Fusion polypeptide and application thereof

Abstract

The invention relates to a fusion polypeptide and application thereof, and particularly, the fusion polypeptide comprises a part capable of blocking a PD-1 / PD-L1 signal, CD86 or a functional active fragment thereof and / or a part capable of activating an immune response. The invention also provides application of the polypeptide.

Description

A fusion polypeptide and its use Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a fusion polypeptide and its use. Background Art

[0002] Immune checkpoints are a class of immune-related molecules that contain inhibitory signaling molecules. They primarily function by regulating immune responses in peripheral tissues, including those involved in immune defense, immune tolerance, and immune tissue damage. Targeted immune checkpoint-related therapeutic approaches aim to modulate immune responses, including both innate and adaptive immune responses, to intervene in the immune system and achieve disease treatment. Tumor cells can exploit immune checkpoint inhibitory signaling pathways to achieve immune evasion. Binding of programmed death-1 (PD-1) to its ligand, programmed death receptor ligand-1 (PD-L1), is a key event in suppressing anti-tumor immune responses. PD-L1 is expressed in a variety of tumor cells, including colon cancer, lung cancer, ovarian cancer, and various myeloma tumors, and its expression is significantly correlated with the prognosis of various cancers. PD-L1 binding to PD-1 can lead to T cell apoptosis, immune signaling inhibition, cell exhaustion, and the secretion of immunosuppressive factors. Consequently, tumor-infiltrating T cells become ineffective, thereby helping tumor cells evade immune surveillance.

[0003] Currently, marketed blocking antibodies targeting PD-L1 include atezolizumab developed by Roche, avelumab jointly developed by Merck KGaA and Pfizer, and durvalumab developed by AstraZeneca. Blocking antibodies vying for market approval against PD-1 include nivolumab developed by Bristol-Myers Squibb (BMS), permbrolizumab developed by Merck, camrelizumab developed by Hengrui Medicine, tislelizumab developed by BeiGene, and sintilimab developed by Innovent Biologics. While these antibodies have demonstrated efficacy in cancer treatment, their average efficacy rate is only around 20%, and a significant number of cancer patients remain unresponsive to anti-PD-L1 and anti-PD-1 antibodies. Therefore, enhancing the effectiveness of cancer treatment remains a critical issue that urgently needs to be addressed.

[0004] Summary of the Invention

[0005] Given the current limitations on the effectiveness of PD-1 / PD-L1 pathway blockade in regulating T cell activation for tumor treatment, this application proposes the development of novel agents that synergize with PD-1 / PD-L1 pathway blockade to enhance T cell activation for clinical tumor treatment. The development of novel agents that synergize with PD-1 / PD-L1 pathway blockade, enhance T cell activation, and / or improve antigen-presenting cell activation for clinical tumor treatment has the potential to bring greater benefits to more cancer patients.

[0006] The present application provides a fusion polypeptide, which may comprise (i) a fusion polypeptide derived from an anti-programmed death receptor protein ligand-1 (PD-L1) antibody / anti-programmed death receptor protein-1 (PD-1) antibody-related binding antigen fragment, or / and (ii) an immunoglobulin Fc domain, or / and (iii) a fusion polypeptide of a CD80 extracellular domain (ECD), or / and (iv) a fusion polypeptide composed of a LAG3 extracellular domain, and the present application provides a polynucleotide expressing the fusion polypeptide; the present application provides a method for inducing and / or enhancing immunity using the fusion polypeptide and a method for treating a disease (e.g., cancer).

[0007] The present application provides a class of dual and / or multifunctional fusion polypeptides that simultaneously interfere with, inhibit or block the PD-1 / PD-L1 signal transduction pathway and / or co-stimulate antigen-presenting cell activation and / or co-stimulate T cell activation. The fusion polypeptides can effectively stimulate T cells to enhance immune responses and / or stimulate antigen-presenting cells, and can have a potential tumor-killing effect, thereby improving the therapeutic effect in the treatment of diseases caused by suppressed T cell function, such as tumors, and in particular in tumor patients who have no response or weak response to anti-PD-1 antibodies and / or anti-PD-L1 antibodies.

[0008] On the one hand, the present application provides a fusion polypeptide comprising a first domain and a second domain, wherein the first domain is capable of blocking PD-1 / PD-L1 signaling, and the second domain comprises CD86 or a functionally active fragment thereof.

[0009] In one embodiment of the fusion polypeptide, the first domain is capable of binding to PD-L1 and / or PD-1.

