Fusion proteins and their applications

Abstract

The present invention relates to a fusion protein comprising an anti-T cell surface molecule antibody, interleukin-2, and immunoglobulin Fc. The fusion protein is a heterodimer or homodimer. The present invention further relates to the use of the fusion protein in the preparation of an antitumor drug.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedical technology, and specifically to a fusion protein consisting of a T cell antibody and interleukin-2 and its applications. [Background technology]

[0002] Cytokines play an important role in cancer immunotherapy. However, due to their extremely short half-lives, limited tumor targeting, and significant side effects, the role of natural cytokines in cancer treatment has generally been limited [1]. IL-2 is currently approved for the treatment of patients with renal carcinoma and melanoma, and is the first cytokine approved for the treatment of oncology [2-4]. More than one-third of patients with renal cell carcinoma (RCC) are diagnosed at an advanced stage, with a predicted 5-year survival rate of approximately 12% [5]. Over the past decade, several targeted therapies have been approved for metastatic RCC (mRCC) that delay cancer progression, but current treatments still fail to induce complete responses (CRs) in most patients [6]. Due to the well-known immunogenicity of RCC, IL-2 immunotherapy is the only treatment known to induce CRs with durable efficacy in mRCC [7]. However, CRs are achieved in only 7%–9% of patients [4]. IL-2 is known to cause severe short-term toxicity, and all patients treated with IL-2 must be hospitalized for observation [8, 9]. Therefore, improving the therapeutic function of IL-2 has become a major challenge in cancer treatment. Current IL-2 therapy has several major problems: 1) limited targeting of tumor tissue, especially tumor-specific T cells; 2) severe side effects due to targeting of lymphoid tissue and endothelium; 3) preferential activation of Tregs, limiting CTL-mediated antitumor effects; and 4) a short half-life (<30 min) due to its small volume and targeting of lymphoid tissues. Summary of the Invention [Problem to be solved by the invention]

[0003] Highly immunogenic tumor tissues typically contain large numbers of T cells, but their dysfunction limits their ability to control tumors. PD1+ and TIM3+ TILs are considered terminally differentiated and dysfunctional TILs

[10] . Anti-PD1 / PDL1 therapy can release the brakes on T cell responses and partially restore their function, but only a minority of patients achieve complete responses

[11] . We have observed that cytokines play an important role in PD1 antibody therapy

[12] . This raises the possibility that targeting TILs with cytokines could overcome PD1 resistance. Most studies have focused on targeting cytokines to tumor cells [13-15]. While this increases cytokine retention in tumor tissues, the accessibility of these cytokines to effector T cells remains limited. It is unclear whether tumor cells or stromal cells are the most ideal targets for T cell-associated cytokines, as most tumor cells are not in contact with T cells and cytokine endocytosis by tumor cells may reduce TIL accessibility to cytokines. However, due to the high expression of cytokine receptors on Treg cells, it is challenging to deliver sufficient cytokines to CD8+ T cells within the tumor while avoiding systemic toxicity. [Means for solving the problem]

[0004] (Summary of the Invention) To target intratumor tumor-specific CD8+ T cells rather than Tregs, novel fusion proteins are designed by linking cytokines (preferably low-affinity cytokines) to antibodies (e.g., anti-TIM3 / anti-LAG3 antibodies). The low binding affinity of the novel fusion proteins to peripheral CD8+ T cells and Treg cells significantly reduces peripheral consumption and toxic side effects. The novel fusion protein therapy primarily targets intratumor CD8+ T cells rather than Treg cells. For example, if the CD8+ T cells are highly PD1 / TIM3 expressing, the novel fusion protein can reactivate tumor-specific PD1+TIM3+CD8+ TILs and generate long-term memory responses to prevent recurrence.

[0005] Specifically, the present invention first relates to a fusion protein, wherein the fusion protein is a heterodimer, and the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by IF, FI, AIF, AFI, IAF, IFA, FIA, or FAI; (2) a second monomer, the second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by AF, FA, AIF, AFI, IAF, IFA, FIA, or FAI, wherein the first monomer and the second monomer are different, where I represents interleukin 2 (IL2), F represents immunoglobulin Fc, and A represents an anti-T cell surface molecule antibody; the anti-T cell surface molecule antibody is in Fab or scFv form; The first monomer and the second monomer are connected by dimerization of a single Fc chain to form the heterodimer.

[0006] In some embodiments, the heterodimer includes the following combination of first and second monomers: In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by IF; (2) a second monomer, The second monomer includes second monomers represented by A-F from the N-terminus to the C-terminus.

[0007] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by IF; (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by FA.

[0008] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by IF; (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by AIF.

[0009] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by IF; (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by IAF.

[0010] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by IF; (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by FIA.

[0011] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by IF; (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by FAI.

[0012] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by FI, (2) a second monomer, The second monomer includes second monomers represented by A-F from the N-terminus to the C-terminus.

[0013] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by FI, (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by FA.

[0014] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by FI, (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by AIF.

[0015] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by FI, (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by IAF.

[0016] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by FI, (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by FIA.

[0017] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by FI, (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by FAI.

[0018] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by AIF; (2) a second monomer, The second monomer includes second monomers represented by A-F from the N-terminus to the C-terminus.

[0019] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by AIF; (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by FA.

[0020] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by AIF; (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by IAF.

[0021] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by AIF; (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by FIA.

[0022] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by AIF; (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by FAI.

[0023] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by IAF; (2) a second monomer, The second monomer includes second monomers represented by A-F from the N-terminus to the C-terminus.

[0024] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by IAF; (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by FA.

[0025] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by IAF; (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by AIF.

[0026] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by IAF; (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by FIA.

[0027] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by IAF; (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by FAI.

[0028] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by FIA, (2) a second monomer, The second monomer includes second monomers represented by A-F from the N-terminus to the C-terminus.

[0029] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by FIA, (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by FA.

[0030] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by FIA, (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by AIF.

[0031] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by FIA, (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by IAF.

[0032] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by FIA, (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by FAI.

[0033] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by FAI; (2) a second monomer, The second monomer includes second monomers represented by A-F from the N-terminus to the C-terminus.

[0034] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by FAI; (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by FA.

[0035] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by FAI; (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by AIF.

[0036] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by FAI; (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by IAF.

[0037] In some embodiments, the heterodimer comprises: (1) a first monomer, The first monomer comprises, from the N-terminus to the C-terminus, a first monomer represented by FAI; (2) a second monomer, The second monomer includes, from the N-terminus to the C-terminus, a second monomer represented by FIA.

[0038] Preferably, the immunoglobulin Fc is a native immunoglobulin Fc or an immunoglobulin Fc in which the ADCC effect has been knocked out by genetic mutation, and more preferably, the immunoglobulin Fc is a single Fc chain of human IgG (preferably human IgG1, human IgG2, human IgG3, or human IgG4), and preferably, the amino acid sequence of the immunoglobulin Fc in which the ADCC effect has been knocked out by genetic mutation is shown in SEQ ID NO: 2.

