

[0497] Interleukin-2 receptor α (IL-2Rα) truncated variant; and

[0498] Cuttable connectors;

[0499] The IL-2Rα truncated body is the IL-2Rα truncated body described in the first aspect of this application.

[0500] In some embodiments, the half-life extender is selected from...

[0501] (i) Antibody Fc region or human serum albumin

[0502] (ii) wild-type Fc region, mutant Fc region, wild-type Fc monomer, mutant Fc monomer, wild-type Fc dimer or mutant Fc dimer, wild-type single-stranded Fc, or mutant single-stranded Fc, preferably, the mutant single-stranded Fc, mutant Fc monomer or mutant Fc dimer is a mutant single-stranded Fc, mutant Fc monomer or mutant Fc dimer modified by site-directed mutagenesis;

[0503] (iii) A single-chain Fc containing an amino acid mutation, a first single-chain Fc and / or a second single-chain Fc;

[0504] (iv) Single-stranded Fc, first single-stranded Fc and / or second single-stranded Fc containing Knob, Hole or LALA mutations;

[0505] (v) Contains an amino acid sequence as shown in SEQ ID NO:36.

[0506] In some embodiments, the half-life extender is an antibody Fc region selected from: wild-type Fc region, mutant Fc region, wild-type Fc monomer, mutant Fc monomer, wild-type Fc dimer, or mutant Fc dimer. In some embodiments, the mutant Fc monomer or mutant Fc dimer is a site-directed mutagenesis-modified mutant Fc monomer or mutant Fc dimer. In some embodiments, the half-life extender comprises the amino acid sequence shown in SEQ ID NO:36.

[0507] In some embodiments, the fusion protein comprises: (i) a first polypeptide chain comprising a first single-chain Fc and an IL-2Rα truncated variant; and (ii) a second polypeptide chain comprising a second single-chain Fc and an IL-2 wild-type or mutant protein. In some embodiments, the IL-2 wild-type or mutant protein is linked to the second single-chain Fc via a cleavable linker, and the IL-2Rα truncated variant is linked to the first single-chain Fc via an uncleavable linker. In some embodiments, the IL-2 wild-type or mutant protein is linked to the second single-chain Fc via an uncleavable linker, and the IL-2Rα truncated variant is linked to the first single-chain Fc via a cleavable linker.

[0508] In some embodiments, the fusion protein further comprises a first target-binding polypeptide and / or a second target-binding polypeptide.

[0509] In some embodiments, the fusion protein is selected from a combination of any of the following polypeptide chains:

[0510] (1) A first polypeptide chain, the first polypeptide chain comprising (preferably from N-terminus to C-terminus) the following in sequence: a Fab segment of a first target-binding polypeptide, a first single-chain Fc, and an IL-2Rα truncated form; a second polypeptide chain, the second polypeptide chain comprising (preferably from N-terminus to C-terminus) the following in sequence: a Fab segment of a first target-binding polypeptide, a second single-chain Fc, and an IL-2 wild-type or mutant protein, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker;

[0511] (2) A first polypeptide chain, which sequentially comprises (preferably from N-terminus to C-terminus): a first target-binding polypeptide with an scFv structure, a first single-chain Fc, and an IL-2Rα truncated form; a second polypeptide chain, which sequentially comprises: a heavy chain variable region of the first target-binding polypeptide, a heavy chain Fd segment of the second target-binding polypeptide, a second single-chain Fc, and an IL-2 wild-type or mutant protein; a third polypeptide chain, which sequentially comprises (preferably from N-terminus to C-terminus): a light chain variable region of the first target-binding polypeptide and a light chain of the second target-binding polypeptide, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker;

[0512] (3) Two first polypeptide chains, comprising: a light chain of a first target-binding polypeptide; a second polypeptide chain, comprising (preferably from N-terminus to C-terminus): a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide of scFv structure, a first single-chain Fc, and an IL-2Rα truncated form; a third polypeptide chain, comprising (preferably from N-terminus to C-terminus): a heavy chain Fd segment of the first target-binding polypeptide, a second single-chain Fc, and an IL-2 wild-type or mutant protein, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker;

[0513] (4) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; a second polypeptide chain, the second polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a first single-chain Fc, and an IL-2Rα truncated form; and a third polypeptide chain, the third polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a second single-chain Fc, and an IL-2 wild-type or mutant protein, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker.

[0514] In the polypeptide chains described in (1)-(4) above, VH and VL in the target-binding polypeptide with the scFv structure can be connected by a flexible linker; and the first target-binding polypeptide and the second target-binding polypeptide can also be connected by a flexible linker. The flexible linkers between VH and VL and between the first and second target-binding polypeptides may be the same or different. The flexible linker is selected from the antibody flexible linker region or contains an amino acid sequence composed of glycine and serine; preferably, the antibody flexible linker region is an elbow region or a hinge region; and preferably, the flexible linker has 5-15 amino acids.

[0515] In this application, the elbow region, located within the Fab segment, is the bend between the heavy chain variable region (VH) and the heavy chain constant region 1 (CH1), and between the light chain variable region (VL) and the light chain constant region (CL). Preferably, the flexible connecting segment can be selected from amino acid sequences of different lengths in the heavy chain elbow region and the light chain elbow region, such as ASTKGP (HC-γS), ASTKGPSVFPLAP (HC-γL), TVAAP (LC-κS), TVAAPSVFIFPP (LC-κL), QPKAAP (LC-λS), or QPKAAPSVTLFPP (LC-λL).

[0516] In this application, the hinge region, located between the Fab and Fc segments, is the amino acid sequence between the heavy chain constant region 1 (CH1) and constant region 2 (CH2). Preferably, the flexible linker can be selected from amino acid sequences of different lengths in the hinge region, such as PNLLGGP (HC-hS), PAPNLLGGP (HC-hL), PAPNLLGGP (LC-hS), or PTISPAPNLLGGP (LC-hL).

[0517] In some embodiments, the single-chain Fc, the first single-chain Fc, and / or the second single-chain Fc contain amino acid mutations; preferably, the amino acid mutations are Knob, Hole, or LALA mutations.

[0518] Ninthly, this application provides an activatable IL-2 fusion protein, characterized in that it comprises a target-binding polypeptide capable of specifically binding to a target, a truncated form of interleukin-2 receptor α (IL-2Rα), and an IL-2 wild-type polypeptide or mutant.

[0519] In some embodiments, the IL-2Rα truncated form is a molecule with the C-terminus of the IL-2Rα extracellular domain truncated.

[0520] In some embodiments, the IL-2Rα truncated derivative comprises the C-terminal amino acids 168-219aa, 169-219aa, 170-219aa, 171-219aa, 172-219aa, 173-219aa, 174-219aa, 175-219aa, 176-219aa, 177-219aa, and 178-219aa from the IL-2Rα extracellular domain. Amino acid sequences missing from 179-219aa, 180-219aa, 181-219aa, 182-219aa, 183-219aa, 184-219aa, 185-219aa, 186-219aa, 187-219aa, 188-219aa, 189-219aa, 190-219aa, 191-219aa, or 192-219aa.

[0521] In some embodiments, the IL-2Rα truncated portion is selected from: SEQ ID NO:4-28.

[0522] In some embodiments, the IL-2Rα truncated portion is selected from: SEQ ID NO:23.

[0523] In some implementations, the IL-2Rα truncated variant contains one or more mutations in addition to truncation.

[0524] In some implementations, the IL-2 is wild-type or mutant.

[0525] In some implementations, the IL-2 mutation is: R81E, L85T, or L85I.

[0526] In some embodiments, the sequence of the IL-2 is selected from SEQ ID NO:1-2.

[0527] In some embodiments, the IL-2Rα truncated section and the IL-2 are connected by a cuttable connector.

[0528] In some embodiments, the half-life extender of the fusion protein is linked to the IL-2Rα truncated via an uncuttable linker.

[0529] In some embodiments, the half-life extender of the fusion protein is linked to IL-2 via an incisional linker.

[0530] In a tenth aspect, this application provides an activatable IL-2 fusion protein, characterized in that it comprises a half-life extender, an IL-2Rα truncated form, and an IL-2 wild-type polypeptide or mutant.

[0531] In some embodiments, the half-life extender is selected from antibody fragments or human serum albumin. In some embodiments, the half-life extender is the Fc region of an antibody. In some embodiments, the half-life extender comprises: SEQ ID NO:36.

[0532] In some embodiments, the IL-2Rα truncated form is a molecule with the C-terminus of the IL-2Rα extracellular domain truncated.

[0533] In some embodiments, the IL-2Rα truncated derivative comprises the C-terminal amino acids 168-219aa, 169-219aa, 170-219aa, 171-219aa, 172-219aa, 173-219aa, 174-219aa, 175-219aa, 176-219aa, 177-219aa, and 178-219aa from the IL-2Rα extracellular domain. Amino acid sequences missing from 179-219aa, 180-219aa, 181-219aa, 182-219aa, 183-219aa, 184-219aa, 185-219aa, 186-219aa, 187-219aa, 188-219aa, 189-219aa, 190-219aa, 191-219aa, or 192-219aa.

[0534] In some embodiments, the IL-2Rα truncated portion is selected from: SEQ ID NO:4-28.

[0535] In some embodiments, the IL-2Rα truncated portion is selected from: SEQ ID NO:23.

[0536] In some implementations, the IL-2Rα truncated variant contains one or more mutations in addition to truncation.

[0537] In some implementations, the IL-2 is wild-type or mutant.

[0538] In some implementations, the IL-2 mutation is: R81E, L85T, or L85I.

[0539] In some embodiments, the sequence of the IL-2 is selected from SEQ ID NO:1-2.

[0540] In some embodiments, the IL-2Rα truncated section and the IL-2 are connected by a cuttable connector.

[0541] In some embodiments, the half-life extender of the fusion protein is linked to the IL-2Rα truncated via an uncuttable linker.

[0542] In some embodiments, the half-life extender of the fusion protein is linked to IL-2 via an incisional linker.

[0543] ●The target-binding polypeptide, the first target-binding polypeptide, and / or the second target-binding polypeptide described in any one of the sixth to ninth aspects of this application.

[0544] In some embodiments, the target-binding peptide described in the sixth aspect of this application, the first target-binding peptide and / or the second target-binding peptide described in the seventh aspect of this application, the first target-binding peptide and / or the second target-binding peptide described in the eighth aspect of this application, and / or the target-binding peptide described in the ninth aspect of this application, can specifically bind to the target, thereby enhancing the targeting of the fusion protein.

[0545] In some embodiments, the target-binding polypeptide, the first target-binding polypeptide, and / or the second target-binding polypeptide are antibodies or functional fragments thereof; preferably, the antibody or functional fragment thereof is selected from Fab, scFv, or nanobody.

[0546] In some embodiments, the target-binding polypeptide, the first target-binding polypeptide, and / or the second target-binding polypeptide are selected from T-cell target-binding polypeptides and tumor cell target-binding polypeptides. In some embodiments, the target of the T-cell target-binding polypeptide is selected from CD3 and / or PD-1, and the target of the tumor cell target-binding polypeptide is selected from one or more of CLDN18.2, PD-L1, VEGFR, and HER2.

[0547] In some embodiments, the target-binding peptide, the first target-binding peptide, and / or the second target-binding peptide can bind to one or more targets; in some embodiments, the target-binding peptide, the first target-binding peptide, and / or the second target-binding peptide can bind to one of the following targets: CLDN18.2, PD-1, PD-L1, VEGFR, HER2. In some embodiments, the target-binding peptide, the first target-binding peptide, and / or the second target-binding peptide can bind to one of the following target combinations: PD-1 / VEGF combination, or CLDN18.2 / CD3 combination.

[0548] ●IL-2 as described in any one of the sixth to tenth aspects of this application

[0549] In some embodiments, the IL-2 wild-type protein described in any one of the sixth to tenth aspects of this application comprises the amino acid sequence shown in SEQ ID NO:1, or comprises an amino acid sequence having at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%) identity with the amino acid sequence shown in SEQ ID NO:1. In some embodiments, the amino acid sequence of the IL-2 wild-type protein is as shown in SEQ ID NO:1, or has at least 95% (e.g., at least 95%, 96%, 97%, 98%, 99%) identity with the amino acid sequence shown in SEQ ID NO:1.

[0550] In some embodiments, the IL-2 mutant protein contains one or more amino acid mutations compared to the wild-type IL-2 protein; preferably, the IL-2 mutant protein contains one or more of the following mutations: R81E, N88D, L85T, or L85I, for example, containing one, two, or all three site mutations (R81E, N88D, and L85T or L85I). In some embodiments, the IL-2 mutant protein contains N88D. In some embodiments, the IL-2 mutant protein contains the amino acid sequence shown in SEQ ID NO:2. In some embodiments, the amino acid sequence of the IL-2 mutant protein is shown in SEQ ID NO:2. In some embodiments, the IL-2 mutant protein contains the amino acid sequence shown in SEQ ID NO:65. In some embodiments, the amino acid sequence of the IL-2 mutant protein is shown in SEQ ID NO:65.

[0551] ●The cuttable joint described in any one of aspects six through ten of this application

[0552] In some embodiments, the cleavable connector described in any one of the sixth to tenth aspects of this application is capable of being cleaved by tumor-associated proteases.

[0553] In some embodiments, the cleavable adapter is cleaved by a matrix metalloproteinase. In some embodiments, the matrix metalloproteinase includes MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, and MMP14.

[0554] In some embodiments, the cleavable connector is cleaved by caspase. In some embodiments, the caspase includes: caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, and caspase 12.

[0555] In some embodiments, the cleavable adapter comprises an amino acid sequence as shown in any one of SEQ ID NO:32-35. In some embodiments, the cleavable adapter is selected from: SEQ ID NO:32-35.