[0010] In one embodiment of the fusion polypeptide, the first domain comprises HCDR1, HCDR2 and / or HCDR3 of an antibody heavy chain, wherein the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17.

[0011] In one embodiment of the fusion polypeptide, the first domain comprises the heavy chain variable region VH of an antibody heavy chain, and the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 17.

[0012] In one embodiment of the fusion polypeptide, the first domain comprises an antibody heavy chain, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17.

[0013] In one embodiment of the fusion polypeptide, the first domain comprises LCDR1, LCDR2 and / or LCDR3 of the light chain of an antibody light chain, and the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 18.

[0014] In one embodiment of the fusion polypeptide, the first domain comprises an antibody or an antigen-binding fragment thereof. In one embodiment of the fusion polypeptide, the first domain comprises a light chain variable region VL of an antibody light chain, wherein the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO: 18.

[0015] In one embodiment of the fusion polypeptide, the first domain comprises an antibody light chain, and the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 18.

[0016] In one embodiment of the fusion polypeptide, the antibody is selected from the group consisting of a recombinant antibody, a single domain antibody, a heavy chain antibody, a chimeric antibody, and a bispecific antibody.

[0017] In one embodiment of the fusion polypeptide, the antigen binding fragment is selected from one or more of the following groups: Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, VHH and dAb.

[0018] In one embodiment of the fusion polypeptide, the first domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of Sugemalimab, Durvalumab, Atezolizumab, Avelumab, Envafolimab, Permbrolizumab and Nivolumab.

[0019] In one embodiment of the fusion polypeptide, the first domain comprises Sugemalimab or an antigen-binding fragment thereof.

[0020] In one embodiment of the fusion polypeptide, the second domain is selected from the group consisting of human-derived CD86 or a functionally active fragment thereof and mouse-derived CD86 or a functionally active fragment thereof.

[0021] In one embodiment of the fusion polypeptide, the second domain is capable of binding to CD28 and / or CTLA4.

[0022] In one embodiment of the fusion polypeptide, the second domain comprises the IgV domain of CD86 or a functionally active fragment thereof.

[0023] In one embodiment of the fusion polypeptide, the second domain comprises the extracellular domain of CD86 or a functionally active fragment thereof.

[0024] In one embodiment of the fusion polypeptide, the second domain comprises the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0025] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly linked to the second domain.

[0026] In one embodiment of the fusion polypeptide, the heavy chain of the first domain is directly or indirectly linked to the second domain.

[0027] In one embodiment of the fusion polypeptide, the C-terminus of the heavy chain of the first domain is directly or indirectly linked to the N-terminus of the second domain.

[0028] In one embodiment of the fusion polypeptide, the N-terminus of the heavy chain of the first domain is directly or indirectly linked to the C-terminus of the second domain.

[0029] In one embodiment of the fusion polypeptide, the light chain of the first domain is directly or indirectly linked to the second domain.

[0030] In one embodiment of the fusion polypeptide, the C-terminus of the light chain of the first domain is directly or indirectly linked to the N-terminus of the second domain.

[0031] In one embodiment of the fusion polypeptide, the N-terminus of the light chain of the first domain is directly or indirectly linked to the C-terminus of the second domain.

[0032] In one embodiment of the fusion polypeptide, the indirect linkage comprises linkage via a linker.

[0033] In one embodiment of the fusion polypeptide, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.

[0034] :58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90 NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86 and SEQ ID NO:87.

[0035] In one embodiment, the fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, and SEQ ID NO: 77.

[0036] On the other hand, the present application provides a fusion polypeptide comprising a first domain, a second domain and a third domain, wherein the first domain can block PD-1 / PD-L1 signaling, the second domain comprises CD86 or a functionally active fragment thereof, and the third domain can activate the innate immune response.

[0037] In one embodiment of the fusion polypeptide, the third domain is capable of binding to an MHC II molecule on an antigen presenting cell.

[0038] In one embodiment of the fusion polypeptide, the third domain comprises LAG3 or a functionally active fragment thereof.

[0039] In one embodiment of the fusion polypeptide, the first domain is capable of binding to PD-L1 and / or PD-1.

[0040] In one embodiment of the fusion polypeptide, the first domain comprises HCDR1, HCDR2 and / or HCDR3 of an antibody heavy chain, wherein the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17.

[0041] In one embodiment of the fusion polypeptide, the first domain comprises the heavy chain variable region VH of an antibody heavy chain, and the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17.

[0042] In one embodiment of the fusion polypeptide, the first domain comprises an antibody heavy chain, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17.