[0039] In some embodiments, when the anti-T cell surface molecule antibody is in scFv form, the interleukin-2 is linked to the N-terminus of the scFv, or when the anti-T cell surface molecule antibody is in Fab form, the interleukin-2 is linked to the N-terminus of CH1 or CL in the Fab, or the interleukin-2 is linked to the C-terminus of CL in the Fab.

[0040] The present invention further relates to a fusion protein, wherein the fusion protein is a homodimer, and the monomers of the homodimer comprise, from the N-terminus to the C-terminus, IFA, AFI, IAF, AIF, FIA, and FAI, wherein I represents interleukin-2, F represents immunoglobulin Fc, and A represents an anti-T cell surface molecule, and the anti-T cell surface molecule antibody is in the form of Fab or scFv, and the monomers are connected by dimerization of a single Fc chain to form the homodimer.

[0041] In some embodiments, when the anti-T cell surface molecule antibody is in scFv form, the interleukin-2 is attached to the N-terminus or C-terminus of the scFv, or when the anti-T cell surface molecule antibody is in Fab form, the interleukin-2 is attached to the N-terminus of CH1 or CL in the Fab, or the interleukin-2 is attached to the C-terminus of CL in the Fab.

[0042] In some embodiments, the interleukin-2, the anti-T cell surface molecule antibody, or the immunoglobulin Fc are connected directly or via a linker, preferably selected from (G4S)n, where n=0, 1, or 2.

[0043] In some embodiments, the T cell surface molecule includes, but is not limited to, TIM3, LAG3, PD1, ICOS, GITR, CD27, CD137, 2B4, OX40, 4-1BB, TIGIT, CTLA-4, VISTA, and CD8. Preferably, the T cell surface molecule is TIM3 or LAG3, and the anti-T cell surface molecule antibody is an anti-TIM3 antibody (aTIM3) or an anti-LAG3 antibody (aLAG3).

[0044] In some embodiments, the anti-T cell surface molecule antibody is a humanized antibody or a fully humanized antibody.

[0045] In some embodiments, the Interleukin-2 sequence is set forth in SEQ ID NO: 1 or a variant of the sequence set forth in SEQ ID NO: 1, and preferably the variant includes any one or any combination of the following mutation sites: R38L, F42A, H16K, D20K, R38A, F42K, and K43E.

[0046] In some embodiments, the heterodimer includes the following monomers: (1) A first monomer, the first monomer being, in order from the N-terminus, i) a wild-type IL-2 protein whose sequence is set forth in SEQ ID NO: 1 or a mutant of said wild-type IL-2 protein, preferably a mutant of said wild-type IL-2 protein comprising any one or any combination of mutation sites of R38L, F42A, H16K, D20K, R38A, F42K and K43E; ii) the necessary connector structure (such as the (G4S)n connector sequence), preferably the connector sequence of which is set forth in SEQ ID NO: 6; iii) comprising an Fc of an IgG having the sequence set forth in SEQ ID NO: 2, or an Fc of a No-ADCC mutant IgG having the sequence set forth in SEQ ID NO: 3, or a knob mutant Fc having the sequence set forth in SEQ ID NO: 4, or a hole mutant Fc having the sequence set forth in SEQ ID NO: 5; (2) A second monomer, wherein the second monomer is selected from the group consisting of: i) the second monomer is a) an anti-TIM3 antibody Fab region consisting of a polypeptide (anti-TIM3 antibody VL-CL) whose sequence is set forth in SEQ ID NO: 7 and a polypeptide (anti-TIM3 antibody VH-CH1) whose sequence is set forth in SEQ ID NO: 8, or an anti-TIM3 single-chain antibody (ScFv) whose sequence is set forth in SEQ ID NO: 9; b) an Fc of an IgG having the sequence set forth in SEQ ID NO: 2, or an Fc of a No-ADCC mutant IgG having the sequence set forth in SEQ ID NO: 3, or an Fc of a knob mutant having the sequence set forth in SEQ ID NO: 4, or an Fc of a hole mutant having the sequence set forth in SEQ ID NO: 5; ii) the second monomer is a) an antibody Fab region consisting of a polypeptide having the sequence shown in SEQ ID NO: 13 (anti-LAG3 antibody VL-CL) and a polypeptide having the sequence shown in SEQ ID NO: 14 (anti-LAG3 antibody VH-CH1), or a polypeptide having the sequence shown in SEQ ID NO: 15 (anti-LAG3 scFv); b) an Fc of an IgG having the sequence set forth in SEQ ID NO: 2, or an Fc of a No-ADCC mutant IgG having the sequence set forth in SEQ ID NO: 3, or an Fc of a knob mutant having the sequence set forth in SEQ ID NO: 4, or an Fc of a hole mutant having the sequence set forth in SEQ ID NO: 5; iii) the second monomer is a) a Fab construct of an anti-LAG3 antibody consisting of a polypeptide (aLAG3 VH-CH1-Fc(knob)) whose sequence is set forth in SEQ ID NO: 16 and a polypeptide (aLAG3 VL-CL) whose sequence is set forth in SEQ ID NO: 13; or b) It comprises a polypeptide whose sequence is set forth in SEQ ID NO: 17 or SEQ ID NO: 22 (aLAG3 ScFv-Fc(knob)).

[0047] In some embodiments, the heterodimer comprises: a first monomer which is a polypeptide (laIL2-Fc) whose sequence is set forth in SEQ ID NO: 10; (1) a second monomer consisting of a polypeptide having the sequence set forth in SEQ ID NO: 11 (anti-TIM3 antibody VH-CH1-Fc) and a polypeptide having the sequence set forth in SEQ ID NO: 7 (anti-TIM3 antibody light chain VL-CL); or (2) a second monomer which is a polypeptide having the sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 22 (aTIM3 ScFv-Fc).

[0048] In some embodiments, the monomers of the homodimer are, in order from the N-terminus: (1) a wild-type IL-2 protein having a sequence set forth in SEQ ID NO: 1, or a mutant of the wild-type IL-2 protein containing any one or any combination of mutation sites selected from R38L, F42A, H16K, D20K, R38A, F42K, and K43E; (2) Preferably, the necessary connecting structure (such as the (G4S)n connecting sequence) whose connecting sequence is set forth in SEQ ID NO: 6; (3) Fab or ScFv of an anti-TIM3 antibody that is a humanized antibody or a fully humanized antibody; (4) An antibody Fc that is a fully human wild-type Fc or a No-ADCC mutant Fc.

[0049] In some embodiments, the monomers of the homodimer are: (1) a polypeptide shown in SEQ ID NO: 18 (TIM3-InFc-laIL2: aTIM3(ScFv)-InFc-laIL2), or (2) a polypeptide shown in SEQ ID NO: 19 (laIL2-InFc-TIM3: laIL2-Fc-aTIM3(ScFv)), or (3) a polypeptide represented by SEQ ID NO: 20 (TIM3-laIL2-InFc:aTIM3(ScFv)-Fc-laIL2), or (4) A polypeptide shown in SEQ ID NO: 21 (laIL2-TIM3-InFc: laIL2-aTIM3(ScFv)-Fc).