[0556] In some embodiments, the cleavable linker comprises an amino acid sequence: XXXD, where X represents any single amino acid. In some embodiments, the amino acid sequence comprised by the cleavable linker is selected from: DEVD, DEVDG, DQTDG, VEID, VEVD, VEIDN, IEDD, LETD, LQTDG, LEHD, LEHDS, YVAD, WEHD, or YVHDA.

[0557] In some embodiments, the cleavable linker comprises glycine / serine. In some embodiments, when the IL2 and the IL2Rα truncated form are sequentially linked, the cleavable linker for linking the IL2 and the half-life extender or the target-binding peptide comprises four G4S molecules. In some embodiments, when the IL2 is linked to the first / second target-binding peptide or the first / second single-chain Fc, the cleavable linker for linking the IL2 and the second / first target-binding peptide or the second / first single-chain Fc comprises two G4S molecules.

[0558] ●The indivisible joint described in any one of aspects six through ten of this application

[0559] In some embodiments, the uncuttable connector described in any one of the sixth to tenth aspects of this application is not cleaved by tumor-associated proteases.

[0560] In some embodiments, the target-binding polypeptide of the fusion protein is linked to an IL-2Rα truncated polypeptide via an uncleavable linker. In some embodiments, the IL-2Rα truncated polypeptide is linked to the N-terminus of the heavy chain of the Fab-type antibody. In some embodiments, the IL-2Rα truncated polypeptide is linked to the C-terminus of the heavy chain of the Fab-type antibody. In some embodiments, the IL-2Rα truncated polypeptide is linked to the N-terminus of the light chain of the Fab-type antibody. In some embodiments, the IL-2Rα truncated polypeptide is linked to the C-terminus of the light chain of the Fab-type antibody. In some embodiments, the IL-2Rα truncated polypeptide is linked to the N-terminus of the heavy chain of the scFv-type antibody. In some embodiments, the IL-2Rα truncated polypeptide is linked to the C-terminus of the light chain of the scFv-type antibody. In some embodiments, the IL-2Rα truncated polypeptide is linked to the N-terminus of the heavy chain of the nanobody-type antibody. In some embodiments, the IL-2Rα truncated polypeptide is linked to the C-terminus of the light chain of the nanobody-type antibody.

[0561] In some embodiments, the target-binding peptide of the fusion protein is linked to IL-2 via a non-cleavable linker. In some embodiments, IL-2 is linked to the N-terminus of the heavy chain of the Fab-type antibody. In some embodiments, IL-2 is linked to the C-terminus of the heavy chain of the Fab-type antibody. In some embodiments, IL-2 is linked to the N-terminus of the light chain of the Fab-type antibody. In some embodiments, IL-2 is linked to the C-terminus of the light chain of the Fab-type antibody. In some embodiments, IL-2 is linked to the N-terminus of the heavy chain of the scFv-type antibody. In some embodiments, IL-2 is linked to the C-terminus of the light chain of the scFv-type antibody. In some embodiments, IL-2 is linked to the N-terminus of the heavy chain of the nanobody-type antibody. In some embodiments, IL-2 is linked to the C-terminus of the light chain of the nanobody-type antibody.

[0562] In some embodiments, the half-life extender of the fusion protein is linked to the IL-2Rα truncated via an uncuttable linker. In some embodiments, the half-life extender of the fusion protein is linked to IL-2 via an uncuttable linker.

[0563] In some embodiments, the uncuttable connector is a glycine / serine connector.

[0564] In some embodiments, when the IL2 and the IL2Rα truncated form are sequentially linked, the uncleavable linker connecting the IL2Rα truncated form and the half-life extender or the target-binding peptide is a 4-G4S linker. In some embodiments, when the IL2 is linked to the first / second target-binding peptide or the first / second single-chain Fc, the uncleavable linker connecting the IL2Rα truncated form and the second / first target-binding peptide or the second / first single-chain Fc is a 3-G4S linker.

[0565] In some embodiments, the uncuttable connector is (G4S). n n is an integer, either 3 or 4.

[0566] In some embodiments, the indivisible connector comprises: SEQ ID NO:39. In some embodiments, the indivisible connector is SEQ ID NO:39.

[0567] ●The fusion protein described in any one of aspects six through ten of this application

[0568] In some embodiments, the fusion protein comprises a first chain and a second chain, the first chain and the second chain comprising the following amino acid sequences or sequences having at least 95% identity with the following amino acid sequences, wherein:

[0569] The amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:40; or

[0570] The amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:41; or

[0571] The amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:42; or

[0572] The amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:43; or

[0573] The amino acid sequence of the first chain is shown in SEQ ID NO:49, and the amino acid sequence of the second chain is shown in SEQ ID NO:50; or

[0574] The amino acid sequence of the first chain is shown in SEQ ID NO:53, and the amino acid sequence of the second chain is shown in SEQ ID NO:54; or

[0575] The amino acid sequence of the first chain is shown in SEQ ID NO:57, and the amino acid sequence of the second chain is shown in SEQ ID NO:58; or

[0576] The amino acid sequence of the first chain is shown in SEQ ID NO:59, and the amino acid sequence of the second chain is shown in SEQ ID NO:60; or

[0577] The amino acid sequence of the first chain is shown in SEQ ID NO:61, and the amino acid sequence of the second chain is shown in SEQ ID NO:62; or

[0578] The amino acid sequence of the first chain is shown in SEQ ID NO:63, and the amino acid sequence of the second chain is shown in SEQ ID NO:64; or

[0579] The amino acid sequence of the first chain is shown in SEQ ID NO:93, and the amino acid sequence of the second chain is shown in SEQ ID NO:94; or

[0580] The amino acid sequence of the first chain is shown in SEQ ID NO:95, and the amino acid sequence of the second chain is shown in SEQ ID NO:96; or

[0581] The amino acid sequence of the first chain is shown in SEQ ID NO:97, and the amino acid sequence of the second chain is shown in SEQ ID NO:98; or

[0582] The amino acid sequence of the first chain is shown in SEQ ID NO:99, and the amino acid sequence of the second chain is shown in SEQ ID NO:100; or

[0583] The amino acid sequence of the first chain is shown in SEQ ID NO:101, and the amino acid sequence of the second chain is shown in SEQ ID NO:102; or

[0584] The amino acid sequence of the first chain is shown in SEQ ID NO:103, and the amino acid sequence of the second chain is shown in SEQ ID NO:104; or

[0585] The amino acid sequence of the first chain is shown in SEQ ID NO:105, and the amino acid sequence of the second chain is shown in SEQ ID NO:106; or

[0586] The amino acid sequence of the first chain is shown in SEQ ID NO:107, and the amino acid sequence of the second chain is shown in SEQ ID NO:108; or

[0587] The amino acid sequence of the first chain is shown in SEQ ID NO:109, and the amino acid sequence of the second chain is shown in SEQ ID NO:110.

[0588] In some embodiments, the fusion protein comprises a first chain, a second chain, and a third chain, wherein the first chain, the second chain, and the third chain comprise the following amino acid sequences or sequences having at least 95% identity with the following amino acid sequences, wherein:

[0589] The amino acid sequence of the first chain is shown in SEQ ID NO:66, the amino acid sequence of the second chain is shown in SEQ ID NO:67, and the amino acid sequence of the third chain is shown in SEQ ID NO:68; or

[0590] The amino acid sequence of the first chain is shown in SEQ ID NO:69, the amino acid sequence of the second chain is shown in SEQ ID NO:70, and the amino acid sequence of the third chain is shown in SEQ ID NO:71; or

[0591] The amino acid sequence of the first chain is shown in SEQ ID NO:72, the amino acid sequence of the second chain is shown in SEQ ID NO:73, and the amino acid sequence of the third chain is shown in SEQ ID NO:74; or

[0592] The amino acid sequence of the first chain is shown in SEQ ID NO:75, the amino acid sequence of the second chain is shown in SEQ ID NO:76, and the amino acid sequence of the third chain is shown in SEQ ID NO:77; or

[0593] The amino acid sequence of the first chain is shown in SEQ ID NO:78, the amino acid sequence of the second chain is shown in SEQ ID NO:79, and the amino acid sequence of the third chain is shown in SEQ ID NO:80; or

[0594] The amino acid sequence of the first chain is shown in SEQ ID NO:81, the amino acid sequence of the second chain is shown in SEQ ID NO:82, and the amino acid sequence of the third chain is shown in SEQ ID NO:83; or

[0595] The amino acid sequence of the first chain is shown in SEQ ID NO:84, the amino acid sequence of the second chain is shown in SEQ ID NO:85, and the amino acid sequence of the third chain is shown in SEQ ID NO:86; or

[0596] The amino acid sequence of the first chain is shown in SEQ ID NO:87, the amino acid sequence of the second chain is shown in SEQ ID NO:88, and the amino acid sequence of the third chain is shown in SEQ ID NO:89.

[0597] In some embodiments, the fusion protein described in any one of aspects six through eight of this application is a dimer. In some embodiments, the fusion protein is a homodimer. In some embodiments, the fusion protein is a heterodimer. In some embodiments, the heterodimer comprises a first monomer and a second monomer. In some embodiments, the first monomer comprises one or two polypeptide chains; and / or the second monomer comprises one or two polypeptide chains.

[0598] In some embodiments, the heterodimer has the following structure: the first monomer comprises a target-binding peptide and a half-life extender, and the second monomer comprises a target-binding peptide, a half-life extender, IL-2, and a truncated form of IL-2Rα.

[0599] Furthermore, the first monomer comprises a first chain and a second chain: the first chain comprises a target-binding polypeptide light chain, and the second chain comprises a target-binding polypeptide heavy chain and a half-life extender; the second monomer comprises a third chain and a fourth chain: the third chain comprises a target-binding polypeptide heavy chain, a half-life extender, IL-2 and IL-2Rα truncated forms, and the fourth chain comprises a target-binding polypeptide light chain.

[0600] Furthermore, the first monomer comprises a first chain and a second chain: the first chain comprises a target-binding polypeptide light chain, and the second chain comprises a target-binding polypeptide heavy chain and a half-life extender; the second monomer comprises a third chain and a fourth chain: the third chain comprises, in sequence, a target-binding polypeptide heavy chain, a half-life extender, IL-2 and IL-2Rα truncated forms, and the fourth chain comprises a target-binding polypeptide light chain.

[0601] Furthermore, the first monomer comprises a first chain and a second chain: the first chain comprises a target-binding polypeptide light chain, and the second chain comprises a target-binding polypeptide heavy chain and a half-life extender; the second monomer comprises a third chain and a fourth chain: the third chain comprises, in sequence, a target-binding polypeptide heavy chain, a half-life extender, an IL-2Rα truncated form and IL-2, and the fourth chain comprises a target-binding polypeptide light chain.

[0602] In some embodiments, the heterodimer has the following structure: the first monomer comprises a target-binding peptide and a half-life extender, and the second monomer comprises IL-2, an IL-2Rα truncated form, and a half-life extender.

[0603] Furthermore, the first monomer comprises a first chain and a second chain: the first chain comprises a target-binding polypeptide light chain, the second chain comprises a target-binding polypeptide heavy chain and a half-life extender; the second monomer comprises a third chain: the third chain comprises IL-2, an IL-2Rα truncated form and a half-life extender.

[0604] Furthermore, the first monomer comprises a first chain and a second chain: the first chain comprises a target-binding polypeptide light chain, the second chain comprises a target-binding polypeptide heavy chain and a half-life extender; the second monomer comprises a third chain: the third chain comprises IL-2, an IL-2Rα truncated form and a half-life extender in sequence.

[0605] Furthermore, the first monomer comprises a first chain and a second chain: the first chain comprises a target-binding polypeptide light chain, and the second chain comprises a target-binding polypeptide heavy chain and a half-life extender; the second monomer comprises a third chain: the third chain comprises, in sequence, an IL-2Rα truncated form, IL-2, and a half-life extender.

[0606] In one aspect, this application provides a polynucleotide comprising the fusion protein described in any one of aspects six through ten of this application.

[0607] In a twelfth aspect, this application provides an expression vector comprising the polynucleotide described in the eleventh aspect of this application.

[0608] In some embodiments, the expression vector is a plasmid, granule, bacteriophage, or viral vector, preferably a plasmid. For example, the polynucleotide encoding the fusion protein can be cloned into a suitable expression vector, which can be any suitable recombinant expression vector and can be used for transformation or transfection of any suitable host. Suitable vectors include those designed for propagation and amplification, or for expression, or both, such as plasmids and viruses.

[0609] In a thirteenth aspect, this application provides a host cell comprising the polynucleotide described in the eleventh aspect of this application or the expression vector described in the twelfth aspect of this application.

[0610] In some implementations, the expression vector is transformed into a host cell for further expression or cloning within the host cell.

[0611] The host cell refers to a cell in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include transformants and transformed cells, which include primary transformed cells and their derived progeny, regardless of passage number. Progeny cells may not be entirely identical to parent cells in terms of nucleic acid content and may contain mutations.

[0612] In some embodiments, the host cell is a eukaryotic cell, a prokaryotic cell, or a bacterial cell.

[0613] In a fourteenth aspect, this application provides a method for preparing a fusion protein, comprising culturing the host cells described in aspect thirteen of this application to prepare the fusion protein described in any one of aspects six to ten of this application.

[0614] In some embodiments, the method further includes purifying or separating the produced fusion protein. The purification or separation methods are conventional in the art.

[0615] In a fifteenth aspect, this application provides a pharmaceutical composition comprising the fusion protein described in any one of aspects six through ten of this application.

[0616] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, carrier, or diluent.

[0617] In a sixteenth aspect, this application provides a method for treating or alleviating a disease or symptom, comprising: administering to a patient in need a therapeutically effective amount of the fusion protein described in any one of aspects six through ten of this application, the polynucleotide described in aspect eleven of this application, the expression vector described in aspect twelf of this application, the host cell described in aspect thirteen of this application, or the pharmaceutical composition described in aspect fourteen of this application.

[0618] In some implementations, the patient in need is a human.