[0043] In one embodiment of the fusion polypeptide, the first domain comprises LCDR1, LCDR2 and / or LCDR3 of the light chain of an antibody light chain, and the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 18.

[0044] In one embodiment of the fusion polypeptide, the first domain comprises the light chain variable region VL of an antibody light chain, and the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 18.

[0045] In one embodiment of the fusion polypeptide, the first domain comprises an antibody light chain, and the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 18.

[0046] In one embodiment of the fusion polypeptide, the first domain comprises an antibody or an antigen-binding fragment thereof.

[0047] In one embodiment of the fusion polypeptide, the antibody is selected from the group consisting of a recombinant antibody, a single domain antibody, a heavy chain antibody, a chimeric antibody, and a bispecific antibody.

[0048] In one embodiment of the fusion polypeptide, the antigen-binding fragment is selected from one or more of the following groups: Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, VHH and dAb.

[0049] In one embodiment of the fusion polypeptide, the first domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of Sugemalimab, Durvalumab, Atezolizumab, Avelumab, Envafolimab, Permbrolizumab and Nivolumab.

[0050] In one embodiment of the fusion polypeptide, the first domain comprises Sugemalimab or an antigen-binding fragment thereof.

[0051] In one embodiment of the fusion polypeptide, the second domain is selected from the group consisting of human-derived CD86 or a functionally active fragment thereof and mouse-derived CD86 or a functionally active fragment thereof.

[0052] In one embodiment of the fusion polypeptide, the second domain is capable of binding to CD28 and / or CTLA4.

[0053] In one embodiment of the fusion polypeptide, the second domain comprises the IgV domain of CD86 or a functionally active fragment thereof.

[0054] In one embodiment of the fusion polypeptide, the second domain comprises the extracellular domain of CD86 or a functionally active fragment thereof.

[0055] In one embodiment of the fusion polypeptide, the second domain comprises the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0056] In one embodiment of the fusion polypeptide, the third domain is selected from the group consisting of human-derived LAG3 or a functionally active fragment thereof and mouse-derived LAG3 or a functionally active fragment thereof.

[0057] In one embodiment of the fusion polypeptide, the third domain comprises the extracellular domain of LAG3 or a functionally active fragment thereof.

[0058] In one embodiment of the fusion polypeptide, the third domain comprises IgD1, IgD2, IgD3 and / or IgD4 of LAG3 or a functionally active fragment thereof.

[0059] In one embodiment of the fusion polypeptide, the third domain comprises IgD1, IgD1-IgD2, IgD1-IgD2-IgD3, and / or IgD1-IgD2-IgD3-IgD4 of LAG3 or a functionally active fragment thereof.

[0060] In one embodiment of the fusion polypeptide, the third domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.

[0061] In one embodiment of the fusion polypeptide, the first domain and the second domain are directly or indirectly linked.

[0062] In one embodiment of the fusion polypeptide, the heavy chain of the first domain is directly or indirectly linked to the second domain.

[0063] In one embodiment of the fusion polypeptide, the C-terminus of the heavy chain of the first domain is directly or indirectly linked to the N-terminus of the second domain.

[0064] In one embodiment of the fusion polypeptide, the N-terminus of the heavy chain of the first domain is directly or indirectly linked to the C-terminus of the second domain.

[0065] In one embodiment of the fusion polypeptide, the light chain of the first domain is directly or indirectly linked to the second domain.

[0066] In one embodiment of the fusion polypeptide, the C-terminus of the light chain of the first domain is directly or indirectly linked to the N-terminus of the second domain.

[0067] In one embodiment of the fusion polypeptide, the N-terminus of the light chain of the first domain is directly or indirectly linked to the C-terminus of the second domain.

[0068] In one embodiment of the fusion polypeptide, the C-terminus of the heavy chain of the first domain is connected to the N-terminus of the second domain, and the C-terminus of the light chain of the first domain is connected to the N-terminus of the second domain.

[0069] In one embodiment of the fusion polypeptide, the N-terminus of the heavy chain of the first domain is connected to the C-terminus of the second domain, and the N-terminus of the light chain of the first domain is connected to the C-terminus of the second domain.

[0070] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly linked to the third domain.

[0071] In one embodiment of the fusion polypeptide, the first domain heavy chain is directly or indirectly linked to the third domain.

[0072] In one embodiment of the fusion polypeptide, the C-terminus of the heavy chain of the first domain is directly or indirectly connected to the N-terminus of the third domain.