[0050] In some embodiments, the present invention relates to yet another fusion protein, wherein the fusion protein combines an anti-LAG3 antibody with IL2, wherein the fusion protein is a heterodimer, and the heterodimer includes the following monomers: (1) A first monomer, the first monomer being, in order from the N-terminus, 1) a wild-type IL-2 protein having a sequence set forth in SEQ ID NO: 1, or a mutant of the wild-type IL-2 protein containing any one or any combination of mutation sites selected from R38L, F42A, D20K, R38A, F42K, and K43E; 2) Preferably, the necessary connecting structure (such as the (G4S)n connecting sequence) whose connecting sequence is set forth in SEQ ID NO: 6; 3) Fc of IgG shown in SEQ ID NO: 2, or Fc of No-ADCC mutant IgG shown in SEQ ID NO: 3, or knob mutant Fc shown in SEQ ID NO: 4, or hole mutant Fc shown in SEQ ID NO: 5; (2) A second monomer, the second monomer comprising: i) an antibody Fab region consisting of a polypeptide (anti-LAG3 antibody light chain VL-CL) whose sequence is set forth in SEQ ID NO: 13 and a polypeptide (anti-LAG3 antibody heavy chain VH-CH1) whose sequence is set forth in SEQ ID NO: 14, or a polypeptide (anti-LAG3 scFv) whose sequence is set forth in SEQ ID NO: 15; ii) The Fc of an IgG having the sequence shown in SEQ ID NO: 2, or the Fc of a No-ADCC mutant IgG having the sequence shown in SEQ ID NO: 3, or the knob mutant Fc having the sequence shown in SEQ ID NO: 4, or the hole mutant Fc having the sequence shown in SEQ ID NO: 5.

[0051] In some embodiments, the heterodimer comprises: a first monomer which is the polypeptide set forth in SEQ ID NO: 10 (laIL2-Fc); (1) a second monomer consisting of a polypeptide having the sequence set forth in SEQ ID NO: 16 (aLAG3 VH-CH1-Fc) and a polypeptide having the sequence set forth in SEQ ID NO: 13 (anti-LAG3 antibody light chain VL-CL); or (2) a second monomer that is the polypeptide (aLAG3 scFv-Fc) whose sequence is set forth in SEQ ID NO: 17.

[0052] The present invention further relates to a fusion protein, said fusion protein being a homodimer, wherein the monomers of said homodimer are A monomer in which one molecule of interleukin 2 (IL2), one single Fc chain, and one molecule of anti-TIM3 antibody or anti-LAG3 antibody Fab are linked directly or via a linker, or It is characterized by being a monomer in which one molecule of interleukin 2 (IL2), one single Fc chain, and one molecule of anti-TIM3 antibody or anti-LAG3 single chain antibody (scFv) are linked directly or via a linker.

[0053] In some embodiments, the monomers of the homodimer are, in order from the N-terminus: (1) a wild-type IL-2 protein having a sequence set forth in SEQ ID NO: 1, or a mutant of the wild-type IL-2 protein containing any one or any combination of mutation sites selected from R38L, F42A, H16K, D20K, R38A, F42K, and K43E; (2) Preferably, the necessary connecting structure (such as the (G4S)n connecting sequence) whose connecting sequence is set forth in SEQ ID NO: 6; (3) Fab or scFv of an anti-LAG3 antibody that is a humanized antibody or a fully humanized antibody; (4) An antibody Fc that is a fully human wild-type Fc or a No-ADCC mutant Fc.

[0054] In some embodiments, the monomers of the homodimer are: (1) a polypeptide shown in SEQ ID NO: 22 (LAG3-InFc-laIL2: aLAG3(ScFv)-InFc-laIL2), or (2) a polypeptide represented by SEQ ID NO: 23 (laIL2-InFc-LAG3: laIL2-Fc-aLAG3(ScFv)), or (3) a polypeptide represented by SEQ ID NO: 24 (LAG3-laIL2-InFc:aLAG3(ScFv)-Fc-laIL2); or (4) A polypeptide shown in SEQ ID NO: 25 (laIL2-LAG3-InFc: laIL2-aLAG3(ScFv)-Fc).

[0055] The present invention further relates to the following applications of said fusion protein: (1) Preparation of antitumor drugs; (2) Preparation of an antitumor kit containing T cells for immune checkpoint inhibitor or T cell adoptive transfer therapy; (3) Preparation of antitumor drugs to overcome resistance to immune checkpoint inhibitors; (4) Preparation of antitumor drugs to overcome non-response to T cell adoptive transfer therapy; Preferably, the immune checkpoint inhibitors are anti-PD-L1 antibody, anti-PD1 antibody and anti-CTLA4 antibody; Preferably, the T cells are anti-tumor T cells, more preferably, the T cells are anti-tumor CAR T cells or structural analogs thereof.

[0056] Another aspect of the present invention further relates to a kit comprising the above-described fusion protein of the present invention, and preferably further comprising one or more anti-tumor agents selected from the group consisting of an anti-PD-L1 antibody, an anti-PD1 antibody, an anti-TIGIT antibody, and a chemotherapeutic agent, preferably a tyrosine kinase inhibitor, nitrogen mustard, doxorubicin, Pts, or paclitaxel.

[0057] Yet another aspect of the present invention relates to the first or second monomer of said fusion protein, or to mRNA or DNA encoding the monomers of said fusion protein.

[0058] In specific embodiments, the tumor is selected from lung cancer, colorectal cancer, gastric cancer, melanoma, renal cancer, lymphoma such as B-cell lymphoma, prostate cancer, thyroid cancer, breast cancer, pancreatic cancer, liver cancer, cervical cancer, ovarian cancer, or esophageal cancer.

[0059] The structure of an exemplary fusion protein of the invention is shown in FIG. [Brief explanation of the drawings]

[0060] [Figure 1] LAG3-laIL2 has lower affinity than LAG3-IL2 and peripheral Tregs. [Figure 2] TIM3-laIL2 has lower affinity than TIM3-IL2 and peripheral Tregs. [Figure 3] TIM3-laIL2 can bind to intratumoral CD8 cells. [Figure 4] LAG3-laIL2 can bind to intratumoral CD8 cells. [Figure 5] TIM3-laIL2 can effectively control the growth of MC38 tumors. [Figure 6] LAG3-laIL2 can effectively control the growth of MC38 tumors. [Figure 7] The antitumor effect of TIM3-laIL2 is significantly superior to that of combined treatment with Anti-TIM3 and laIL2. [Figure 8] The antitumor effect of LAG3-laIL2 is significantly superior to that of the combined treatment of Anti-LAG3 and laIL2. [Figure 9] The antitumor effect of TIM3-laIL2 is independent of CD4 T cells. [Figure 10] The antitumor effect of TIM3-laIL2 is dependent on CD8 T cells. [Figure 11] TIM3-laIL2 can cooperate with anti-PDL1 to control tumors. [Figure 12] LAG3-laIL2 can cooperate with anti-PDL1 to control tumors. [Figure 13] LAG3-InFc-laIL2 can effectively control the growth of MC38 tumors. [Figure 14] LAG3-InFc-laIL2 can effectively control the growth of CFPAC1 tumors. [Figure 15] 1 is a structural diagram of an exemplary fusion protein, where A is a heterodimeric form and B is a homodimeric form. DETAILED DESCRIPTION OF THE INVENTION

[0061] Experimental materials 1. Bacterial species and plasmids Bacterial species: Top10 E.coli, DH5α E.coli competent cells (Beijing Quankin Biotechnology Co., Ltd.) Plasmids: pEE6.4-IgGκ-hlgG1 (Lonza) containing the mouse IgGκ signal peptide and human IgG1 Fc sequence was used to express the antibody.