[0619] In a seventeenth aspect, this application provides the use of the fusion protein described in any one of aspects six through ten of this application, the polynucleotide described in aspect eleven of this application, the expression vector described in aspect twelf of this application, the host cell described in aspect thirteen of this application, or the pharmaceutical composition described in aspect fourteen of this application in treating or alleviating a disease or symptom.

[0620] Eighteenthly, this application also provides the use of the fusion protein described in any one of aspects six through ten of this application, the polynucleotide described in aspect eleven of this application, the expression vector described in aspect twelf of this application, the host cell described in aspect thirteen of this application, or the pharmaceutical composition described in aspect fourteen of this application in the preparation of a medicament for treating or alleviating a disease or symptom.

[0621] In some implementations, the aforementioned disease or condition is cancer. For example, the aforementioned disease or condition is selected from: acute myeloid leukemia, adrenocortical carcinoma, B-cell lymphoma, urothelial carcinoma of the bladder, ductal carcinoma of the breast, lobular carcinoma of the breast, esophageal cancer, castration-resistant prostate cancer (CRPC), cervical cancer, cholangiocarcinoma, chronic myeloid leukemia, colorectal adenocarcinoma, colorectal cancer (CRC), esophageal cancer, gastric adenocarcinoma, glioblastoma multiforme, squamous cell carcinoma of the head and neck, Hodgkin's lymphoma / primary mediastinal B-cell lymphoma, hepatocellular carcinoma (HCC), renal stenosis... Chromoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, low-grade glioma, lung adenocarcinoma, squamous cell lung carcinoma, melanoma (MEL), mesothelioma, non-squamous NSCLC, ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma, paraganglioma and pheochromocytoma, prostate cancer, renal cell carcinoma (RCC), sarcoma, cutaneous melanoma, head and neck squamous cell carcinoma, T-cell lymphoma, thymoma, papillary thyroid carcinoma, uterine carcinosarcoma, endometrioid carcinoma of the uterine body or uveal melanoma.

[0622] In a nineteenth aspect, this application also provides a monoclonal antibody anti-Claudin18.2.

[0623] In some embodiments, the monoclonal antibody anti-Claudin18.2 comprises a CDR sequence as shown in any one of SEQ ID NO:120-125. In some embodiments, the monoclonal antibody anti-Claudin18.2 comprises: LCDR1 as shown in SEQ ID NO:120, LCDR2 as shown in SEQ ID NO:121, LCDR3 as shown in SEQ ID NO:122, HCDR1 as shown in SEQ ID NO:123, HCDR2 as shown in SEQ ID NO:124, and HCDR3 as shown in SEQ ID NO:125.

[0624] In some embodiments, the heavy chain sequence of the monoclonal antibody is as shown in SEQ ID NO:119 or has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity with SEQ ID NO:119; the light chain amino acid sequence is as shown in SEQ ID NO:118 or has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity with SEQ ID NO:119.

[0625] In some embodiments, the monoclonal antibody anti-Claudin18.2 comprises a CDR sequence as shown in any one of SEQ ID NO:126-131. In some embodiments, the monoclonal antibody anti-Claudin18.2 comprises: LCDR1 as shown in SEQ ID NO:126, LCDR2 as shown in SEQ ID NO:127, LCDR3 as shown in SEQ ID NO:128, HCDR1 as shown in SEQ ID NO:129, HCDR2 as shown in SEQ ID NO:130, and HCDR3 as shown in SEQ ID NO:131.

[0626] In some embodiments, the heavy chain sequence of the monoclonal antibody is as shown in SEQ ID NO:133 or has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity with SEQ ID NO:133; the light chain amino acid sequence is as shown in SEQ ID NO:132 or has at least 85% (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%) identity with SEQ ID NO:132.

[0627] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the fusion protein, preparation method and use of this application, and are not intended to limit the scope of the invention.

[0628] Example

[0629] The following description, in conjunction with specific embodiments, illustrates the content of this application, but the scope of this application is not limited thereto. Unless otherwise specified, the reagents and instruments used in the following embodiments are all conventional reagents and instruments in the art and can be obtained commercially. The methods used are all conventional experimental methods, and those skilled in the art can undoubtedly implement the described schemes and obtain corresponding results based on the embodiments.

[0630] Example 1: Protein preparation of the fusion protein

[0631] 1.1 Preparation of fusion protein

[0632] In one embodiment, this application includes a method for reducing the toxicity of a fusion protein, comprising administering an effective amount of an interleukin-2 (IL-2) fusion protein. The IL-2 fusion protein comprises: a target-binding polypeptide; an IL-2 wild-type or mutant protein; a cleavable linker to IL-2; and a truncated form of interleukin-2 receptor α (IL-2Rα), wherein the IL-2Rα truncated form comprises an amino acid sequence selected from: SEQ ID NO:3, with a C-terminus truncated to a length of 29 to 52 amino acids.

[0633] In one embodiment, this application includes a method for reducing the toxicity of a fusion protein, comprising administering an effective amount of an interleukin-2 (IL-2) fusion protein. The IL-2 fusion protein comprises: a target-binding polypeptide; an IL-2 wild-type or mutant protein; a cleavable linker to IL-2; and a truncated form of interleukin-2 receptor α (IL-2Rα), wherein the IL-2Rα truncated form comprises an amino acid sequence selected from: SEQ ID NO:3, with a C-terminus truncated to a length of 29 to 53 amino acids.

[0634] In one embodiment, this application includes a method for reducing the toxicity of a fusion protein, comprising administering an effective amount of an interleukin-2 (IL-2) fusion protein. The IL-2 fusion protein comprises: a target-binding polypeptide; an IL-2 wild-type or mutant protein; and a truncated form of interleukin-2 receptor α (IL-2Rα), wherein the IL-2Rα truncated form comprises an amino acid sequence selected from: SEQ ID NO:3, with a C-terminus truncated to a length of 29 to 53 amino acids.

[0635] Preferably, the target-binding polypeptide is selected from T cell target-binding polypeptides and tumor cell target-binding polypeptides.

[0636] Preferably, the target-binding peptide, the first target-binding peptide, and / or the second target-binding peptide can bind to the following targets: PD-1 / VEGF combination, or CLDN18.2 / CD3 combination.

[0637] Preferably, the target-binding peptide is selected from the following targets: CLDN18.2, PD-1, PD-L1, VEGFR, HER2.

[0638] Preferably, the target of the T cell target-binding peptide is selected from one or more of CD3 and programmed death receptor 1 (PD-1, an immune checkpoint molecule on the surface of T cells), and the target of the tumor cell target-binding peptide is selected from one or more of CLDN18.2, PD-L1, VEGFR, and HER2.

[0639] In one embodiment, the humanized monoclonal antibody anti-Claudin18.2 against CLDN18.2 is sequentially fused with an IL-2Rα truncated variant, a cleavable linker, and IL-2 to obtain the fusion protein CLDN18.2-IL2Rα-IL2; the humanized monoclonal antibody anti-Claudin18.2 against CLDN18.2 is sequentially fused with IL-2, a cleavable linker, and an IL-2Rα truncated variant to obtain the fusion protein CLDN18.2-IL2-IL2Rα. In some embodiments, the heavy chain amino acid sequence of the humanized monoclonal antibody against CLDN18.2, anti-Claudin18.2 (BH, Before humanization), is shown in SEQ ID NO:119, the light chain amino acid sequence is shown in SEQ ID NO:118, and the CDR sequence is shown in any one of SEQ ID NO:120-125; or, the heavy chain amino acid sequence of the humanized monoclonal antibody against CLDN18.2, anti-Claudin18.2 (AH, After humanization), is shown in SEQ ID NO:133, the light chain amino acid sequence is shown in SEQ ID NO:132, and the CDR sequence is shown in any one of SEQ ID NO:126-131. This general construction logic has broad applicability. Antibodies targeting single targets such as PD-1, PD-L1, VEGFR, HER2, or bispecific combinations such as PD-1 / VEGF and CLDN18.2 / CD3 can all be prepared using the above method to prepare the corresponding fusion proteins.

[0640] The structure of the IL-2 fusion protein of this application is shown in Figures 17A-17M.

[0641] Based on the above embodiments, fusion proteins as shown in Table 1 were obtained by using different compositions, linkage sequences and antibody forms. The amino acid sequences of each fusion protein are detailed in the sequence listing below.

[0642] Table 1. Molecular structure of the fusion protein

[0643] In Table 1:

[0644] (1) In the embodiments of the present invention, “IL-2Rα(ΔCnaa)” refers to a truncated IL-2Rα fragment of a specific length. Compared with the 212 amino acid extracellular domain fragment of IL-2Rα, its fragment length is shortened by n amino acids at the C-terminus, that is, the 212aa-naa truncated IL-2Rα fragment. For example, the "IL-2Rα(ΔC40aa) or IL-2Rα(ΔC46aa)" labeling of fusion proteins 3-19, 3-23, 3-31, 5-2, 5-4, 5-7, and 5-9 refers to truncated IL-2Rα fragments with a length of 172aa or 166aa. Compared to the 212-amino acid IL-2Rα extracellular domain fragment (such as SEQ ID NO:111, SEQ ID NO:112, or IL-2Rα in fusion protein 3), these fragments are truncated by 40 or 46 amino acids at the C-terminus, resulting in 212aa-40aa or 212aa-46aa IL-2Rα truncated fragments. Compared to the 219-amino acid IL-2Rα extracellular domain fragment (such as SEQ ID NO:3), this truncated length is 47 or 53 amino acids shorter at the C-terminus.

[0645] (2) Uncleavable Liker is denoted as UL, and Cleavable Linker is denoted as CL.

[0646] 1.2 Determination of purity of fusion protein

[0647] To examine whether IL-2Rα truncation alters the aggregation of the fusion protein and affects its molecular stability, the specific experimental method is as follows:

[0648] (1) Experimental methods and reagents

[0649] The procedure and reagents used for Protein A affinity chromatography are shown in Table 2.

[0650] Table 2 Affinity Chromatography Operation Procedure

[0651] (2) Purity determination of fusion protein

[0652] The fusion protein obtained by the above purification method was analyzed by SEC-HPLC, and the purity is as follows:

[0653] Table 3. Determination of purity of fusion protein by SEC-HPLC

[0654] Example 2: In vitro protein / cell binding ability of truncated fusion protein

[0655] This embodiment aims to examine whether the binding affinity of a fusion protein to the antibody target changes when the IL-2 activity is masked by an IL-2Rα truncated variant, particularly when this activity-masking combination proIL2 (IL-2-IL2Rα truncated variant) is fused with different antibodies (such as PD-1 antibody, PD-L1 antibody, VEGFR antibody, HER2 antibody, CLDN18.2 antibody, VEGF-PD1 antibody, or PD1-VEGF antibody) or incorporated into the fusion protein in different linkages (homogeneous or heterodimer). The in vitro binding affinity of the fusion protein was assessed using PD-1 protein (SinoBiological, 10377-H08H), VEGF protein (SinoBiological, 11066-HNAH), CLDN18.2 protein (Acro, CL2-H52P7), PD-L1 protein (SinoBiological, 10084-H08H), and VEGFR protein (SinoBiological, 10012-H08H).

[0656] 2.1 In vitro binding activity assay of fusion proteins linked via homodimers

[0657] (1) Fusion protein 4

[0658] To examine whether the binding ability of the antibody to the target site (CLDN18.2) changes after the fusion protein is fused with IL-2 and a truncated IL-2Rα variant (which is truncated by 40 amino acids at the C-terminus compared to the 212-amino acid IL-2Rα extracellular domain fragment; in fact, it is truncated by 47 amino acids at the C-terminus compared to SEQ ID NO:3), the in vitro binding ability of the fusion protein was detected using the MC38-CLDN18.2 cell line.

[0659] Number of stained cells 2×10 5 Cells / well; initial protein concentration was 40 μg / mL, diluted 1:5, and then diluted sequentially in 8 gradients; after incubating the protein and cells at 4°C for 30 min, wash once with 200 μL FACS buffer; calculate the amount of secondary antibody PE anti-human IgG Fc (BIolegend, 366904) to be used at 0.3 μL / well, add 9 μL of secondary antibody to 3 mL FACS buffer, mix well, and then add 100 μL / well of the diluted secondary antibody to the corresponding well. After incubating at 4°C in the dark for 30 min, wash twice with 200 μL FACS buffer; resuspend the cells in 200 μL FACS buffer and perform fluorescence detection on a Sino Cyte flow cytometer; plot the protein binding curve using GraphPad software.

[0660] Fusion protein 3 contains the full-length IL-2Rα extracellular domain (212 amino acids in length) and is named fusion protein 3 or fusion protein 3 (full-length); fusion protein 4 contains a truncated IL-2Rα variant (40 amino acids shorter at the C-terminus than the 212 amino acid IL-2Rα extracellular domain) and is named fusion protein 4 or fusion protein 4 (△C40aa); the structures of fusion proteins 3 and 4 are shown in Figure 17B.

[0661] As shown in Figure 1A, in fusion protein 4 containing the truncated IL-2Rα, the binding ability of the antibody to the target was essentially unaffected by the fused proIL2, and its binding ability with the standalone antibody (aCLDN) and fusion protein 3 was essentially the same. This indicates that the fusion protein of the present invention, which uses the truncated IL-2Rα (rather than the full-length extracellular domain of IL-2Rα) to mask the activity of IL-2, does not affect the protein-binding ability of the antibody in the fusion protein.

[0662] (2) Fusion proteins 24-3, 22-6, 33-3, 33-4, and 35-2

[0663] Furthermore, the binding affinity of the fusion protein to various targets (PD-L1, VEGFR, HER2) was examined after the fusion protein was fused with the IL-2Rα truncated variant of IL-2 and 172aa.