[0073] In one embodiment of the fusion polypeptide, the N-terminus of the heavy chain of the first domain is directly or indirectly connected to the C-terminus of the third domain.

[0074] In one embodiment of the fusion polypeptide, the first domain light chain is directly or indirectly linked to the third domain.

[0075] In one embodiment of the fusion polypeptide, the C-terminus of the light chain of the first domain is directly or indirectly connected to the N-terminus of the third domain.

[0076] In one embodiment of the fusion polypeptide, the N-terminus of the light chain of the first domain is directly or indirectly connected to the C-terminus of the third domain.

[0077] In one embodiment of the fusion polypeptide, the second domain and the third domain are directly or indirectly linked.

[0078] In one embodiment of the fusion polypeptide, the C-terminus of the second domain is directly or indirectly connected to the N-terminus of the third domain.

[0079] In one embodiment of the fusion polypeptide, the N-terminus of the second domain is directly or indirectly connected to the C-terminus of the third domain.

[0080] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly linked to the second domain, and the second domain is directly or indirectly linked to the third domain.

[0081] In one embodiment of the fusion polypeptide, the C-terminus of the first domain is directly or indirectly connected to the N-terminus of the second domain, and the C-terminus of the second domain is directly or indirectly connected to the N-terminus of the third domain.

[0082] In one embodiment of the fusion polypeptide, the heavy chain C-terminus of the first domain is directly or indirectly connected to the N-terminus of the second domain, and the C-terminus of the second domain is directly or indirectly connected to the N-terminus of the third domain.

[0083] In one embodiment of the fusion polypeptide, the C-terminus of the light chain of the first domain is directly or indirectly connected to the N-terminus of the second domain, and the C-terminus of the second domain is directly or indirectly connected to the N-terminus of the third domain.

[0084] In one embodiment of the fusion polypeptide, the N-terminus of the first domain is directly or indirectly connected to the C-terminus of the second domain, and the N-terminus of the second domain is directly or indirectly connected to the C-terminus of the third domain.

[0085] In one embodiment of the fusion polypeptide, the N-terminus of the heavy chain of the first domain is directly or indirectly connected to the C-terminus of the second domain, and the N-terminus of the second domain is directly or indirectly connected to the C-terminus of the third domain.

[0086] In one embodiment of the fusion polypeptide, the N-terminus of the light chain of the first domain is directly or indirectly connected to the C-terminus of the second domain, and the N-terminus of the second domain is directly or indirectly connected to the C-terminus of the third domain.

[0087] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly linked to the third domain, and the third domain is directly or indirectly linked to the second domain.

[0088] In one embodiment of the fusion polypeptide, the C-terminus of the first domain is directly or indirectly connected to the N-terminus of the third domain, and the C-terminus of the third domain is directly or indirectly connected to the N-terminus of the second domain.

[0089] In one embodiment of the fusion polypeptide, the C-terminus of the heavy chain of the first domain is directly or indirectly connected to the N-terminus of the third domain, and the C-terminus of the third domain is directly or indirectly connected to the N-terminus of the second domain.

[0090] In one embodiment of the fusion polypeptide, the C-terminus of the light chain of the first domain is directly or indirectly connected to the N-terminus of the third domain, and the C-terminus of the third domain is directly or indirectly connected to the N-terminus of the second domain.

[0091] In one embodiment of the fusion polypeptide, the N-terminus of the first domain is directly or indirectly connected to the C-terminus of the third domain, and the N-terminus of the third domain is directly or indirectly connected to the C-terminus of the second domain.

[0092] In one embodiment of the fusion polypeptide, the N-terminus of the heavy chain of the first domain is directly or indirectly connected to the C-terminus of the third domain, and the N-terminus of the third domain is directly or indirectly connected to the C-terminus of the second domain.

[0093] In one embodiment of the fusion polypeptide, the N-terminus of the light chain of the first domain is directly or indirectly connected to the C-terminus of the third domain, and the N-terminus of the third domain is directly or indirectly connected to the C-terminus of the second domain.

[0094] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly linked to the second domain, and the first domain is directly or indirectly linked to the third domain.

[0095] In one embodiment of the fusion polypeptide, the C-terminus of the first domain is directly or indirectly connected to the N-terminus of the second domain, and the N-terminus of the first domain is directly or indirectly connected to the C-terminus of the third domain.

[0096] In one embodiment of the fusion polypeptide, the C-terminus of the heavy chain of the first domain is directly or indirectly connected to the N-terminus of the second domain, and the N-terminus of the heavy chain of the first domain is directly or indirectly connected to the C-terminus of the third domain.