[0062] pEE6.4-IgGκ-hlgG1-Fc-hole and pEE6.4-IgGκ-hlgG1-Fc-knob were used to express heterodimeric proteins.

[0063] pEE6.4-TIM3 VH-CH1-Fc-knob and pEE6.4-TIM3 VL-CL were used to express the TIM3 antibody portion of the heterodimeric protein, and pEE6.4-laIL2-Fc-hole was used to express the laIL2 portion of the heterodimeric protein.

[0064] 2. Experimental Animals Wild-type C57BL / 6 and BALB / c mice were purchased from Beijing Weitong Lihua Experimental Animal Center, China, and NCG mice were purchased from Jisui Yaokang Biotechnology Co., Ltd. Unless otherwise specified, 8- to 10-week-old mice were used in all experiments. All mice were housed in a specific pathogen-free (SPF) barrier environment.

[0065] 3. Cell lines MC38 (ATCC) is a C57 background murine colorectal cancer cell line cultured in DMEM complete medium (containing 10% inactivated fetal bovine serum, 2 mmol / l L-glutamine, 0.1 mmol / l non-essential amino acids, 100 U penicillin, and 100 μg / mL streptomycin).

[0066] A20 (ATCC) is a BALB / c background murine B-cell lymphoma cell line cultured in RPMI1640 complete medium (containing 10% inactivated fetal bovine serum, 2 mmol / L L-glutamine, 0.1 mmol / L non-essential amino acids, 100 U penicillin, and 100 μg / mL streptomycin).

[0067] FreeStyle (商標) The 293F cell line (Invitrogen) is a suspension cell derived from the HEK293 cell line, and is also available as SMM293-TII or CD OptiCHO (商標) In culture, it is mainly used for transient transfection to express fusion proteins.

[0068] The IL2 gene sequence containing the R38L and F42A mutations is shown in SEQ ID NO: 32 and is designated laIL2.

[0069] The proteins used in the experiments were designed using SnapGene software and synthesized by Tongyong Biological Company.

[0070] The following are proteins used in the examples, where the anti-TIM3 antibody is in Fab form and the anti-LAG3 antibody is in scFv form, specifically as follows: 1. TIM3-laIL2 is composed of the following first and second monomers: (1) a polypeptide comprising an IL2 fusion polypeptide and an Fc fragment, the polypeptide comprising the R38L and F42A mutations, whose sequence is set forth in SEQ ID NO: 10; (2) A second monomer consisting of aTIM3-VL-CL shown in SEQ ID NO: 26 and aTIM3-VH-CH1-InFc(hole) shown in SEQ ID NO: 27.

[0071] 2. TIM3-IL2 is composed of the following first and second monomers: (1) a polypeptide obtained by fusing wild-type IL2 and an Fc fragment, the sequence of which is set forth in SEQ ID NO: 31; (2) A second monomer consisting of aTIM3-VL-CL shown in SEQ ID NO: 26 and aTIM3-VH-CH1-InFc(hole) shown in SEQ ID NO: 27.

[0072] 3. LAG3-laIL2 is composed of the following first and second monomers: (1) a polypeptide comprising an IL2 fusion polypeptide containing R38L and F42A mutations and an Fc fragment, the sequence of which is set forth in SEQ ID NO: 10; (2) The second monomer of aLAG3 shown in SEQ ID NO: 17.

[0073] 4. LAG3-IL2 is composed of the following first and second monomers: (1) a polypeptide obtained by fusing wild-type IL2 and an Fc fragment, the sequence of which is set forth in SEQ ID NO: 31; (2) The second monomer of aLAG3 shown in SEQ ID NO: 17.

[0074] 5. PD1-laIL2 is composed of the following first and second monomers: (1) a polypeptide comprising an IL2 fusion polypeptide containing R38L and F42A mutations and an Fc fragment, the sequence of which is set forth in SEQ ID NO: 10; (2) The second monomer of aPD1-ScFv-Fc(hole) shown in SEQ ID NO: 28.

[0075] 6. hTIM3-laIL2 is composed of the following first and second monomers: (1) a polypeptide comprising an IL2 fusion polypeptide containing R38L and F42A mutations and an Fc fragment, the sequence of which is set forth in SEQ ID NO: 10; (2) The second monomer of aTIM3 shown in SEQ ID NO: 12.

[0076] 7. hLAG3-laIL2 is composed of the following first and second monomers: (1) a polypeptide comprising an IL2 fusion polypeptide containing R38L and F42A mutations and an Fc fragment, the sequence of which is set forth in SEQ ID NO: 10; (2) The second monomer of aLAG3 shown in SEQ ID NO: 17.

[0077] 8. hPD1-laIL2 is composed of the following first and second monomers: (1) a polypeptide comprising an IL2 fusion polypeptide containing R38L and F42A mutations and an Fc fragment, the sequence of which is set forth in SEQ ID NO: 10; (2) The second monomer of ahPD1-ScFv-Fc(hole) shown in SEQ ID NO: 29.

[0078] 9. The aTIM3 antibody is composed of aTIM3-VL-CL shown in SEQ ID NO: 26 and aTIM3-VH-CH1-Fc shown in SEQ ID NO: 30.

[0079] 10. The aPDL1 antibody is the commercialized atezolizumab.

[0080] 11. LAG3-InFc-laIL2 is composed of the sequence shown in SEQ ID NO: 22.

[0081] Mouse tumor inoculation and treatment (1) Tumor inoculation and measurement: Establishment of tumor model: 1 × 10 6 MC38 single cells were suspended in 100 μl of PBS and inoculated subcutaneously into the back of C57BL / 6 mice. 6 A20 single cells were suspended in 100 μl of PBS and subcutaneously inoculated into the back of BALB / c mice at a concentration of 1 × 10 6 Single CFPAC1 cells were suspended in 100 μl of PBS and inoculated subcutaneously into the backs of NCG mice. When tumors were re-challenged with the same type of tumor cells, the number of tumor cells inoculated was five times higher than that used in the initial tumor modeling experiment, and the inoculation site was subcutaneous on the other side of the mouse's back.

[0082] The tumor size was monitored twice a week, and the major axis (a), minor axis (b), and height (c) of the tumor were measured using a vernier caliper, and the tumor volume of the mouse was calculated as a×b×c / 2.

[0083] (2) Treatment: The antibody or fusion protein is administered by intraperitoneal injection, and specific dosages are given in the Examples.