[0664] The in vitro binding capacity of fusion proteins 24-3, 22-6, 33-3, and 33-4 was detected using protein assays. First, the proteins were coated overnight with 5 μg / mL of the target protein (PD-L1 protein (SinoBiological, 10084-H08H), PD-1 protein (SinoBiological, 10377-H08H), or VEGFR protein (SinoBiological, 11066-HNAH). After washing and blocking, the initial protein concentration was 60 nmol / L, diluted 1:5, and then serially diluted eight times. The diluted fusion proteins were then added to ELISA plates and incubated at 37°C for 60 min. After washing, HRP-labeled anti-human IgG Fc (Sigma-Aldrich, A0170) was added, and the plates were incubated at 37°C for 30 min. The OD value was then read after color development.

[0665] The in vitro binding ability of fusion protein 35-2 was detected using the SK-BR-3 cell line (Pronosei Biotechnology, CL-0211). The experimental method was the same as described in "(1) Fusion Protein 4".

[0666] Fusion protein 24-3 contains a truncated IL-2Rα (ΔC40aa, compared to a 212-amino acid extracellular domain of IL-2Rα) and a PD-L1 antibody; fusion protein 24-2 contains a truncated IL-2Rα (ΔC30aa, compared to a 212-amino acid extracellular domain of IL-2Rα) and a PD-L1 antibody, differing from fusion protein 24-3 only in the length of the truncated domain; fusion protein 22-6 contains a 172aa truncated IL-2Rα, namely IL-2Rα (ΔC40aa, compared to a 212-amino acid extracellular domain of IL-2Rα) and a PD-L1 antibody. Fusion proteins 24-3, 33-4, and 35-2 contain a 12-amino acid IL-2Rα extracellular domain and a PD-1 antibody; fusion proteins 33-3 and 33-4 contain a truncated IL-2Rα domain (ΔC40aa, compared to a 212-amino acid IL-2Rα extracellular domain) and a VEGFR antibody, the difference between fusion proteins 33-3 and 33-4 being the order in which the truncated IL-2Rα domain is linked to IL-2; fusion protein 35-2 contains a truncated IL-2Rα domain (ΔC40aa, compared to a 212-amino acid IL-2Rα extracellular domain) and a HER2 antibody. The structures of fusion proteins 24-3, 33-3, and 35-2 are shown in Figure 17B.

[0667] The results are shown in Figures 1B-1E. In fusion proteins 24-3 containing the truncated IL-2Rα, the binding ability of the antibody to PD-L1 protein was largely unaffected by the fused proIL2, and its binding ability to the single antibody (Atezolizumab, Yiqiao Shenzhou, 68049-H001) was essentially the same (Figure 1B). In fusion proteins 22-6 containing the truncated IL-2Rα, the binding ability of the antibody to PD-1 protein was largely unaffected by the fused proIL2, and its binding ability to the single antibody (Nivolumab (PD-1 antibody, Yiqiao Shenzhou)) was essentially the same (Figure 1B). The binding ability of the antibody to VEGFR protein was basically the same as that of the fusion protein 68050-H001 (Figure 1C); in the fusion proteins 33-3 and 33-4 containing the truncated IL-2Rα, the binding ability of the antibody to VEGFR protein was basically unaffected by the fusion proIL2, and its binding ability to the single antibody (Ramucirumab, Yiqiao Shenzhou, 68054-H001) was basically the same (Figure 1D); in the fusion protein 35-2 containing the truncated IL-2Rα, the binding ability of the antibody to HER2 protein was basically unaffected by the fusion proIL2 (Figure 1E).

[0668] The above results demonstrate that the fusion protein provided by this invention successfully achieves functional uncoupling by introducing an IL-2Rα truncated variant to mask IL-2 activity: that is, while reducing IL-2 toxicity, it unexpectedly maintains the excellent target-binding activity of the antibody portion. Furthermore, this strategy has good versatility; experimental data confirm that the design of this fusion protein and the IL-2Rα truncated variant can be successfully applied to fusion proteins targeting various different targets, such as anti-PD-L1, anti-VEGFR, and anti-HER2.

[0669] (3) Fusion proteins 5-2, 5-4, 5-7, 5-9, 5-11, 5-13, 5-15 and 5-17

[0670] Furthermore, the effect of the fusion protein on the binding activity of the fusion protein containing the bispecific antibody after fusing with the IL-2Rα truncated variant of IL-2 and 172aa was investigated. Protein assays were performed using the methods described in “(2) Fusion Proteins 24-3, 22-6, 33-3, 33-4, and 35-2” above.

[0671] Among them, fusion protein 5-1 is a VEGF-PD-1 antibody. The difference between fusion protein 5-12 and 5-11 is that fusion protein 5-12 does not undergo ΔC40aa truncation; fusion proteins 5-2, 5-4, 5-7, 5-9, 5-11, 5-13, 5-15 and 5-17 all contain the truncated IL-2Rα variant (ΔC40aa, compared to the 212 amino acid extracellular domain of IL-2Rα) and PD1-VEGF antibody, and the structural schematic diagrams of the VEGF-PD-1 antibody contained therein are shown in Figures 17C-17H and Figure J, respectively.

[0672] As shown in Figures 1F-1G, the binding affinity of fusion proteins 5-2, 5-4, 5-7, and 5-9 containing the IL-2Rα truncated variant (ΔC40aa) to PD-1 protein was essentially the same as that of Nivolumab (PD-1 antibody, Sinocare, 68050-H001) and fusion protein 5-1 (VEGF-PD-1 antibody) (Figure 1F). Furthermore, the binding affinity of fusion proteins 5-11, 5-13, 5-15, and 5-17 containing the IL-2Rα truncated variant (ΔC40aa) to PD-1 protein was also essentially the same as that of Nivolumab (Figure 1G).

[0673] Furthermore, as shown in Table 4 below, the fusion proteins 5-2, 5-4, 5-7, and 5-9, which contain a 172aa truncated form of IL-2Rα (IL-2Rα(ΔC40aa)), exhibit a binding affinity to VEGFA proteins that is essentially consistent with that of fusion protein 5-1 (VEGF antibody-PD-1).

[0674] Table 4

[0675] The above results indicate that using the IL-2Rα truncated form to mask IL-2 activity does not affect the protein binding ability of multi-target antibodies in the fusion protein, and is applicable to multiple targets.

[0676] 2.2 In vitro binding activity assay of fusion proteins linked via heterodimers

[0677] (1) Fusion proteins 22-23, 22-27, 22-38 and 22-39

[0678] The effects of different lengths of IL-2Rα truncated variants and the combined IL-2 mutation (N88D) on the in vitro binding activity of the fusion proteins were investigated. Protein assays were performed using the methods described in “(2) Fusion proteins 24-3, 22-6, 33-3, 33-4, and 35-2” above.

[0679] Among them, fusion proteins 22-23 and 22-27 contain a 172aa truncated IL-2Rα (IL-2Rα(△C40aa), compared to the 212 amino acid extracellular domain fragment of IL-2Rα) and a PD-1 antibody, and 22-27 additionally contains an IL-2 mutation (N88D). The structures of fusion proteins 22-23 and 22-27 are shown in Figure 17A.

[0680] Fusion proteins 22-38 and 22-39 contain a 166aa truncated IL-2Rα (IL-2Rα(△C46aa) compared to the 212 amino acid extracellular domain fragment of IL-2Rα) and a PD-L1 antibody. Fusion protein 22-39 also contains an IL-2 mutation (N88D). The structures of fusion proteins 22-38 and 22-39 are shown in Figure 17A.

[0681] The results are shown in Figures 2A and 2B. The binding abilities of fusion proteins 22-23 and 22-27 to PD-1 protein were essentially consistent with those of nivolumab (Figure 2A); the binding abilities of fusion proteins 22-38 and 22-39 to PD-L1 protein were essentially consistent with those of atezolizumab (Figure 2B). These results indicate that the antibody partial binding activity of fusion proteins with different lengths of IL-2Rα truncated variants (40-46 amino acids shorter at the C-terminus compared to the 212-amino acid IL-2Rα extracellular domain fragment) is essentially consistent with that of the corresponding monoclonal antibodies, and the addition of the IL-2 mutation (N88D) did not affect its binding ability.

[0682] Example 3: In vitro cell binding ability of the truncated fusion protein to receptor-expressing cells

[0683] This embodiment aims to examine whether the binding ability of the fusion protein to the IL-2R receptor changes after fusing IL-2 and IL-2Rα truncated variants. The in vitro binding activity of the IL-2-containing fusion protein to the receptor protein or receptor-expressing cells was detected using IL-2Rα, IL-2Rβγ proteins, or the HEK-Blue-IL2 reporter cell line.

[0684] 3.1 In vitro binding activity assay of fusion proteins linked via homodimers

[0685] (1) Fusion proteins 4, 22-6 and 24-3

[0686] The binding affinity of the IL-2R receptor to the truncated IL-2Rα variant, which fused with IL-2 and 172 amino acids (the truncated IL-2Rα variant is 40 amino acids shorter at the C-terminus compared to the 212 amino acid extracellular domain fragment of IL-2Rα; and substantially 47 amino acids shorter at the C-terminus compared to SEQ ID NO:3), was tested.

[0687] The binding affinity of fusion protein 4 to the IL-2R receptor was detected using the HEK-Blue-IL2 reporter cell line (InvivoGen, hkb-il2-2). The number of stained cells was 3 × 10⁻⁶. 5 Cells / well; initial protein concentration was 50 μg / mL, diluted 1:5, and then serially diluted 8 times; after incubating the protein and cells at 4°C for 30 min, wash once with 200 μL FACS buffer; calculate the amount of secondary antibody PE anti-human IgG Fc (BIolegend, 366904) to be used at 0.3 μL / well, take 12 μL of secondary antibody (clone number QA19A42) and add it to 4 mL FACS buffer, mix well, add 100 μL of the diluted secondary antibody to the corresponding well, incubate at 4°C in the dark for 30 min, and wash twice with 200 μL FACS buffer; resuspend the cells with 200 μL FACS buffer and perform fluorescence detection on a Sino Cyte flow cytometer; plot the protein binding curve using GraphPad software.

[0688] As shown in Figure 3A, compared with IL2-Fc, the binding ability of fusion protein 3 and fusion protein 4 containing the IL-2Rα truncated form to the IL-2R receptor almost disappeared. This indicates that in the fusion protein, the IL-2Rα truncated form still has a good masking effect on IL-2, and its masking ability is close to that of IL-2Rα.

[0689] The binding affinity of fusion proteins 22-6 (ΔC40aa) and 24-3 (ΔC40aa) to the IL-2R receptor was detected using protein assays. First, the proteins were coated overnight with 5 μg / mL of the target protein (IL-2Rβγ or IL-2Rα). After washing and blocking, the initial protein concentration was 60 nmol / L, and the plates were diluted 1:5, followed by eight further dilutions. The diluted fusion proteins were then added to ELISA plates and incubated at 37°C for 60 min. After washing, HRP-labeled anti-human IgG Fc was added, and the plates were incubated at 37°C for 30 min. The OD value was then read after color development.

[0690] Fusion protein 22-6 contains a 172aa truncated IL-2Rα, namely IL-2Rα (ΔC40aa, compared to the 212 amino acid extracellular domain of IL-2Rα) and a PD-1 antibody, as shown in Figure 17B; fusion protein 24-3 contains a 172aa truncated IL-2Rα, namely IL-2Rα (ΔC40aa, compared to the 212 amino acid extracellular domain of IL-2Rα) and a PD-L1 antibody, as shown in Figure 17B; fusion protein 24-2 contains a truncated IL-2Rα (ΔC30aa, compared to the 212 amino acid extracellular domain of IL-2Rα) and a PD-L1 antibody, as shown in Figure 17B.

[0691] As shown in Figures 3B-3C, compared to IL2-Fc, the binding ability of fusion protein 22-6 (ΔC40aa) containing the IL-2Rα truncated variant to the IL-2R receptor was almost completely lost (Figure 3B); similarly, compared to IL2-Fc, the binding ability of fusion protein 24-3 (ΔC40aa) containing the IL-2Rα truncated variant to the IL-2 receptor was almost completely lost (Figure 3C). These results indicate that the IL-2Rα truncated variant has a good masking effect on IL-2 in fusion proteins, and this strategy is applicable to different targets and has universality for fusion proteins carrying different antibodies.

[0692] (3) Fusion protein 3-31

[0693] To further reduce the toxicity of the IL-2Rα-containing fusion protein, the IL-2 of the fusion protein was modified (mutated to N88D) to reduce its binding ability to Rβ / γ. Protein detection was performed using the methods described in “(1) Fusion Proteins 4, 22-6, and 24-3” above. Fusion protein 3-31 is a fusion protein based on the IL-2 fragment of fusion protein 3-23 (ΔC40aa) with an N88D amino acid mutation.

[0694] As shown in Figure 3D, compared to IL2-Fc and fusion protein 23 (ΔC40aa), fusion protein 3-31 (ΔC40aa, N88D) almost completely lost its ability to bind to the IL-2R receptor. This indicates that, under the condition of IL-2Rα being truncated to the same length, the fusion protein containing the N88D mutation has a lower receptor binding capacity than the IL-2 molecule and is expected to have lower in vivo toxicity.

[0695] 3.2 In vitro binding activity assay of fusion proteins linked via heterodimers

[0696] (1) Fusion proteins 3-19 and 3-23

[0697] The detection was performed using proteins, and the experimental method was the same as described above for "(1) Fusion proteins 4, 22-6 and 24-3".

[0698] Among them, fusion proteins 3-17, 3-22 and 3-29 are fusion proteins containing the full-length extracellular domain of IL-2Rα with 212 amino acids, and their structural schematic diagrams are shown in Figures 17A, 17B and 17B, respectively.

[0699] Among them, fusion proteins 3-19 and 3-23 both contain a 172aa truncated IL-2Rα, namely IL-2Rα(△C40aa), which is 40aa shorter at the C-terminus compared to the 212 amino acid extracellular domain of IL-2Rα. Their structural schematic diagrams are shown in Figures 17A and 17B, respectively.