[0097] In one embodiment of the fusion polypeptide, the C-terminus of the light chain of the first domain is directly or indirectly connected to the N-terminus of the second domain, and the N-terminus of the light chain of the first domain is directly or indirectly connected to the C-terminus of the third domain.

[0098] In one embodiment of the fusion polypeptide, the C-terminus of the heavy chain of the first domain is directly or indirectly connected to the N-terminus of the second domain, and the N-terminus of the light chain of the first domain is directly or indirectly connected to the C-terminus of the third domain.

[0099] In one embodiment of the fusion polypeptide, the C-terminus of the light chain of the first domain is directly or indirectly connected to the N-terminus of the second domain, and the N-terminus of the heavy chain of the first domain is directly or indirectly connected to the C-terminus of the third domain.

[0100] In one embodiment of the fusion polypeptide, the N-terminus of the first domain is directly or indirectly connected to the C-terminus of the second domain, and the C-terminus of the first domain is directly or indirectly connected to the N-terminus of the third domain.

[0101] In one embodiment of the fusion polypeptide, the N-terminus of the heavy chain of the first domain is directly or indirectly connected to the C-terminus of the second domain, and the C-terminus of the heavy chain of the first domain is directly or indirectly connected to the N-terminus of the third domain.

[0102] In one embodiment of the fusion polypeptide, the N-terminus of the light chain of the first domain is directly or indirectly connected to the C-terminus of the second domain, and the C-terminus of the light chain of the first domain is directly or indirectly connected to the N-terminus of the third domain.

[0103] In one embodiment of the fusion polypeptide, the N-terminus of the heavy chain of the first domain is directly or indirectly connected to the C-terminus of the second domain, and the C-terminus of the light chain of the first domain is directly or indirectly connected to the N-terminus of the third domain.

[0104] In one embodiment of the fusion polypeptide, the N-terminus of the light chain of the first domain is directly or indirectly connected to the C-terminus of the second domain, and the C-terminus of the heavy chain of the first domain is directly or indirectly connected to the N-terminus of the third domain.

[0105] In one embodiment of the fusion polypeptide, the indirect linkage comprises linkage via a linker.

[0106] In one embodiment of the fusion polypeptide, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.

[0107] In one embodiment, the fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120 NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 171, SEQ ID NO: 173.

[0108] In one embodiment, the fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 104, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 171, and SEQ ID NO: 173.

[0109] On the other hand, the present application provides a CD86 variant polypeptide comprising an amino acid substitution mutation of humanized CD86.

[0110] In one embodiment, the CD86 variant polypeptide comprises an amino acid substitution mutant of the humanized CD86 extracellular domain IgV domain.

[0111] In one embodiment, the CD86 variant polypeptide comprises an amino acid substitution mutant of the IgV domain of CD86, wherein the mutation site of the amino acid substitution mutant of the IgV domain of CD86 comprises one or more amino acid site mutations selected from the group consisting of A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, and H90F. In the present disclosure, unless otherwise explicitly stated, the representation of the mutation site is based on the wild-type sequence. For example, "the mutation site A13I of the amino acid substitution mutant of the IgV domain of CD86" indicates that the A at amino acid site 13 of the wild-type CD86 IgV domain is mutated to I.

[0112] In one embodiment, the CD86 variant polypeptide comprises an amino acid substitution mutant of the IgV domain of CD86, wherein the amino acid substitution mutant comprises a combination selected from the group consisting of Q25I / F33L / H90I, Q25V / F33L / H90I, Q25I / F33V / H90I, Q25V / F33V / H90I, Q25I / F33L / H90V, Q25V / F33L / H90V, Q25I / F33V / H90V, Q25V / F33V / H90V, A13L / Q25I / F33L / H90I, A13I / Q25I / F33L / H90I, A13L / Q25V / F33L / H90I, A13I / Q25V / F33L / H90I, Q25I / F33L / I89L / H90I, Q25I / F33L / M60R / H90I, Q 25V / F33L / I89L / H90I, Q25V / F33L / M60R / H90I, Q25F / F33L / H90I, Q25I / F33L / H90F, Q25I / F33A / H90I, Q25I / H90I, Q25I, H9 0I, Q25V / H90V, Q25V / H90I, Q25F / H90I, Q25F / H90V, A13F / Q25I / F33L / H90I, A13M / Q25I / F33L / H90I, Q25A / F33L / H90I, Q25M / F33L / H90I, Q25I / F33M / H90I, Q25I / F33L / I89V / H90I, Q25I / F33L / I89F / H90I, Q25I / F33L / I89M / H90I, and Q25I / F33L / H90M.