[0084] Depletion of CD4+ and CD8+ T cells in mice CD4+ and CD8+ T cells were depleted by intraperitoneal injection of 200 μg GK1.5 or TIB210 antibody (BioXCell) on the day before IL2 or IL2 fusion protein treatment, followed by injection every 3 days, with the number of injections adjusted according to the treatment cycle. Depletion efficiency was monitored by streaming analysis.

[0085] Example 1. TIM3-laIL2 and LAG3-laIL2 have extremely low peripheral binding, which prevents peripheral side effects. To reduce IL2 toxicity, we reduced the binding capacity of IL2 to IL2Rα and IL2Rβ. We then examined the binding ability of low-affinity IL2 (laIL2) fused with different antibodies to peripheral and intratumoral T cells. Single-cell spleen suspensions were stained with 1 μg / ml of each protein. As shown in Figure 1, LAG3-laIL2 had lower affinity for peripheral splenic Tregs than LAG3-IL2. This may reduce LAG3-laIL2 consumption by peripheral Treg cells. As shown in Figure 2, TIM3-laIL2 had lower affinity for peripheral splenic Tregs than TIM3-IL2. This may reduce TIM3-laIL2 consumption by peripheral Treg cells.

[0086] We also detected the binding of LAG3-laIL2 and TIM3-laIL2 to intratumoral T cells by adding 1 μg / ml of each protein to tumor single-cell suspensions and staining. LAG3-laIL2 and TIM3-laIL2 effectively bound to intratumoral T cells (Figures 3 and 4).

[0087] Example 2. TIM3-laIL2 fusion protein and LAG3-laIL2 fusion protein have superior antitumor activity

[0088] 1. TIM3-laIL2 fusion protein effectively controls MC38 tumors 1 × 10 C57BL / 6 mice 6 MC38 tumor cells were subcutaneously inoculated, and treatment was initiated 13 days after tumor inoculation by intraperitoneal injection of 30 μg of TIM3-laIL2 antibody protein. Tumor size was measured twice a week, and the results showed that the TIM3-laIL2 fusion protein had excellent therapeutic effects (Figure 5).

[0089] 2. The LAG3-laIL2 fusion protein effectively controls MC38 tumors. 1 × 10 C57BL / 6 mice 6 MC38 tumor cells were inoculated subcutaneously, and treatment was initiated 13 days after tumor inoculation by intraperitoneal injection of 30 μg of LAG3-laIL2 antibody protein. Tumor size was measured twice weekly, and the results showed that the LAG3-laIL2 fusion protein had excellent therapeutic effects (Figure 6).

[0090] 3.A20 TIM3-laIL2 fusion protein is significantly more effective than combination therapy in B-cell lymphoma 1.5 × 10 6 A20 tumor cells were subcutaneously inoculated, and treatment was initiated 16 days after tumor inoculation by intraperitoneal injection of 15 μg aTIM3 antibody and 30 μg ahTIM3-laIL2 or 30 μg TIM3-laIL2 antibody protein. Tumor size was measured twice weekly, and the results showed that the TIM3-laIL2 fusion protein had a superior therapeutic effect compared to the combination treatment of aTIM3 antibody and laIL2 (Figure 7).

[0091] 4. In colorectal cancer tumors, the LAG3-laIL2 fusion protein is significantly more effective than monotherapy. 1 × 10 C57BL / 6 mice 6 MC38 tumor cells were subcutaneously inoculated, and treatment was initiated 15 days after tumor inoculation with intraperitoneal injection of 15 μg aLAG3 antibody and 30 μg ahLAG3-laIL2 or 30 μg LAG3-laIL2 antibody protein. Tumor size was measured twice weekly, and the results showed that the LAG3-laIL2 fusion protein had a superior therapeutic effect compared to the combination treatment of aLAG3 antibody and laIL2 (Figure 8).

[0092] Example 3. TIM3-laIL2 can activate CD8 T cells 1. The therapeutic effect of the TIM3-laIL2 fusion protein is independent of CD4 cells To determine which immune cell populations are primarily responsible for TIM3-laIL2 antibody treatment, we performed depletion experiments of different cell populations.

[0093] 1.5 × 10 6 A20 tumor cells were subcutaneously inoculated, and 30 μg of TIM3-laIL2 fusion protein was injected intraperitoneally on day 15. 200 μg of a CD4 T cell-depleting antibody (clone number: GK1.5, purchased from BioXCell) was injected intraperitoneally once every 4 days, for a total of three times, one day before the start of treatment.

[0094] In mouse tumor experiments, the TIM3-laIL2 antibody still had therapeutic efficacy even after CD4 cell depletion, indicating that CD4 cells are not the primary effector cells for the antibody's therapeutic effect (Figure 9).

[0095] 2. The therapeutic effect of TIM3-laIL2 antibody is dependent on CD8 T cells Furthermore, we investigated the role of CD8 T cells in antibody therapy. BALB / c mice were treated with 1.5 × 10 6After subcutaneous inoculation of A20 tumor cells, treatment was initiated on day 15 with intraperitoneal injection of 30 μg TIM3-laIL2 protein. One day before the start of treatment, 200 μg of a CD8 T cell-depleting antibody (clone number: TIB210, purchased from BioXCell) was intraperitoneally injected once every four days for a total of three times.

[0096] CD8 T cell depletion experiments showed that the therapeutic effect was significantly reduced after CD8 T cell depletion, indicating that the therapeutic effect of the TIM3-laIL2 antibody is primarily dependent on CD8 T cells (Figure 10).

[0097] Example 4. TIM3-laIL2 and LAG3-laIL2 can overcome non-response to anti-PD-L1 antibody treatment MC38 tumor-bearing mice had tumors of 150 mm 3 When tumors were smaller than 100 μg / mL, single-agent TIM3-laIL2 or LAG3-laIL2 antibody treatments had excellent tumor clearance effects (Figures 7 and 8). However, when tumors were larger, single-agent treatments only controlled tumor growth and failed to completely clear the tumor (Figure 11). To further enhance the therapeutic effect, we investigated whether combining the fusion protein with an immune checkpoint inhibitor antibody could enhance the therapeutic effect.

[0098] Specific experimental protocol: 1 x 10 C57BL / 6 mice 6 MC38 tumor cells were inoculated subcutaneously, and treatment began 19 days after tumor inoculation with intraperitoneal injection of 100 μg anti-PD-L1 antibody or / and 30 μg TIM3-laIL2 antibody protein. Tumor size was measured twice weekly, and the results showed that the combination treatment had a superior therapeutic effect compared to anti-PD-L1 antibody or TIM3-laIL2 fusion protein treatment alone (Figure 11).

[0099] 1 × 10 C57BL / 6 mice 6MC38 tumor cells were inoculated subcutaneously, and treatment began 19 days after tumor inoculation with intraperitoneal injection of 100 μg anti-PD-L1 antibody or / and 30 μg LAG3-laIL2 antibody protein. Tumor size was measured twice weekly, and the results showed that the combination treatment had a superior therapeutic effect compared to anti-PD-L1 antibody or LAG3-laIL2 fusion protein treatment alone (Figure 12).