[0700] As shown in Figure 4A, the binding affinity of fusion proteins 3-19 (heterodimer) and 3-23 (homodimer) to the IL-2 receptor protein was lower than that of IL2-Fc, but their binding affinity to the IL-2R receptor was essentially the same as that of fusion proteins 3-17 (heterodimer), 3-22 (homodimer), and 3-29 (homodimer) containing the full-length IL-2Rα fragment. This indicates that fusion proteins containing the IL-2Rα truncated form of this invention have the same good masking effect as fusion proteins containing the full-length IL-2Rα fragment.

[0701] (2) Fusion proteins 22-23, 22-27, 3-19, 3-30, 22-38, 22-39, 3-51 and 3-52

[0702] Furthermore, the effects of different lengths of IL-2Rα truncated variants and the combined IL-2 mutation (N88D) on the in vitro binding activity of the fusion proteins were investigated. Protein assays were performed using the methods described in “(1) Fusion proteins 4, 22-6, and 24-3” above.

[0703] Among them, fusion proteins 22-23 and 22-27 contain a 172aa truncated IL-2Rα, namely IL-2Rα(△C40aa) and PD-1 antibody, and 22-27 additionally contains an IL-2 mutation (N88D). The structures of fusion proteins 22-23 and 22-27 are shown in Figure 17A.

[0704] Among them, fusion proteins 3-19 and 3-30 contain a 172aa truncated IL-2Rα, namely IL-2Rα(△C40aa) and CLDN18.2 antibody, and 3-30 additionally contains an IL-2 mutation (N88D). The structures of fusion proteins 3-19 and 3-30 are shown in Figure 17A.

[0705] Fusion proteins 22-38 and 22-39 contain a 166aa truncated IL-2Rα, namely IL-2Rα(△C46aa) and PD-L1 antibody. Fusion protein 22-39 also contains an IL-2 mutation (N88D). The structures of fusion proteins 22-38 and 22-39 are shown in Figure 17A.

[0706] Among them, fusion proteins 3-51 and 3-52 contain a 166aa truncated IL-2Rα, namely IL-2Rα(△C46aa) and CLDN18.2 antibody, and fusion protein 3-52 additionally contains an IL-2 mutation (N88D). The structures of fusion proteins 3-51 and 3-52 are shown in Figure 17A.

[0707] As shown in Figures 4B-4E, compared to IL2-Fc, the binding affinity of IL-2Rα truncated variants containing 166aa-172aa, namely IL-2Rα(△C40aa-△C46aa) fusion proteins 22-23, 3-19, 22-38, and 3-51 to the IL-2Rβγ receptor is reduced; and after the introduction of the N88D mutation, the binding affinity of fusion proteins 22-27, 3-30, 22-39, and 3-52 to the IL-2Rβγ receptor is further reduced.

[0708] The above results indicate that even when applied to fusion proteins containing different targets, IL-2Rα truncated variants of different lengths exhibit similar masking effects; furthermore, under the condition of IL-2Rα truncated to the same length, the fusion protein containing the N88D mutation has a lower IL-2Rβγ receptor binding capacity relative to the IL-2 molecule, and is expected to have lower in vivo toxicity.

[0709] Example 4: Changes in the in vitro bioactivity of the fusion protein

[0710] In this embodiment, to examine whether the IL-2 activity of the IL-2 fusion protein is restored after cleavage by tumor-associated proteases, the CTLL2 cell line (Pronosai, CL-0331) was used to detect changes in IL-2 activity in the fusion protein.

[0711] 4.1 Changes in the in vitro bioactivity of fusion proteins linked via homodimers

[0712] (1) Changes in IL-2 activity before and after enzyme digestion of fusion protein 4

[0713] The changes in the in vitro bioactivity of the IL-2Rα truncated variant, IL-2Rα(ΔC40aa), which combines IL-2 and 172aa, were examined.

[0714] Collect the grown cells into centrifuge tubes, centrifuge at 700 rpm for 5 minutes, discard the supernatant, resuspend in 10 ml of culture medium, and count the cells; take 100 μl of the resuspended cells, centrifuge at 700 rpm for 5 minutes, discard the supernatant, and adjust the cell volume to 1 x 10⁻⁶ cells / mL with culture medium. 5 / mL-5x10 4 / mL, add 100μL to each well of a 96-well plate using a pipette; dilute the sample, the current concentration of IL2-Fc is 2mg / mL, dilute to 300nmol / mL (corresponding to a mass concentration of 24.68μg / mL), i.e., 81 times, take 10μL and add it to 800μL of complete culture medium, then take another 100μL and add it to 400μL, perform a 5-fold serial dilution, for a total of 10 concentration gradients; the other two samples are diluted 10.8 and 10 times respectively and then serially diluted; add 100μL of the diluted sample to each well of cells, and incubate in a 37℃ CO2 incubator for 48 hours; add 20μL of CCK8 (Solarbio, CA1210) to each well, and continue to incubate for 3.5 hours, and read the OD450 value.

[0715] As shown in Figure 5A, in fusion protein 4 containing the truncated IL-2Rα, the activity of IL-2 decreased compared to IL2-Fc after masking. However, the activity was restored after enzyme digestion (addition of MMP, RD, and 918-MP-010). Fusion protein 4 had a similar masking effect to fusion protein 3, and its activity decreased by about 100 times compared to IL2-Fc.

[0716] (2) Cell proliferation of fusion protein 22-6-CTLL2

[0717] Furthermore, the changes in the in vitro bioactivity of the fusion protein after fusing IL-2 and 172aa into the truncated IL-2Rα, namely IL-2Rα(△C40aa), were examined.

[0718] The experiment was conducted according to the following method:

[0719] 1) Wash cells in the logarithmic growth phase with PBS, digest them into single cells, and adjust the concentration to 5 x 10⁻⁶ cells / mL. 4 / mL, plated, 100μL per well is 5000 cells / well.

[0720] 2) Dilute the fusion protein and control protein with culture medium to an initial concentration of 60 nM, and then dilute them sequentially down to 10 gradients at a ratio of 1:5;

[0721] 3) Mix the diluted fusion protein or control protein with the cells separately, and incubate the mixture in a cell culture incubator for 48 h. Add 20 μL of CCK8 detection solution to each well, mix well, and incubate at 37°C for 15-30 min in the dark. Measure the absorbance at 450 nm using a microplate reader.

[0722] Fusion protein 22-6 contains a 172aa truncated IL-2Rα, namely IL-2Rα(ΔC40aa) and PD-1 antibody, and its structure is shown in Figure 17B; fusion protein 24-3 contains a 172aa truncated IL-2Rα, namely IL-2Rα(ΔC40aa) and PD-L1 antibody, and its structure is shown in Figure 17B.

[0723] As shown in Figure 5B, the fusion protein 22-6 (ΔC40aa) containing the 172aa truncated IL-2Rα showed decreased activity compared to IL2-Fc, indicating that the fusion protein containing the truncated IL-2Rα of this invention has a good masking effect and is expected to have lower toxicity compared to untruncated IL-2Rα.

[0724] (3) Fusion proteins 5-2, 5-4, 5-7 and 5-9—CTLL2 cell proliferation

[0725] Furthermore, the changes in the in vitro bioactivity of the fusion protein containing the bispecific antibody were examined. The experimental method was the same as described above in "(2) Fusion protein 22-6-CTLL2 cell proliferation".

[0726] Among them, fusion proteins 5-2, 5-4, 5-7 and 5-9 each contain a 172aa truncated IL-2Rα, namely IL-2Rα(△C40aa) and PD1-VEGF antibody, and their structural schematic diagrams are shown in Figures 17C, 17D, 17E and 17F, respectively.

[0727] As shown in Figure 5C, the fusion proteins 5-2 (ΔC40aa), 5-4 (ΔC40aa), 5-7 (ΔC40aa), and 5-9 (ΔC40aa) containing the 172aa IL-2Rα truncated variant exhibited decreased activity compared to IL2-Fc. This indicates that among fusion proteins containing bispecific antibodies, the fusion proteins containing the IL-2Rα truncated variant of this invention all possess good masking ability against IL2.

[0728] (4) Cell proliferation of fusion proteins 3-23 and 3-31-CTLL2

[0729] Furthermore, the effect of the IL-2Rα truncated variant combined with the IL-2 mutation (N88D) on the in vitro bioactivity of the fusion protein was examined. The experimental methods were the same as described in "(2) Cell proliferation of fusion protein 22-6-CTLL2".

[0730] Fusion proteins 3-23 and 3-31 both contain a 172aa truncated IL-2Rα, namely IL-2Rα(△C40aa) and CLDN18.2 antibody. Fusion protein 3-31 additionally contains an IL-2 mutation (N88D). The structures of fusion proteins 3-23(△C40aa) and 3-31(△C40aa, N88D) are shown in Figure 17B.

[0731] As shown in Figure 5D, the fusion proteins 3-23 (ΔC40aa) and 3-31 (ΔC40aa, N88D) showed decreased activity compared to IL2-Fc, and their ability to promote CTLL2 cell proliferation was basically consistent with that of the fusion protein 3-22 (212aa of IL-2Rα). This indicates that the fusion proteins containing the truncated form of IL-2Rα of the present invention, as well as the fusion proteins with the N88D mutation, have good IL2 masking ability and lower expected toxicity, just like the fusion proteins containing the full-length IL-2Rα.

[0732] 4.2 Changes in the in vitro bioactivity of fusion proteins linked via heterodimers

[0733] (1) Cell proliferation of fusion proteins 3-19 and 3-23-CTLL2

[0734] The experimental method was the same as described above in “(2) Fusion protein 22-6-CTLL2 cell proliferation”.

[0735] Among them, fusion proteins 3-17 and 3-29 are fusion proteins containing the full-length extracellular domain of IL-2Rα with 212 amino acids, and their structural schematic diagrams are shown in Figures 17A and 17B, respectively.

[0736] Among them, fusion proteins 3-19 and 3-23 both contain a 172-amino acid truncated form of IL-2Rα, namely IL-2Rα(△C40aa), and their structural schematic diagrams are shown in Figures 17A and 17B, respectively.

[0737] As shown in Figure 6A, the binding affinity of fusion proteins 3-19 (heterodimer) and 3-23 (homodimer), containing different structures of the IL-2Rα truncated variant, to the IL-2R receptor protein was significantly lower than that of IL2-Fc; however, their ability to promote CTLL2 cell proliferation was essentially the same as that of fusion proteins 3-17 (heterodimer) and 3-29 (homodimer), which contain the full-length IL-2Rα fragment. This indicates that, in different structures, fusion proteins containing the IL-2Rα truncated variant of this invention exhibit good IL2 masking activity, similar to fusion proteins containing the full-length IL-2Rα fragment.

[0738] (2) Fusion proteins 3-19, 3-30, 22-38, 22-39, 3-51 and 3-52—CTLL2 cell proliferation

[0739] The effects of different lengths of IL-2Rα truncated variants and the combined use of IL-2 mutation (N88D) on the in vitro binding activity of the fusion protein were investigated. The experimental methods were the same as described in "(2) Cell proliferation of fusion protein 22-6-CTLL2".

[0740] Among them, fusion proteins 3-19 and 3-30 contain a 172aa truncated IL-2Rα, namely IL-2Rα(△C40aa) and CLDN18.2 antibody, and 3-30 additionally contains an IL-2 mutation (N88D). The structures of fusion proteins 3-19 and 3-30 are shown in Figure 17A.

[0741] Fusion proteins 22-38 and 22-39 contain a 166aa truncated IL-2Rα, IL-2Rα (ΔC46aa), and a PD-L1 antibody. Fusion protein 22-39 also contains an IL-2 mutation (N88D). The structures of fusion proteins 22-38 and 22-39 are shown in Figure 17A.

[0742] Among them, fusion proteins 3-51 and 3-52 contain a 166aa truncated IL-2Rα, IL-2Rα (ΔC46aa) and CLDN18.2 antibody, and fusion protein 3-52 additionally contains an IL-2 mutation (N88D). The structures of fusion proteins 3-51 and 3-52 are shown in Figure 17A.

[0743] As shown in Figures 6B-6D, the fusion proteins 3-19 (△C40aa), 3-30 (△C40aa, N88D), 22-38 (△C46aa), 22-39 (△C46aa, N88D), 3-51 (△C46aa), and 3-52 (△C46aa, N88D) showed decreased activity compared to IL2-Fc. This indicates that the fusion proteins containing IL-2Rα truncated variants of different lengths of the present invention, as well as the fusion proteins of the present invention with further N88D mutations, are expected to have lower toxicity.

[0744] (3) Fusion proteins 22-23 and 22-27—HEK cells

[0745] The experiment was conducted according to the following method:

[0746] Cells in the logarithmic growth phase were washed with PBS and digested into single cells, with the concentration adjusted to 1.5 x 10⁻⁶. 6 / mL, plated, 100μL per well is 1.5x10 5 Cells / pores.

[0747] The fusion protein and control protein were diluted with culture medium to an initial concentration of 60 nM, and then down-diluted 10 times at a ratio of 1:5.

[0748] Mix the diluted fusion protein or control protein with the cells separately, and incubate the mixture in a cell culture incubator for 24 hours. Take 20 μL of supernatant from each well, add 180 μL of Quanti Blue assay solution (Invivogen, rep-qbs), mix well, and incubate at 37°C in the dark for 30-60 minutes. Measure the absorbance at 630 nm using a microplate reader.

[0749] Among them, fusion proteins 22-23 and 22-27 contain a 172aa truncated IL-2Rα, namely IL-2Rα(△C40aa) and PD-1 antibody, and 22-27 additionally contains an IL-2 mutation (N88D). The structures of fusion proteins 22-23 and 22-27 are shown in Figure 17A.