[0113] In one embodiment, the CD86 variant polypeptide comprises the CD86 IgV domain amino acid substitution mutant Q25I / F33L / H90I.

[0114] In one embodiment, the CD86 variant polypeptide comprises an amino acid substitution mutant of the extracellular domain of humanized CD86.

[0115] In one embodiment, the CD86 variant polypeptide comprises an amino acid substitution mutant of the extracellular domain of CD86, and the amino acid substitution mutant of the extracellular domain of CD86 comprises one or more mutations in amino acid sites selected from the group consisting of A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, and H90F.

[0116] In one embodiment, the CD86 variant polypeptide comprises an amino acid substitution mutant of the extracellular domain of CD86, wherein the amino acid substitution mutant comprises a combination selected from the group consisting of Q25I / F33L / H90I, 25V / F33L / H90I, Q25I / F33V / H90I, Q25V / F33V / H90I, Q25I / F33L / H90V, Q25V / F33L / H90V, Q25I / F33V / H90V, Q25V / F33V / H90V, A13L / Q25I / F33L / H90I, A13I / Q25I / F33L / H90I, A 13L / Q25V / F33L / H90I, A13I / Q25V / F33L / H90I, Q25I / F33L / I89L / H90I, Q25I / F33L / M60R / H90 I. Q25V / F33L / I89L / H90I, Q25V / F33L / M60R / H90I, Q25F / F33L / H90I, Q25I / F33L / H90F, Q25I / F33A / H90I, Q25I / H90I, Q25I, H90I, Q25V / H90V, Q25V / H90I, Q25F / H90I, Q25F / H90V, A13F / Q 90I, Q25I / F33L / H90I, A13M / Q25I / F33L / H90I, Q25A / F33L / H90I, Q25M / F33L / H90I, Q25I / F33M / H90I, Q25I / F33L / I89V / H90I, Q25I / F33L / I89F / H90I, Q25I / F33L / I89M / H90I, and Q25I / F33L / H90M.

[0117] In one embodiment, the CD86 variant polypeptide comprises the extracellular domain amino acid substitution mutant Q25I / F33L / H90I of CD86.

[0118] In one embodiment, the CD86 variant polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 178 to SEQ ID NO: 243.

[0119] On the other hand, the present application provides a fusion polypeptide, which comprises a first domain and a second domain, wherein the first domain comprises the CD86 variant polypeptide described in the present application, and the second domain comprises an antibody or an antigen-binding fragment thereof or an immunoglobulin domain.

[0120] In one embodiment of the fusion polypeptide, the second domain comprises an antibody or an antigen-binding fragment thereof.

[0121] In one embodiment of the fusion polypeptide, the second domain is capable of binding to one or more of the following targets: PD-L1, PD-L2, PD-1, OX40, 4-1BB, ICOS, TIGIT, CTLA4, LAG3, CD3, VEGF, VEGFR, CD47, HGF, Trop2, EpCAM, CCR8, CCR4, CCR5, GPRC5D, BCMA, CD19, CD20, HER-2neu, DLL1, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CD123, CD33, CD30, CD38, NKG2A, Nkp36 and Tim3.

[0122] In one embodiment of the fusion polypeptide, the second domain is capable of binding to PD-L1, PD-1 and / or PD-L2.

[0123] In one embodiment of the fusion polypeptide, the second domain comprises HCDR1, HCDR2 and / or HCDR3 of an antibody heavy chain, wherein the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17.

[0124] In one embodiment of the fusion polypeptide, the second domain comprises the heavy chain variable region VH of an antibody heavy chain, and the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 17.

[0125] In one embodiment of the fusion polypeptide, the second domain comprises an antibody heavy chain, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17.

[0126] In one embodiment of the fusion polypeptide, the second domain comprises LCDR1, LCDR2 and / or LCDR3 of an antibody light chain, wherein the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 18.

[0127] In one embodiment of the fusion polypeptide, the second domain comprises a light chain variable region VL of an antibody light chain, and the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 18.

[0128] In one embodiment of the fusion polypeptide, the second domain comprises an antibody light chain, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 18.

[0129] In one embodiment of the fusion polypeptide, the second domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of Sugemalimab, Durvalumab, Atezolizumab, Avelumab, Envafolimab, Permbrolizumab and Nivolumab.