[0100] Example 4. LAG3-InFc-laIL2 fusion protein has superior antitumor activity

[0101] 1. In MC38 colorectal cancer tumors, the LAG3-InFc-laIL2 fusion protein is significantly more effective than combination therapy. 1 × 10 C57BL / 6 mice 6 MC38 tumor cells were inoculated subcutaneously, and treatment was performed on days 9 and 12 after tumor inoculation by intraperitoneal injection of 15 μg aLAG3 antibody and 15 μg laIL2-Fc or 30 μg LAG3-InFc-laIL2 antibody protein, respectively. Tumor size was measured twice a week, and the results showed that the LAG3-laIL2 fusion protein had a superior therapeutic effect compared to the combination treatment of aLAG3 antibody and laIL2 (Figure 13).

[0102] 2. In human CFPAC1 tumors, the TIM3-InFc-laIL2 fusion protein is significantly more effective than combination therapy. 1 × 10 cells into NCG mice 6 CFPAC1 (ATCC) tumor cells were subcutaneously inoculated, and 1 × 10 6 PBMCs were injected intravenously. Treatment was performed on days 17 and 20, with 15 μg aLAG3 antibody and 15 μg laIL2 or 30 μg LAG3-InFc-laIL2 antibody protein injected intraperitoneally, respectively. Tumor size was measured twice weekly, and the results showed that the LAG3-InFc-laIL2 fusion protein had a superior therapeutic effect compared to the combination treatment (Figure 14).

[0103] The above examples are only intended to help those skilled in the art understand the essence of the present invention, and are not intended to limit the protection scope of the present invention.

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Fishman, M., et al., Overall survival by clinical risk category for high dose interleukin-2 (HD IL-2) treated patients with metastatic renal cell cancer (mRCC): data from the PROCLAIM(SM) registry. J Immunother Cancer, 2019. 7(1): p. 84. 8. Acquavella, N., et al., Toxicity and activity of a twice daily high-dose bolus interleukin 2 regimen in patients with metastatic melanoma and metastatic renal cell cancer. J Immunother, 2008. 31(6): p. 569-76. 9. Alwan, L.M., et al., Comparison of acute toxicity and mortality after two different dosing regimens of high-dose interleukin-2 for patients with metastatic melanoma. Target Oncol, 2014. 9(1): p. 63-71. 10. Apetoh, L., et al., Consensus nomenclature for CD8(+) T cell phenotypes in cancer. Oncoimmunology, 2015. 4(4): p. e998538. 11. Vesely, M.D., T. Zhang, and L. Chen, Resistance Mechanisms to Anti-PD Cancer Immunotherapy. Annu Rev Immunol, 2022. 40: p. 45-74. 12. 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Sequence Listing Wild-type IL-2 (SEQ ID NO:1) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT Human IgG Fc single chain (SEQ ID NO:2) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Fc of IgG with No-ADCC mutation (SEQ ID NO:3) KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK knob mutant Fc (SEQ ID NO:4) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSALTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hole mutant Fc (SEQ ID NO:5) DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFKLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK G4S linker sequence (SEQ ID NO:6) GGGGS Anti-TIM3 antibody VL-CL (SEQ ID NO:7) AIQLTQSPSSLSASVGDRVTITCRASESVEYYGTSLMQWYQQKPGKAPKLLIYAASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQSRKDPSTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Anti-TIM3 antibody VH-CH1 (SEQ ID NO:8) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGDIYPGNGDTSYNQKFKGRVTITADKSTSTVYMELSSLRSEDTAVYYCARVGGAFPMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC TIM3 single-chain antibody (ScFv) (SEQ ID NO:9) AIQLTQSPSSLSASVGDRVTITCRASESVEYYGTSLMQWYQQKPGKAPKLLIYAASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQSRKDPSTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGDIYPGNGDTSYNQKFKGRVTITADKSTSTVYMELSSLRSEDTAVYYCARVGGAFPMDYWGQGTTVTVSS laIL2-Fc(hole) (SEQ ID NO:10) APTSSSTKKTQLQLEHLLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSDKTHTCPPCPPAEAPEAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFKLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Anti-TIM3 antibodyVH-CH1-Fc(knob) (SEQ ID NO:11) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGDIYPGNGDTSYNQKFKGRVTITADKSTSTVYMELSSLRSEDTAVYYCARVGGAFPMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSALTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK aTIM3 ScFv-Fc(knob) (SEQ ID NO:12) AIQLTQSPSSLSASVGDRVTITCRASESVEYYGTSLMQWYQQKPGKAPKLLIYAASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQSRKDPSTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGDIYPGNGDTSYNQKFKGRVTITADKSTSTVYMELSSLRSEDTAVYYCARVGGAFPMDYWGQGTTVTVSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSALTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Anti-LAG3 antibody VL-CL (SEQ ID NO:13) EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGISSRATGIPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQANNFPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Anti-LAG3 antibody VH-CH1 (SEQ ID NO:14) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAASTLRVPNPPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC Anti-LAG3 scFv (SEQ ID NO:15) EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGISSRATGIPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQANNFPLTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAASTLRVPNPPYWGQGTLVTVSS aLAG3 VH-CH1-Fc(knob) (SEQ ID NO:16) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAASTLRVPNPPYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSALTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK aLAG3 ScFv-Fc(knob) (SEQ ID NO:17) EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGISSRATGIPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQANNFPLTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAASTLRVPNPPYWGQGTLVTVSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSALTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK TIM3-InFc-laIL2:aTIM3(ScFv)-InFc-laIL2 (SEQ ID NO:18) AIQLTQSPSSLSASVGDRVTITCRASESVEYYGTSLMQWYQQKPGKAPKLLIAASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQSRKDPSTFGGGTKVEIKGGGGGGGGGGSQVQLVQSGAEVKKKPGSSVKVKVKVKVKSGSG YTFTSYNMHWVRQAPGQGLEWMGDIYPGNGDTSYNQKFKGRVTITADKSTSTVYMELSSLEDTAVYYCARVGGAFPMDYWGQGTTVSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVVVVDVSHEDPEWKFNWVDYVD GVEVHNACTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDGSFFLYSKLTVDKSRWQQQNVQQVQGHAL HNHYTQKSLSLSPGKGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTLMLTAKFYMPKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLEKGSETTFMCEYADETATIVEFLNRWITFCQSIIST laIL2-InFc-TIM3:laIL2-InFc-aTIM3(ScFv) (SEQ ID NO:19) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTLMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQNKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSDKTHTCPPCPAPEAA GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEWKFNWYVDGVEVHNACTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNCALGAPIEKTISKKQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSGDIPAWEN NYKTTPPPVLDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSAIQLTQSPSSLSASVGDRVTITCRASESVEYGTSLMQWYQQKPGKAPKLLIAASNVESGVPSRFSGSGCGGTDFTLTTISL FCQQSRKDPSTFGGGTKVEIKGGGGSGGGGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGDIYPGNGDTSYNQKFKGRVTITADKSTSTVYMELSSLEDTAVYCVYCVYGGFPTVGGFPTVGGFTTV TIM3-laIL2-InFc:aTIM3(ScFv)-laIL2-InFc (SEQ ID NO:20) AIQLTQSPSSLSASVGDRVTITCRASESVEYYGTSLMQWYQQKPGKAPKLLIAASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQS RKDPSTFGGGTKVEIKGGGGGGGGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGDIYPGNGDTSYNQKFK GRVTITADKSTSTVYMELSSLRSEDTAVYYCARVGGAFPMDYWGQGTTVTVSSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLT LMLTAKFYMPCKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLEKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT GGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNACTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAP IEKTISCAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPPGK laIL2-TIM3-InFc:laIL2-aTIM3(ScFv)-Fc (SEQ ID NO:21) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTLMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSAIQLTQSPSSLSAS VGDRVERVETSIY WITH ARGEY WITLINGING ANDINGING ANDINGS ANDINGS AND ANDINGS AND ANDINGINGS FCQQSRCDGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGES ANward Affairs PGQGLEWMGDIYPGNGDTSYNQFKGRVTITADKSTSTVYMELSSLRSEDTAVYYCARVGGAFPMDYWGQGTTVTVSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVVVVDVSHEDPEWKFNWYVDGVVHNAKTKPREEK QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNCALGAPIEKTISKKQQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQQSLKSLGKSL LAG3-InFc-laIL2:aLAG3(ScFv)-InFc-laIL2 (SEQ ID NO:22) EIVLTQSPGTLSSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGISSRATGIPDRFSGSGSGTDFTLTISSLQPEDFATYYCQANNFPLTFGGGTKLEIKGGGGGGGGGGSQVQLVQSGAEVKKPKPGSSVKVSCKASGGTF SSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAASTLRVPNPPYWGQGTLVSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVVVDVSHEDPEWKFNWYVDGV EVHNACTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDGSFFLYSKLTVDKSRWQFSCSQGNVHALH NHYTQKSLSLSPGKGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTLMLTAKFYMPKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLEKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT laIL2-InFc-LAG3:laIL2-InFc-aLAG3(ScFv) (SEQ ID NO:23) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTLMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSDKTHTCPPCPAPEA AGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEWKFNWYVDGVEVHNACTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNCALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDAVESQPAVW ENNYCTTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSYQQKPGQAPRLLIYGISSRATGIPDRFSGSGSGTDFTLTISSLQPEDFATYC QQANNFPLTFGGGTKLEIKGGGGGGGGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLEDTAVYYCAASTLRVPNPPYWGQTLVGTVSS LAG3-laIL2-InFc:aLAG3(ScFv)-laIL2-InFc (SEQ ID NO:24) EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGISSRATGIPDRFSGSGSGTDFTLTISSLQPEDFATYYCQANNFPLTFGGGTKLEIKGGGGGGGGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFS SYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAASTLRVPPNPPYWGQGTLVSSGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTLMLTAKFYMPKKATELKHLQCL EEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVVVVDVSHEDPEWKFNWYVDGVVHNACTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNCALGAPIEKTISKKQQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQQSLKSLGKSL laIL2-LAG3-InFc:laIL2-aLAG3(ScFv)-InFc (SEQ ID NO:25) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTLMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGISSRATGIPDRFSGSGSGTDFTLTISSLQPEDFATYYCQQANNFPLTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAASTLRVPNPPYWGQGTLVTVSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK aTIM3-VL-CL (SEQ ID NO:26) DIQMTQSPASLSASLGETVTIQCRASEDIYSGLAWFQQKPGKSPQLLIYGASSLQDGVPSRFSGSGSGTQYSLKISSMQTEDEGVYFCQQGLKYPPTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC aTIM3-VH-CH1-Fc(hole) (SEQ ID NO:27) EVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISYEGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWEDWGQGVMVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSALTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK aPD1-ScFv-Fc(hole) (SEQ ID NO:28) SYELTQPPSASVNVGETVKITCSGDQLPKYFADWFHQRSDQTILQVIYDDNKRPSGIPERISGSSSGTTATLTIRDVRAEDGDYYCFSGYVDSDSKLYVFGSGTQLTVLGGGGSGGGGSGGGGSEVRLLESGGGLVKPEGSLKLSCVASGFTFSDYFMSWVRQAPGKGLEWVAHIYTKSYNYATYYSGSVKGRFTISRDDSRSMVYLQMNLRTEDTATYYCTRDGSGYPSLDFW GQGTQVTVSSDKTHTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSALTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK ahPD1-ScFv-Fc(hole) (SEQ ID NO:29) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSALTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK aTIM3-VH-CH1-Fc (SEQ ID NO:30) EVQLVESGGGLVQPGRSLKLSCAASGFTFSDFYMAWVRQAPKKGLEWVASISYEGSSTYYGDSVMGRFTISRDNAKSTLYLQMNSLRSEDTATYYCARQREANWEDWGQGVMVTVSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPPAEAPEAGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK IL2-Fc(hole) (SEQ ID NO:31) APTSSSTKKTQLQLEHLLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTGGGGSDKTHTCPPCPPAEAPEAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFKLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 1aIL2 (SEQ ID NO:32) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