[0750] As shown in Figure 6E, a certain level of IL-2 masking activity was detected on reporter cells (top figure), and this masking activity could be restored to wild-type IL2 levels with the help of the antibody (bottom figure), similar to PD1-IL2. This result indicates that the fusion protein containing the truncated IL-2Rα variant of this invention, and the fusion protein further incorporating the N88D mutation, have good masking ability for IL2 in non-target enriched regions, i.e., low toxicity is expected, while also exhibiting good IL2 activation activity in target enriched regions.

[0751] Example 5: In vitro toxicity assessment of the fusion protein containing the truncated form (detection of cytokine release from the fusion protein onto PBMCs).

[0752] 5.1 In vitro toxicity assessment of fusion proteins linked via homodimers

[0753] (1) Fusion proteins 22-6, 33-3 and 35-2

[0754] The effects of different lengths of IL-2Rα truncated variants and different target sites on the in vitro toxicity of the fusion protein were evaluated. Specifically, the effects of fusion protein 22-6, 33-3, and 35-2 on IFN-γ secretion levels were detected using human PBMCs to assess the in vitro toxicity of the fusion protein.

[0755] The experiment was conducted according to the following method:

[0756] Revive frozen PBMCs (Miaoshun Biotechnology, catalog number PB25C). After resuspending the cells in complete culture medium, stabilize them in a cell culture incubator for 1-2 hours; dilute the sample by diluting the sample to be tested with complete culture medium to 600 nmol / L; collect PBMCs, count them, and adjust the concentration to 4 × 10⁻⁶. 6 / mL; Add 50μL to each well of a 96-well plate, i.e., 2×10 5 Cells / well; Add 50 μL of the prepared sample to the corresponding well, i.e., the final sample concentration is 100 nM / L; Add 100 μL of 1640 complete medium to the Cell only group; Add CD3 (Invitrogen, 2944626) to the positive control wells at a final concentration of 1 μg / mL and CD28 (Invitrogen, 2798473) at a final concentration of 2.5 μg / mL. Gently tap to mix, incubate at 37°C, 5% CO2 for 24 hours, collect the supernatant, and detect the secretion of IFNγ using a human inflammatory factor detection kit (BD, catalog number 551809).

[0757] The fusion protein 22-6 contains a 172aa truncated IL-2Rα, namely IL-2Rα(ΔC40aa) and PD-1 antibody, the structure of which is shown in Figure 17B.

[0758] The fusion protein 33-3 contains a 172aa truncated IL-2Rα, namely IL-2Rα(ΔC40aa) and VEGFR antibody, the structure of which is shown in Figure 17B.

[0759] The fusion protein 35-2 contains a 172aa truncated IL-2Rα, namely IL-2Rα(ΔC40aa) and HER2 antibody, the structure of which is shown in Figure 17B.

[0760] As shown in Figures 7A and 7B, compared to IL2-Fc, the secretion level of IFN-γ was significantly reduced after co-incubation of each fusion protein with PBMC in vitro. This indicates that the activation ability of each fusion protein to PBMC is weakened, resulting in lower toxicity. This result demonstrates that the IL-2Rα truncated variants of different lengths of the present invention can reduce the in vitro toxicity of the fusion proteins of the present invention and are applicable to a variety of different targets.

[0761] (2) Fusion proteins 5-2, 5-4, 5-7, 5-9, 5-11, 5-13, 5-15 and 5-17

[0762] Furthermore, the in vitro toxicity of the fusion proteins containing bispecific antibodies was investigated. The experimental methods were the same as described above for fusion proteins 22-6, 33-3, and 35-2 (1).

[0763] Among them, fusion proteins 5-2, 5-4, 5-7, 5-9, 5-11, 5-13, 5-15 and 5-17 all contain a 172aa truncated IL-2Rα, namely IL-2Rα(△C40aa) and PD1-VEGF antibody. The schematic diagrams of the VEGF-PD-1 antibody structures contained therein are shown in Figures 17C-17J, respectively.

[0764] As shown in Figure 7C, compared with IL2-Fc, the secretion level of IFN-γ was significantly reduced after each fusion protein was co-incubated with PBMC in vitro. This indicates that the activation ability of each fusion protein to PBMC is weakened and that it has lower toxicity.

[0765] (3) Fusion proteins 3-23 and 3-31

[0766] Furthermore, the effects of different lengths of IL-2Rα truncated variants combined with IL-2 mutation (N88D) on the in vitro toxicity of the fusion proteins were investigated. The experimental methods were the same as described above for "(1) Fusion proteins 22-6, 33-3 and 35-2".

[0767] Fusion proteins 3-23 and 3-31 both contain a 172aa truncated IL-2Rα, namely IL-2Rα(ΔC40aa) and CLDN18.2 antibody. Fusion protein 3-31 also contains an IL-2 mutation (N88D). The structures of fusion proteins 3-23 and 3-31 are shown in Figure 17B.

[0768] As shown in Figure 7B, compared to IL-2-Fc and fusion protein 3-22 (IL-2Rα not truncated), the secretion level of IFN-γ was significantly reduced after co-incubation with PBMCs in vitro with fusion protein 3-23 (ΔC40aa). Further introduction of the N88D mutant fusion protein 3-31 (ΔC46aa, N88D) resulted in a further decrease in IFN-γ secretion levels. This result demonstrates that fusion proteins containing different lengths of truncated IL-2Rα variants of the present invention exhibit low in vitro toxicity; and that under the same IL-2Rα truncation length, the fusion protein containing the N88D mutant exhibits even lower in vitro toxicity.

[0769] 5.2 In vitro toxicity assessment of fusion proteins linked via heterodimers

[0770] (1) Fusion proteins 22-23, 22-27, 3-19, 3-30, 22-38, 22-39, 3-51 and 3-52

[0771] The effects of IL-2Rα truncated variants of different lengths and the combined effect of IL-2 mutation (N88D) on the in vitro toxicity of the fusion proteins were investigated. The experimental methods were the same as described above for "(1) Fusion proteins 22-6, 33-3 and 35-2".

[0772] Among them, fusion proteins 22-23 and 22-27 contain a 172aa truncated IL-2Rα, namely IL-2Rα(△C40aa) and PD-1 antibody, and 22-27 additionally contains an IL-2 mutation (N88D). The structures of fusion proteins 22-23 and 22-27 are shown in Figure 17A.

[0773] Among them, fusion proteins 3-19 and 3-30 contain a truncated 172aa IL-2Rα, namely IL-2Rα (△C40aa) and CLDN18.2 antibody, and 3-30 additionally contains an IL-2 mutation (N88D). The structures of fusion proteins 3-19 and 3-30 are shown in Figure 17A.

[0774] Fusion proteins 22-38 and 22-39 contain a 166aa truncated IL-2Rα, namely IL-2Rα(△C46aa) and PD-L1 antibody. Fusion protein 22-39 also contains an IL-2 mutation (N88D). The structures of fusion proteins 22-38 and 22-39 are shown in Figure 17A.

[0775] Among them, fusion proteins 3-51 and 3-52 contain a 166aa truncated IL-2Rα, namely IL-2Rα(△C46aa) and CLDN18.2 antibody, and fusion protein 3-52 additionally contains an IL-2 mutation (N88D). The structures of fusion proteins 3-51 and 3-52 are shown in Figure 17A.

[0776] As shown in Figures 8A-8D, compared with IL-2-Fc, the secretion levels of IFN-γ were significantly reduced after co-incubating with PBMCs in vitro with fusion proteins 22-23 (△C40aa), 3-19 (△C40aa), 22-38 (△C46aa), and 3-51 (△C46aa) containing different lengths of the IL-2Rα truncated variants of this invention and fused with different target sites. Furthermore, the introduction of N88D mutant fusion proteins 22-27 (△C40aa, N88D), 3-30 (△C40aa, N88D), 22-39 (△C46aa, N88D), and 3-52 (△C46aa, N88D) further reduced the secretion levels of IFN-γ. This result demonstrates that fusion proteins containing different lengths of the IL-2Rα truncated variants of the present invention all exhibit low in vitro toxicity, and that fusion proteins containing the N88D mutation exhibit even lower in vitro toxicity when the IL-2Rα truncated to the same length.

[0777] Example 6: In vivo activity changes and safety assays of the truncated fusion protein

[0778] In this embodiment, to test the antitumor efficacy and safety of the truncated fusion protein, a subcutaneous tumor model was established using cell lines such as MC38-CLDN18.2, and the antitumor efficacy of the candidate molecule was tested in vivo.

[0779] 1e6 cells / mouse were subcutaneously inoculated with cells exhibiting a survival rate greater than 90% and in the logarithmic growth phase; tumor growth rate was assessed when the tumor volume was between 100-120 mm. 3 The mice were divided into groups of eight, and each group received an intraperitoneal injection of a different drug twice, with a three-day interval between doses. Tumor volume and body weight were measured every 3-4 days. The tumor volume in the control group was measured until it reached 1500 mmHg. 3 The experiment was terminated, and the animals were euthanized.

[0780] 6.1 In vivo activity and safety assays of fusion proteins linked via homodimers

[0781] (1) Fusion protein 4

[0782] Mice were divided into 5 groups and injected intraperitoneally with different drugs. The results are shown in Figure 9. In two experiments with different dosages (60+60μg, 100+100μg), the results showed that fusion protein 4 containing IL-2Rα with a 47aa truncated at the C-terminus and fusion protein 9 containing IL-2Rα with a 37aa truncated at the C-terminus had similar antitumor effects compared to fusion protein 3 and fusion protein 8, but without weight loss, indicating that they have lower potential in vivo toxicity and good safety.

[0783] (2) Fusion protein 22-6

[0784] Mice were divided into three groups and administered different drugs via intraperitoneal injection. The results are shown in Figure 10. Compared with Nivolumab, the fusion protein 22-6 showed better anti-tumor effects after two administrations (100+100μg), and its body weight did not change significantly compared with the control group (injected with PBS), indicating that it has lower potential in vivo toxicity and good safety.

[0785] (3) Fusion proteins 24-3 and 24-2

[0786] Mice were divided into three groups and administered different drugs via intraperitoneal injection. The results are shown in Figure 11. Compared with Atezolizumab, the fusion proteins 24-3 and 24-2 showed better antitumor effects after two administrations (100+100μg), and their body weight did not change significantly compared with the control group (injected with PBS), indicating that they have lower potential in vivo toxicity and good safety.

[0787] (4) Fusion proteins 33-3 and 33-4

[0788] Mice were divided into three groups and injected intraperitoneally with different drugs. The results are shown in Figure 12. Both fusion proteins 33-3 and 33-4 showed significant antitumor activity, and fusion protein 33-3 was more effective. At the same time, the mice did not lose weight significantly during the treatment, indicating that they have low potential in vivo toxicity and good safety.

[0789] (5) Fusion protein 35-2

[0790] Mice were divided into three groups and injected intraperitoneally with different drugs. The results are shown in Figure 13. Compared with anti-HER2 (Anti-HER2 SinoBiological, 68046-H002), the fusion protein 35-2 showed better anti-tumor effect after two administrations (100+100μg), and its body weight did not change significantly compared with the control group (injected with PBS), indicating that it has lower potential in vivo toxicity and good safety.

[0791] 6.2 In vivo activity changes and safety assays of fusion proteins linked via heterodimers

[0792] (1) Fusion protein 3-52

[0793] Mice were divided into two groups and injected intraperitoneally with different drugs. The results are shown in Figure 14. Compared with the control group (injected with PBS), the fusion protein 3-52 showed excellent anti-tumor effects after three administrations (100+100+100μg), and its body weight did not change significantly compared with the control group (injected with PBS), indicating that it has lower in vivo toxicity and good safety.

[0794] (2) Fusion proteins 22-38 and 22-39

[0795] Mice were divided into two groups and injected intraperitoneally with different drugs. The results are shown in Figure 15. Compared with the control group (injected with PBS), the fusion proteins 22-38 and 22-39 showed good anti-tumor effects after three administrations (100+100μg), and the body weight did not change significantly compared with the control group (injected with PBS). The combination of N88D and 22-39 showed that it had lower in vivo toxicity and good safety.

[0796] Example 7: In vitro enzyme digestion experiment of fusion protein

[0797] This embodiment aims to examine whether the in vitro enzymatic digestion efficiency of the fusion protein containing the truncated IL-2Rα fragment is affected compared to that of the fusion protein containing the full-length IL-2Rα fragment (212aa).

[0798] In vitro enzyme digestion was performed at a molar ratio of MMP14 enzyme to protein of 1:5. The experimental protein sample was 2 μg, and the digestion was carried out overnight at 37°C (in a water bath). The prepared samples were then analyzed by SDS-PAGE to detect the MMP14 digestion results.

[0799] As shown in Figure 16, the fusion protein 3-23 containing the truncated IL-2Rα fragment and the fusion protein 3-29 containing the full-length IL-2Rα fragment have basically the same enzymatic digestion efficiency, indicating that the fusion protein containing the truncated IL-2Rα fragment has excellent in vitro enzymatic digestion efficiency, and thus its good biological activity in vivo is expected.

[0800] Sequence List:

[0801] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. A truncated form of interleukin-2 receptor α (IL-2Rα) comprising 166 to 190 or 167 to 190 amino acids continuously from the N-terminus of the extracellular domain of wild-type IL-2Rα.

2. The IL-2Rα truncated form according to claim 1, wherein the IL-2Rα truncated form comprises 166 to 182 or 167 to 182 amino acids continuously from the N-terminus of the extracellular domain of wild-type IL-2Rα; Preferably, the IL-2Rα truncated form comprises 166 to 172 or 167 to 172 amino acids consecutively starting from the N-terminus of the extracellular domain of wild-type IL-2Rα.