[0130] In one embodiment of the fusion polypeptide, the second domain comprises Sugemalimab or an antigen-binding fragment thereof.

[0131] In one embodiment of the fusion polypeptide, the second domain is capable of binding to CD3.

[0132] In one embodiment of the fusion polypeptide, the second domain comprises a CD3B219 antibody or an antigen-binding fragment thereof.

[0133] In one embodiment of the fusion polypeptide, the second domain comprises HCDR1, HCDR2 and / or HCDR3 of an antibody heavy chain, wherein the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 616.

[0134] In one embodiment of the fusion polypeptide, the second domain comprises the heavy chain variable region VH of an antibody, and the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 616.

[0135] In one embodiment of the fusion polypeptide, the second domain comprises an antibody heavy chain, and the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 616.

[0136] In one embodiment of the fusion polypeptide, the second domain comprises LCDR1, LCDR2 and / or LCDR3 of an antibody light chain, wherein the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 617.

[0137] In one embodiment of the fusion polypeptide, the second domain comprises the light chain variable region VL of an antibody, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 617.

[0138] In one embodiment of the fusion polypeptide, the second domain comprises an antibody light chain, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 617.

[0139] In one embodiment of the fusion polypeptide, the antigen-binding fragment is selected from one or more of the following groups: Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, VHH and dAb.

[0140] In one embodiment of the fusion polypeptide, the second domain comprises an immunoglobulin domain.

[0141] In one embodiment of the fusion polypeptide, the second domain comprises an immunoglobulin IgG antibody.

[0142] In one embodiment of the fusion polypeptide, the immunoglobulin IgG antibody comprises one or more selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b and mouse IgG3.

[0143] In one embodiment of the fusion polypeptide, the second domain comprises an immunoglobulin Fc domain.

[0144] In one embodiment of the fusion polypeptide, the second domain comprises the Fc domain of an immunoglobulin IgG antibody.

[0145] In one embodiment of the fusion polypeptide, the second domain comprises the Fc domain of an immunoglobulin IgG antibody, and the Fc domain of the immunoglobulin IgG antibody comprises a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgG1 Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain or a mouse IgG3 Fc domain.

[0146] In one embodiment of the fusion polypeptide, the second domain comprises the Fc domain of human immunoglobulin IgG4.

[0147] In one embodiment of the fusion polypeptide, the second domain is selected from the group consisting of amino acid sequences shown in SEQ ID NO: 4 to SEQ ID NO: 18, SEQ ID NO: 29 to SEQ ID NO: 30, SEQ ID NO: 634 to SEQ ID NO: 639, SEQ ID NO: 616 and SEQ ID NO: 617.

[0148] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly linked to the second domain.

[0149] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly connected to the N-terminus of the second domain.

[0150] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly connected to the C-terminus of the second domain.

[0151] In one embodiment of the fusion polypeptide, the indirect linkage comprises linkage via a linker.

[0152] In one embodiment of the fusion polypeptide, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.

[0153] In one embodiment, the fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 350 to SEQ ID NO: 382, ​​SEQ ID NO: 384 to SEQ ID NO: 416, SEQ ID NO: 418 to SEQ ID NO: 450, SEQ ID NO: 451 to SEQ ID NO: 483, SEQ ID NO: 484 to SEQ ID NO: 615, SEQ ID NO: 618 to SEQ ID NO: 624 and SEQ ID NO: 626 to SEQ ID NO: 632.

[0154] On the other hand, the present application provides a fusion polypeptide comprising a first domain, a second domain and a third domain, wherein the first domain comprises the CD86 variant described in the present application, the second domain comprises an antibody or an antigen-binding fragment thereof or an immunoglobulin antibody, and the third domain is an antibody or an antigen-binding fragment thereof or a functional protein or an active fragment thereof that is the same as or different from the second domain.

[0155] In one embodiment of the fusion polypeptide, the second domain comprises an antibody or an antigen-binding fragment thereof.

[0156] In one embodiment of the fusion polypeptide, the second domain is capable of binding to one or more of the following targets: PD-L1, PD-L2, PD-1, OX40, 4-1BB, ICOS, TIGIT, CTLA4, LAG3, CD3, VEGF, VEGFR, CD47, HGF, Trop2, EpCAM, CCR8, CCR4, CCR5, GPRC5D, BCMA, CD19, CD20, HER-2neu, DLL1, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CD123, CD33, CD30, CD38, NKG2A, Nkp36 and Tim3.