Claims

1. A fusion protein comprising at least one of the following species: a) interleukin 2 (IL2) polypeptide; b) anti-T cell surface molecule antibody, c) Immunoglobulin Fc.

2. The fusion protein of claim 1, wherein the T cell surface molecule includes, but is not limited to, TIM3, LAG3, PD1, ICOS, GITR, CD27, CD137, 2B4, OX40, 4-1BB, TIGIT, CTLA4, VISTA, and CD8.

3. The fusion protein according to claim 1, wherein the immunoglobulin Fc is a natural immunoglobulin Fc or an immunoglobulin Fc whose ADCC effect has been knocked out by genetic mutation, and preferably the immunoglobulin Fc is an Fc of human IgG.

4. the T cell surface molecule is TIM3 or LAG3; The fusion protein according to any one of claims 1 to 3, wherein the anti-T cell surface molecule antibody is an anti-TIM3 antibody or an anti-LAG3 antibody.

5. The fusion protein is a heterodimer, and the heterodimer includes the following monomers: (1) A first monomer, the first monomer being, in order from the N-terminus, 1) a wild-type IL-2 protein whose sequence is set forth in SEQ ID NO: 1 or a mutant of the wild-type IL-2 protein, preferably a mutant of the wild-type IL-2 protein containing any one of R38L, F42A, H16K, D20K, R38A, F42K and K43E mutation sites or any combination of mutation sites; 2) the necessary connecting structure (G4S connecting sequence), preferably the connecting sequence of which is shown in SEQ ID NO: 6; 3) an IgG Fc having a sequence set forth in SEQ ID NO: 2, or an ADCC-knockout mutant IgG Fc (InFc) set forth in SEQ ID NO: 3, or a knob mutant Fc set forth in SEQ ID NO: 4, or a hole mutant Fc set forth in SEQ ID NO: 5; (2) A second monomer, the second monomer comprising: 1) a Fab region of an anti-TIM3 antibody consisting of an anti-TIM3 antibody light chain VL-KCL having the sequence shown in SEQ ID NO: 7 and an anti-TIM3 antibody heavy chain VH&CH1 having the sequence shown in SEQ ID NO: 8, or an anti-TIM3 single-chain antibody (ScFv) whose sequence is set forth in SEQ ID NO: 9; 2) The fusion protein according to any one of claims 1 to 4, characterized in that it comprises an IgG Fc whose sequence is set forth in SEQ ID NO: 2, or an ADCC effect knockout mutant IgG Fc (InFc) whose sequence is set forth in SEQ ID NO: 3, or a knob mutant Fc whose sequence is set forth in SEQ ID NO: 4, or a hole mutant Fc whose sequence is set forth in SEQ ID NO:

5.