3. The IL-2Rα truncated form according to claim 1 or 2, wherein the IL-2Rα truncated form comprises an amino acid sequence as shown in any one of SEQ ID NO: 5-29, or the amino acid sequence of the IL-2Rα truncated form is as shown in any one of SEQ ID NO: 5-29; Preferably, the IL-2Rα truncated form comprises an amino acid sequence as shown in any one of SEQ ID NO:13-29, or the amino acid sequence of the IL-2Rα truncated form is as shown in any one of SEQ ID NO:13-29; More preferably, the IL-2Rα truncated form comprises an amino acid sequence as shown in any one of SEQ ID NO:23-29, or the amino acid sequence of the IL-2Rα truncated form is as shown in any one of SEQ ID NO:23-29.

4. A fusion protein comprising Interleukin-2 (IL-2) wild-type or mutant protein; A cleavable linker that connects to the wild-type or mutant IL-2 protein; Interleukin-2 receptor α (IL-2Rα) truncated bodies connected to the said cleavable connector; and Target-binding peptides; wherein, The IL-2Rα truncated body is the IL-2Rα truncated body described in any one of claims 1-3.

5. The fusion protein according to claim 4, wherein the target-binding polypeptide is linked to the IL-2Rα truncated form via an uncuttable linker, or the target-binding polypeptide is linked to the wild-type or mutant IL-2 protein via an uncuttable linker.

6. The fusion protein according to claim 4 or 5, wherein the fusion protein comprises, in sequence: (i) a target-binding peptide, an IL-2Rα truncated form, and a wild-type or mutant IL-2 protein, wherein the target-binding peptide is linked to the IL-2Rα truncated form via an uncleavable linker; or (ii) a target-binding polypeptide, wild-type or mutant IL-2 protein and a truncated IL-2Rα protein, wherein the target-binding polypeptide is linked to the wild-type or mutant IL-2 protein via an uncuttable linker.

7. A fusion protein comprising: Half-life extenders; Interleukin-2 (IL-2) wild-type or mutant protein; Cleavable linkers to the wild-type or mutant IL-2 protein; and A truncated form of interleukin-2 receptor α (IL-2Rα) connected to the cuttable connector; wherein, The IL-2Rα truncated body is the IL-2Rα truncated body described in any one of claims 1-3.

8. The fusion protein of claim 7, wherein the half-life extender is connected to the IL-2Rα truncated form via an uncuttable linker, or the half-life extender is connected to the wild-type or mutant IL-2 protein via an uncuttable linker.

9. The fusion protein according to claim 7 or 8, wherein the fusion protein is selected from any of the following: (i) a half-life extender, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the half-life extender is connected to the IL-2Rα truncated form via an incuttable linker; (ii) a half-life extender, wild-type or mutant IL-2 protein and IL-2Rα truncated form, wherein the half-life extender is connected to the wild-type or mutant IL-2 protein via an incuttable linker; (iii) A target-binding peptide, a half-life extender, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the half-life extender is linked to the IL-2Rα truncated form via an incleavable linker; or (iv) A target-binding peptide, a half-life extender, a wild-type or mutant IL-2 protein, and an IL-2Rα truncated form, wherein the half-life extender is linked to the wild-type or mutant IL-2 protein via an uncuttable linker.

10. The fusion protein according to any one of claims 7-9, wherein the fusion protein is selected from any one of the following: (1) Two first polypeptide chains, wherein the first polypeptide chains sequentially comprise: a first target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated form and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the single-chain Fc via an uncleavable linker. (2) Two first polypeptide chains, each first polypeptide chain comprising, in sequence: a light chain of a first target-binding polypeptide, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the light chain of the first target-binding polypeptide via an uncleavable linker; and two second polypeptide chains, each second polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a single-chain Fc segment, and a second target-binding polypeptide with an scFv structure; (3) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; and two second polypeptide chains, each second polypeptide chain comprising, in sequence: wild-type or mutant IL-2 protein, a truncated IL-2Rα protein, a heavy chain Fd segment of the first target-binding polypeptide, a single chain Fc segment, and a second target-binding polypeptide with an scFv structure, wherein the wild-type or mutant IL-2 protein is connected to the truncated IL-2Rα protein via a cleavable linker, and the truncated IL-2Rα protein is connected to the heavy chain of the first target-binding polypeptide via an uncleavable linker. (4) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; two second polypeptide chains, each second polypeptide chain comprising, in sequence: a second target-binding polypeptide with an scFv structure, a heavy chain Fd segment of the first target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the single-chain Fc via an uncleavable linker; (5) Two first polypeptide chains, each first polypeptide chain comprising: a light chain variable region of a second target-binding polypeptide and a light chain of the first target-binding polypeptide; two second polypeptide chains, each second polypeptide chain comprising: a heavy chain variable region of a second target-binding polypeptide, a heavy chain Fd segment of the first target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated body, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated body via a cleavable linker, and the IL-2Rα truncated body is connected to the single-chain Fc via an uncleavable linker; (6) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; and two second polypeptide chains, each second polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the single-chain Fc via an uncleavable linker. (7) Two first polypeptide chains, each first polypeptide chain comprising, in sequence: a light chain of a first target-binding polypeptide, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the light chain of the first target-binding polypeptide via an uncleavable linker; and two second polypeptide chains, each second polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), and a single-chain Fc. (8) Two first polypeptide chains, each first polypeptide chain comprising, in sequence: wild-type or mutant IL-2 protein, IL-2Rα truncated form, and light chain of first target-binding polypeptide, wherein the wild-type or mutant IL-2 protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the light chain of first target-binding polypeptide via an uncleavable linker; two second polypeptide chains, each second polypeptide chain comprising, in sequence: heavy chain Fd segment of first target-binding polypeptide, second target-binding polypeptide in the form of nanobody (VHH or V-NAR), and single chain Fc; (9) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; two second polypeptide chains, each second polypeptide chain comprising, in sequence: wild-type or mutant IL-2 protein, an IL-2Rα truncated form, a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), and a single chain Fc, wherein the wild-type or mutant IL-2 protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the heavy chain Fd segment of the first target-binding polypeptide via an uncleavable linker; (10) A first polypeptide chain, comprising in sequence: a first target-binding polypeptide with an scFv structure, a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the single-chain Fc via an uncleavable linker; a second polypeptide chain, comprising in sequence: a heavy chain variable region of the first target-binding polypeptide, a heavy chain Fd segment of the second target-binding polypeptide, and a single-chain Fc; a third polypeptide chain, comprising in sequence: a light chain variable region of the first target-binding polypeptide and a light chain of the second target-binding polypeptide; (11) Two first polypeptide chains, comprising: a light chain of a first target-binding polypeptide; a second polypeptide chain, comprising in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide of scFv structure, a first single chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the first single chain Fc via an uncleavable linker; a third polypeptide chain, comprising in sequence: a heavy chain Fd segment of the first target-binding polypeptide and a second single chain Fc.

11. A fusion protein selected from any of the following: (1) Two first polypeptide chains, wherein the first polypeptide chains sequentially comprise: a first target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated form and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the single-chain Fc via an uncleavable linker. (2) Two first polypeptide chains, each first polypeptide chain comprising, in sequence: a light chain of a first target-binding polypeptide, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the light chain of the first target-binding polypeptide via an uncleavable linker; and two second polypeptide chains, each second polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a single-chain Fc segment, and a second target-binding polypeptide with an scFv structure; (3) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; and two second polypeptide chains, each second polypeptide chain comprising, in sequence: wild-type or mutant IL-2 protein, a truncated IL-2Rα protein, a heavy chain Fd segment of the first target-binding polypeptide, a single chain Fc segment, and a second target-binding polypeptide with an scFv structure, wherein the wild-type or mutant IL-2 protein is connected to the truncated IL-2Rα protein via a cleavable linker, and the truncated IL-2Rα protein is connected to the heavy chain of the first target-binding polypeptide via an uncleavable linker. (4) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; two second polypeptide chains, each second polypeptide chain comprising, in sequence: a second target-binding polypeptide with an scFv structure, a heavy chain Fd segment of the first target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the single-chain Fc via an uncleavable linker; (5) Two first polypeptide chains, each first polypeptide chain comprising: a light chain variable region of a second target-binding polypeptide and a light chain of the first target-binding polypeptide; two second polypeptide chains, each second polypeptide chain comprising: a heavy chain variable region of a second target-binding polypeptide, a heavy chain Fd segment of the first target-binding polypeptide, a single-chain Fc, an IL-2Rα truncated body, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated body via a cleavable linker, and the IL-2Rα truncated body is connected to the single-chain Fc via an uncleavable linker; (6) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; and two second polypeptide chains, each second polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the single-chain Fc via an uncleavable linker. (7) Two first polypeptide chains, each first polypeptide chain comprising, in sequence: a light chain of a first target-binding polypeptide, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the light chain of the first target-binding polypeptide via an uncleavable linker; and two second polypeptide chains, each second polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), and a single-chain Fc. (8) Two first polypeptide chains, each first polypeptide chain comprising, in sequence: wild-type or mutant IL-2 protein, IL-2Rα truncated form, and light chain of first target-binding polypeptide, wherein the wild-type or mutant IL-2 protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the light chain of first target-binding polypeptide via an uncleavable linker; two second polypeptide chains, each second polypeptide chain comprising, in sequence: heavy chain Fd segment of first target-binding polypeptide, second target-binding polypeptide in the form of nanobody (VHH or V-NAR), and single chain Fc; (9) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; two second polypeptide chains, each second polypeptide chain comprising, in sequence: wild-type or mutant IL-2 protein, an IL-2Rα truncated form, a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), and a single chain Fc, wherein the wild-type or mutant IL-2 protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the heavy chain Fd segment of the first target-binding polypeptide via an uncleavable linker; (10) A first polypeptide chain, comprising in sequence: a first target-binding polypeptide with an scFv structure, a single-chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the single-chain Fc via an uncleavable linker; a second polypeptide chain, comprising in sequence: a heavy chain variable region of the first target-binding polypeptide, a heavy chain Fd segment of the second target-binding polypeptide, and a single-chain Fc; a third polypeptide chain, comprising in sequence: a light chain variable region of the first target-binding polypeptide and a light chain of the second target-binding polypeptide; (11) Two first polypeptide chains, comprising: a light chain of a first target-binding polypeptide; a second polypeptide chain, comprising in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide of scFv structure, a first single chain Fc, an IL-2Rα truncated form, and an IL-2 wild-type or mutant protein, wherein the IL-2 wild-type or mutant protein is connected to the IL-2Rα truncated form via a cleavable linker, and the IL-2Rα truncated form is connected to the first single chain Fc via an uncleavable linker; a third polypeptide chain, comprising in sequence: a heavy chain Fd segment of the first target-binding polypeptide and a second single chain Fc; in, The IL-2Rα truncated body is the IL-2Rα truncated body described in any one of claims 1-3.

12. A fusion protein comprising: Half-life extenders; Interleukin-2 (IL-2) wild-type or mutant protein; Interleukin-2 receptor α (IL-2Rα) truncated variant; and Cuttable connectors; in, The IL-2Rα truncated body is the IL-2Rα truncated body described in any one of claims 1-3.

13. The fusion protein of claim 12, wherein the fusion protein comprises: (i) a first polypeptide chain, the first polypeptide chain comprising a first single-chain Fc and an IL-2Rα truncated form; and (ii) A second polypeptide chain comprising a second single-chain Fc and wild-type or mutant IL-2 protein; in, The wild-type or mutant IL-2 protein is connected to the second single-chain Fc via a cleavable linker, and the truncated IL-2Rα protein is connected to the first single-chain Fc via an uncleavable linker; or, the wild-type or mutant IL-2 protein is connected to the second single-chain Fc via an uncleavable linker, and the truncated IL-2Rα protein is connected to the first single-chain Fc via a cleavable linker.

14. The fusion protein according to claim 12 or 13, wherein the fusion protein is selected from any of the following: (1) A first polypeptide chain, the first polypeptide chain comprising, in sequence: a Fab segment of a first target-binding polypeptide, a first single-chain Fc, and an IL-2Rα truncated form; a second polypeptide chain, the second polypeptide chain comprising, in sequence: a Fab segment of the first target-binding polypeptide, a second single-chain Fc, and an IL-2 wild-type or mutant protein, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker; (2) A first polypeptide chain, comprising in sequence: a first target-binding polypeptide with an scFv structure, a first single-chain Fc, and an IL-2Rα truncated form; a second polypeptide chain, comprising in sequence: a heavy chain variable region of the first target-binding polypeptide, a heavy chain Fd segment of the second target-binding polypeptide, a second single-chain Fc, and an IL-2 wild-type or mutant protein; a third polypeptide chain, comprising in sequence: a light chain variable region of the first target-binding polypeptide and a light chain of the second target-binding polypeptide, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker; (3) Two first polypeptide chains, comprising: a light chain of a first target-binding polypeptide; a second polypeptide chain, comprising in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide with an scFv structure, a first single-chain Fc, and an IL-2Rα truncated form; and a third polypeptide chain, comprising in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second single-chain Fc, and an IL-2 wild-type or mutant protein, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker. (4) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; a second polypeptide chain, the second polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a first single-chain Fc, and an IL-2Rα truncated form; and a third polypeptide chain, the third polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a second single-chain Fc, and an IL-2 wild-type or mutant protein, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker.

15. A fusion protein selected from any of the following: (1) A first polypeptide chain, the first polypeptide chain comprising, in sequence: a Fab segment of a first target-binding polypeptide, a first single-chain Fc, and an IL-2Rα truncated form; a second polypeptide chain, the second polypeptide chain comprising, in sequence: a Fab segment of the first target-binding polypeptide, a second single-chain Fc, and an IL-2 wild-type or mutant protein, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker; (2) A first polypeptide chain, comprising in sequence: a first target-binding polypeptide with an scFv structure, a first single-chain Fc, and an IL-2Rα truncated form; a second polypeptide chain, comprising in sequence: a heavy chain variable region of the first target-binding polypeptide, a heavy chain Fd segment of the second target-binding polypeptide, a second single-chain Fc, and an IL-2 wild-type or mutant protein; a third polypeptide chain, comprising in sequence: a light chain variable region of the first target-binding polypeptide and a light chain of the second target-binding polypeptide, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker; (3) Two first polypeptide chains, comprising: a light chain of a first target-binding polypeptide; a second polypeptide chain, comprising in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide with an scFv structure, a first single-chain Fc, and an IL-2Rα truncated form; and a third polypeptide chain, comprising in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second single-chain Fc, and an IL-2 wild-type or mutant protein, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker. (4) Two first polypeptide chains, each first polypeptide chain comprising: a light chain of a first target-binding polypeptide; a second polypeptide chain, the second polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a first single-chain Fc, and an IL-2Rα truncated form; and a third polypeptide chain, the third polypeptide chain comprising, in sequence: a heavy chain Fd segment of the first target-binding polypeptide, a second target-binding polypeptide in the form of a nanobody (VHH or V-NAR), a second single-chain Fc, and an IL-2 wild-type or mutant protein, wherein the IL-2Rα truncated form is connected to the first single-chain Fc via an uncleavable linker, and the IL-2 wild-type or mutant protein is connected to the second single-chain Fc via a cleavable linker. in, The IL-2Rα truncated body is the IL-2Rα truncated body described in any one of claims 1-3.

16. The fusion protein according to any one of claims 7-15, wherein the half-life extender is selected from: (i) Antibody Fc region or human serum albumin (ii) wild-type Fc region, mutant Fc region, wild-type Fc monomer, mutant Fc monomer, wild-type Fc dimer or mutant Fc dimer, wild-type single-stranded Fc, or mutant single-stranded Fc, preferably, the mutant single-stranded Fc, mutant Fc monomer or mutant Fc dimer is a mutant single-stranded Fc, mutant Fc monomer or mutant Fc dimer modified by site-directed mutagenesis; (iii) A single-chain Fc containing an amino acid mutation, a first single-chain Fc and / or a second single-chain Fc; (iv) Single-stranded Fc, first single-stranded Fc and / or second single-stranded Fc containing Knob, Hole or LALA mutations; (v) Contains an amino acid sequence as shown in SEQ ID NO:

36.

17. The fusion protein according to any one of claims 7-9 and 12-13, wherein the fusion protein further comprises a first target-binding polypeptide and / or a second target-binding polypeptide.

18. The fusion protein of claim 17, wherein the target-binding polypeptide, the first target-binding polypeptide, and / or the second target-binding polypeptide are selected from: (i) an antibody or a functional fragment thereof; or (ii) Fab, scFv, or nanobody.

19. The fusion protein according to any one of claims 4-6, 10-11, 14-15 and 17-18, wherein the target-binding polypeptide, the first target-binding polypeptide and / or the second target-binding polypeptide are selected from T cell target-binding polypeptides and tumor cell target-binding polypeptides; Preferably, the target-binding polypeptide, the first target-binding polypeptide, and / or the second target-binding polypeptide can bind to one or more targets; Preferably, the target-binding peptide, the first target-binding peptide, and / or the second target-binding peptide are capable of binding to one of the following targets: CLDN18.2, PD-1, PD-L1, VEGFR, HER2; Preferably, the target-binding peptide, the first target-binding peptide, and / or the second target-binding peptide can bind to one of the following target combinations: PD-1 / VEGF combination, or CLDN18.2 / CD3 combination; Preferably, the target of the T cell target-binding peptide is selected from CD3 and / or PD-1, and the target of the tumor cell target-binding peptide is selected from one or more of CLDN18.2, PD-L1, VEGFR, and HER2.

20. The fusion protein according to any one of claims 5-6, 8-11, 13-15, wherein the uncleavable linker is selected from at least one of the following: (i) Not cleaved by tumor-associated proteases; (ii) is a glycine / serine linker; (iii) When the IL2 and the IL2Rα truncated form are sequentially connected, the non-cleavable linker used to connect the IL2Rα truncated form and the half-life extender or the target-binding peptide is 4 G4S; (iv) When the IL2 is connected to the first / second target-binding polypeptide or the first / second single-chain Fc, the non-cleavable linker for connecting the IL2Rα truncated form and the second / first target-binding polypeptide or the second / first single-chain Fc is 3 G4S; (v) Contains or is an amino acid sequence as shown in SEQ ID NO:

39.

21. The fusion protein according to any one of claims 4-20, wherein the cleavable linker is selected from at least one of the following: (i) It can be cleaved by tumor-associated proteases; (ii) It can be cleaved by matrix metalloproteinases; (iii) It can be cleaved by matrix metalloproteinase 14 (MMP14); (iv) It can be cleaved by caspase; (v) Can be cleaved by caspase, wherein the caspase is selected from one or more of the following: caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11 or caspase 12; (vi) Contains an amino acid sequence as shown in any one of SEQ ID NO:32-35 (vii) contains glycine / serine; (viii) When the IL2 and the IL2Rα truncated form are sequentially connected, the cleavable linker for connecting the IL2 and the half-life extender or the target-binding peptide contains 4 G4S; (ix) When the IL2 is linked to the first / second target-binding peptide or the first / second single-chain Fc, the cleavable linker for linking the IL2 and the second / first target-binding peptide or the second / first single-chain Fc contains two G4S.

22. The fusion protein according to any one of claims 4-21, wherein the wild-type or mutant IL-2 protein is selected from at least one of the following: (i) The wild-type IL-2 protein contains or is an amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO:1; (ii) Compared with the wild-type IL-2 protein, the mutant IL-2 protein contains one or more amino acid mutations; (iii) Compared with the wild-type IL-2 protein, the mutant IL-2 protein contains one or more of the N88D, R81E, and L85T / I mutations; (iv) Compared with the wild-type I-2 protein, the mutant IL-2 protein contains R81E and L85T / I mutations; (v) Compared with the wild-type IL-2 protein, the mutant IL-2 protein contains R81E, L85T / I and N88D mutations; (vi) The IL-2 mutant protein contains or has an amino acid sequence as shown in SEQ ID NO:2 or SEQ ID NO:

65.

23. The fusion protein according to any one of claims 4-22, wherein the fusion protein is selected from (i) or (ii): (i) The fusion protein comprises a first chain and a second chain, wherein the first chain and the second chain comprise the following amino acid sequences or sequences having at least 95% identity with the following amino acid sequences, wherein: The amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:40; or, the amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:41; or, the amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:42; or, the amino acid sequence of the first chain is shown in SEQ ID NO:37, and the amino acid sequence of the second chain is shown in SEQ ID NO:43; or, the amino acid sequence of the first chain is shown in SEQ ID NO:49, and the amino acid sequence of the second chain is shown in SEQ ID NO:50; or, the amino acid sequence of the first chain is shown in SEQ ID NO:53, and the amino acid sequence of the second chain is shown in SEQ ID NO:54; or, the amino acid sequence of the first chain is shown in SEQ ID NO:57, and the amino acid sequence of the second chain is shown in SEQ ID NO:58; or, the amino acid sequence of the first chain is shown in SEQ ID NO:59, and the amino acid sequence of the second chain is shown in SEQ ID NO:

50. The amino acid sequence of the first chain is shown in SEQ ID NO:60; or, the amino acid sequence of the second chain is shown in SEQ ID NO:61, and the amino acid sequence of the second chain is shown in SEQ ID NO:62; or, the amino acid sequence of the first chain is shown in SEQ ID NO:63, and the amino acid sequence of the second chain is shown in SEQ ID NO:64; or, the amino acid sequence of the first chain is shown in SEQ ID NO:93, and the amino acid sequence of the second chain is shown in SEQ ID NO:94; or, the amino acid sequence of the first chain is shown in SEQ ID NO:95, and the amino acid sequence of the second chain is shown in SEQ ID NO:96; or, the amino acid sequence of the first chain is shown in SEQ ID NO:97, and the amino acid sequence of the second chain is shown in SEQ ID NO:98; or, the amino acid sequence of the first chain is shown in SEQ ID NO:99, and the amino acid sequence of the second chain is shown in SEQ ID NO:100; or, the amino acid sequence of the first chain is shown in SEQ ID NO:101, and the amino acid sequence of the second chain is shown in SEQ ID NO:102; or, the amino acid sequence of the first chain is shown in SEQ ID NO:103, and the amino acid sequence of the second chain is shown in SEQ ID NO:

104. As shown in NO:104; or, the amino acid sequence of the first chain is shown in SEQ ID NO:105, and the amino acid sequence of the second chain is shown in SEQ ID NO:106;or the amino acid sequence of the first chain is set forth in SEQ ID NO: 107, and the amino acid sequence of the second chain is set forth in SEQ ID NO: 108; or the amino acid sequence of the first chain is set forth in SEQ ID NO: 109, and the amino acid sequence of the second chain is set forth in SEQ ID NO: 110; (ii) The fusion protein comprises a first chain, a second chain, and a third chain, wherein the first chain, the second chain, and the third chain comprise the following amino acid sequences or sequences having at least 95% identity with the following amino acid sequences, wherein: The amino acid sequence of the first chain is shown in SEQ ID NO:66, the amino acid sequence of the second chain is shown in SEQ ID NO:67, and the amino acid sequence of the third chain is shown in SEQ ID NO:68; or, the amino acid sequence of the first chain is shown in SEQ ID NO:69, the amino acid sequence of the second chain is shown in SEQ ID NO:70, and the amino acid sequence of the third chain is shown in SEQ ID NO:71; or, the amino acid sequence of the first chain is shown in SEQ ID NO:72, the amino acid sequence of the second chain is shown in SEQ ID NO:73, and the amino acid sequence of the third chain is shown in SEQ ID NO:74; or, the amino acid sequence of the first chain is shown in SEQ ID NO:75, the amino acid sequence of the second chain is shown in SEQ ID NO:76, and the amino acid sequence of the third chain is shown in SEQ ID NO:77; or, the amino acid sequence of the first chain is shown in SEQ ID NO:78, the amino acid sequence of the second chain is shown in SEQ ID NO:79, and the amino acid sequence of the third chain is shown in SEQ ID NO:80; or, the amino acid sequence of the first chain is shown in SEQ ID NO:

69. As shown in NO:81, the amino acid sequence of the second chain is as shown in SEQ ID NO:82, and the amino acid sequence of the third chain is as shown in SEQ ID NO:83; or, the amino acid sequence of the first chain is as shown in SEQ ID NO:84, the amino acid sequence of the second chain is as shown in SEQ ID NO:85, and the amino acid sequence of the third chain is as shown in SEQ ID NO:86; or, the amino acid sequence of the first chain is as shown in SEQ ID NO:87, the amino acid sequence of the second chain is as shown in SEQ ID NO:88, and the amino acid sequence of the third chain is as shown in SEQ ID NO:

89.

24. The fusion protein according to any one of claims 4-23, wherein the fusion protein is a dimer; Preferably, the fusion protein is a homodimer or a heterodimer.

25. The fusion protein according to any one of claims 4-24, wherein the fusion protein comprising the truncated form of IL-2Rα has reduced toxicity compared to the fusion protein comprising the untruncated form of IL-2Rα.

26. A polynucleotide encoding an IL-2Rα truncated form as described in any one of claims 1-3, or a fusion protein as described in any one of claims 4-25.

27. An expression vector comprising the polynucleotide as described in claim 26.

28. A host cell comprising the polynucleotide of claim 26 or the expression vector of claim 27.

29. A method for preparing an IL-2Rα truncated form or fusion protein, comprising culturing a host cell as described in claim 28 to prepare an IL-2Rα truncated form as described in any one of claims 1-3, or a fusion protein as described in any one of claims 4-25; Preferably, the method further includes purifying or isolating the produced IL-2Rα truncated form or fusion protein.

30. A pharmaceutical composition comprising the fusion protein as described in any one of claims 4-25; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, carrier, or diluent.

31. A method for treating or alleviating a disease or symptom, comprising: Administer to a patient in need a therapeutically effective amount of the fusion protein of any one of claims 4-25, the polynucleotide of claim 26, the expression vector of claim 27, the host cell of claim 28, or the pharmaceutical composition of claim 30; Preferably, the patient in need is a human being.

32. The method of claim 31, wherein the disease or symptom is cancer; Preferably, the disease or condition is selected from: acute myeloid leukemia, adrenocortical carcinoma, B-cell lymphoma, urothelial carcinoma of the bladder, ductal carcinoma of the breast, lobular carcinoma of the breast, esophageal cancer, castration-resistant prostate cancer (CRPC), cervical cancer, cholangiocarcinoma, chronic myeloid leukemia, colorectal adenocarcinoma, colorectal cancer (CRC), esophageal cancer, gastric adenocarcinoma, glioblastoma multiforme, squamous cell carcinoma of the head and neck, Hodgkin lymphoma / primary mediastinal B-cell lymphoma, hepatocellular carcinoma (HCC), kidney... Chromophobe carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, low-grade glioma, lung adenocarcinoma, squamous cell lung carcinoma, melanoma (MEL), mesothelioma, non-squamous NSCLC, ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma, paraganglioma and pheochromocytoma, prostate cancer, renal cell carcinoma (RCC), sarcoma, cutaneous melanoma, head and neck squamous cell carcinoma, T-cell lymphoma, thymoma, papillary thyroid carcinoma, uterine carcinosarcoma, endometrioid carcinoma of the uterine body or uveal melanoma.

33. Use of the fusion protein of any one of claims 4-25, the polynucleotide of claim 26, the expression vector of claim 27, the host cell of claim 28, or the pharmaceutical composition of claim 30 for the treatment or relief of a disease or condition.

34. Use of the fusion protein of any one of claims 4-25, the polynucleotide of claim 26, the expression vector of claim 27, the host cell of claim 28, or the pharmaceutical composition of claim 30 in the preparation of a medicament for treating or alleviating a disease or condition.

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