6. The heterodimer includes: a first monomer which is a polypeptide (laIL2-Fc(hole)) whose sequence is set forth in SEQ ID NO: 10; 1) an anti-TIM3 antibody Fab construct consisting of an anti-TIM3 antibody light chain VL-KCL having the sequence shown in SEQ ID NO: 7 and an anti-TIM3 antibody VH-CH1-Fc (knob) having the sequence shown in SEQ ID NO: 11; or 2) a second monomer that is a polypeptide (aTIM3-ScFv-Fc(knob)) whose sequence is set forth in SEQ ID NO: 12 or SEQ ID NO:

22.

7. The second monomer is 1) an antibody Fab region consisting of an anti-LAG3 antibody light chain VL-CL having the sequence shown in SEQ ID NO: 13 and an anti-LAG3 antibody heavy chain VH-CH1 having the sequence shown in SEQ ID NO: 14; or 2) an anti-LAG3 single chain antibody (ScFv) whose sequence is set forth in SEQ ID NO: 15; 3) The fusion protein according to claim 5, characterized in that it comprises an Fc of an IgG whose sequence is set forth in SEQ ID NO: 2, or an Fc (InFc) of a No-ADCC mutant IgG whose sequence is set forth in SEQ ID NO: 3, or a knob mutant Fc whose sequence is set forth in SEQ ID NO: 4, or a hole mutant Fc whose sequence is set forth in SEQ ID NO:

5.

8. The second monomer is (1) A Fab construct of an anti-LAG3 antibody consisting of a polypeptide (aLAG3 VH-CH1-Fc(knob)) whose sequence is set forth in SEQ ID NO: 16 and an anti-LAG3 antibody light chain variable region whose sequence is set forth in SEQ ID NO: 13, or (2) The fusion protein according to claim 6, characterized in that the sequence is a polypeptide (aLAG3 ScFv-Fc (knob)) shown in SEQ ID NO: 17 or SEQ ID NO:

22.

9. the fusion protein is a homodimer, The monomers of the homodimer are A monomer in which one molecule of interleukin 2 is linked to one molecule of anti-TIM3 antibody (aTIM3) or anti-LAG3 antibody (aLAG3) Fab in any manner; or The fusion protein according to any one of claims 1 to 4, characterized in that it is a monomer in which one molecule of interleukin-2 and one molecule of anti-LAG3 antibody (aLAG3) or (ScFv) of anti-LAG3 antibody (aLAG3) are linked by any method.

10. The monomers of the homodimer are, in order from the N-terminus, (1) Fab / ScFv of an anti-TIM3 antibody (aTIM3) or an anti-LAG3 antibody (aLAG3), wherein the Fab is a Fab of a humanized antibody or a Fab of a fully humanized antibody, and the ScFv is a ScFv of a humanized antibody or a ScFv of a fully humanized antibody; and (2) an antibody Fc that is a fully human wild-type Fc or a No-ADCC variant Fc (InFc); (3) Preferably, the necessary connecting structure (such as a G4S connecting sequence) whose connecting sequence is set forth in SEQ ID NO: 6; (4) The fusion protein according to claim 9, characterized in that it comprises wild-type interleukin 2 as shown in SEQ ID NO: 1, or a mutant of the wild-type interleukin 2 containing any one of the mutation sites R38L, F42A, H16K, D20K, R38A, F42K and K43E, or any combination of the mutation sites.

11. The monomers of the homodimer are (1) A polypeptide represented by SEQ ID NO: 18 (TIM3-InFc-laIL2: aTIM3(ScFv)-InFc-laIL2), or (2) A polypeptide represented by SEQ ID NO: 19 (laIL2-InFc-TIM3: laIL2-Fc-aTIM3 (ScFv)), or (3) A polypeptide represented by SEQ ID NO: 20 (TIM3-laIL2-InFc: aTIM3(ScFv)-Fc-laIL2), or (4) A polypeptide represented by SEQ ID NO: 21 (laIL2-TIM3-InFc: laIL2-aTIM3(ScFv)-Fc), or (5) A polypeptide represented by SEQ ID NO: 22 ((LAG3-InFc-laIL2: aLAG3(ScFv)-InFc-laIL2), or (6) A polypeptide represented by SEQ ID NO: 23 (laIL2-InFc-LAG3: laIL2-Fc-aLAG3 (ScFv)), or (7) A polypeptide represented by SEQ ID NO: 24 (LAG3-laIL2-InFc: aLAG3(ScFv)-Fc-laIL2), or (8) The fusion protein according to claim 9, which is a polypeptide represented by SEQ ID NO: 25 (laIL2-LAG3-InFc: laIL2-aLAG3(ScFv)-Fc).

12. 12. An application of the fusion protein according to any one of claims 1 to 11, said application comprising: (1) Preparation of antitumor drugs; (2) Preparation of an antitumor drug in combination with an immune checkpoint inhibitor; (3) Preparation of antitumor drugs to overcome resistance to immune checkpoint inhibitors; (4) Preparation of antitumor drugs in combination with T cell adoptive transfer; (5) Preparation of an antitumor drug to overcome unresponsiveness to T cell adoptive transfer; Preferably, the tumor is selected from lung cancer, colorectal cancer, gastric cancer, melanoma, renal cancer, lymphoma such as B-cell lymphoma, prostate cancer, thyroid cancer, breast cancer, pancreatic cancer, liver cancer, cervical cancer, ovarian cancer or esophageal cancer.

13. 13. The application of claim 12, wherein the immune checkpoint inhibitor is an anti-PDL1 antagonist, preferably, the anti-PDL1 antagonist is an anti-PDL1 antibody.

14. 13. The application according to claim 12, wherein the T cells are anti-tumor T cells, more preferably the T cells are anti-tumor CAR T cells or structural analogs thereof.

15. A drug or pharmaceutical composition comprising the fusion protein of any one of claims 1 to 12.

16. A kit comprising the fusion protein of any one of claims 1 to 12, and preferably further comprising one or more anti-tumor drugs selected from the group consisting of an anti-PD-L1 antibody, an anti-PD1 antibody, an anti-TIGIT antibody, and a chemotherapeutic drug, preferably a tyrosine kinase inhibitor, nitrogen mustard, doxorubicin, Pts, paclitaxel, etc.

17. An mRNA or DNA encoding the first or second monomer of the fusion protein according to any one of claims 5 to 8, or the monomer of the fusion protein according to any one of claims 9 to 11.

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