[0157] In one embodiment of the fusion polypeptide, the second domain is capable of binding to PD-L1, PD-1 and / or PD-L2.

[0158] In one embodiment of the fusion polypeptide, the second domain comprises HCDR1, HCDR2 and / or HCDR3 of an antibody heavy chain, wherein the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17.

[0159] In one embodiment of the fusion polypeptide, the second domain comprises the heavy chain variable region VH of an antibody heavy chain, and the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 17.

[0160] In one embodiment of the fusion polypeptide, the second domain comprises an antibody heavy chain, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17.

[0161] In one embodiment of the fusion polypeptide, the second domain comprises LCDR1, LCDR2 and / or LCDR3 of a light chain of an antibody light chain, wherein the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 18.

[0162] In one embodiment of the fusion polypeptide, the second domain comprises a light chain variable region VL of an antibody light chain, and the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 18.

[0163] In one embodiment of the fusion polypeptide, the second domain comprises an antibody light chain, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 18.

[0164] In one embodiment of the fusion polypeptide, the second domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of Sugemalimab, Durvalumab, Atezolizumab, Avelumab, Envafolimab, Permbrolizumab and Nivolumab.

[0165] In one embodiment of the fusion polypeptide, the second domain comprises Sugemalimab or an antigen-binding fragment thereof.

[0166] In one embodiment of the fusion polypeptide, the second domain is capable of binding to CD3.

[0167] In one embodiment of the fusion polypeptide, the second domain comprises a CD3B219 antibody or an antigen-binding fragment thereof.

[0168] In one embodiment of the fusion polypeptide, the second domain comprises HCDR1, HCDR2 and / or HCDR3 of an antibody heavy chain, wherein the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 616.

[0169] In one embodiment of the fusion polypeptide, the second domain comprises the heavy chain variable region VH of an antibody, and the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 616.

[0170] In one embodiment of the fusion polypeptide, the second domain comprises an antibody heavy chain, and the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO: 616.

[0171] In one embodiment of the fusion polypeptide, the second domain comprises LCDR1, LCDR2 and / or LCDR3 of an antibody light chain, wherein the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 617.

[0172] In one embodiment of the fusion polypeptide, the second domain comprises the light chain variable region VL of an antibody, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO: 617.

[0173] In one embodiment of the fusion polypeptide, the second domain comprises an antibody light chain comprising the amino acid sequence of SEQ ID NO: 617. In one embodiment of the fusion polypeptide, the antibody is selected from the group consisting of an immunoglobulin antibody, a recombinant antibody, a chimeric antibody, a heavy chain antibody, a single domain antibody, and a bispecific antibody.

[0174] In one embodiment of the fusion polypeptide, the antigen-binding fragment is selected from one or more of the following groups: Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, VHH and dAb.

[0175] In one embodiment of the fusion polypeptide, the second domain comprises an immunoglobulin domain.

[0176] In one embodiment of the fusion polypeptide, the second domain comprises an immunoglobulin IgG antibody, and the immunoglobulin IgG antibody comprises one or more selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b and mouse IgG3.

[0177] In one embodiment of the fusion polypeptide, the second domain comprises an immunoglobulin Fc domain.

[0178] In one embodiment of the fusion polypeptide, the second domain comprises the Fc domain of an immunoglobulin IgG antibody.

[0179] In one embodiment of the fusion polypeptide, the second domain comprises the Fc domain of an immunoglobulin IgG antibody, and the Fc domain of the immunoglobulin IgG antibody comprises a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgG1 Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain or a mouse IgG3 Fc domain.

[0180] In one embodiment of the fusion polypeptide, the second domain comprises the Fc domain of human immunoglobulin IgG4.

[0181] In one embodiment of the fusion polypeptide, the third domain is a functional protein or an active fragment thereof, and the functional protein or the active fragment thereof can activate the innate immune response.

[0182] In one embodiment of the fusion polypeptide, the third domain is capable of binding to an MHC II molecule on an antigen presenting cell.

[0183] In one embodiment of the fusion polypeptide, the third domain comprises LAG3 or a functionally active fragment thereof.

[0184] In one embodiment of the fusion polypeptide, the third domain is selected from the group consisting of human-derived LAG3 or a functionally active fragment thereof and mouse-derived LAG3 or a functionally active fragment thereof.

[0185] In one embodiment of the fusion polypeptide, the third domain comprises the extracellular domain of LAG3 or a functionally active fragment thereof.

[0186] In one embodiment of the fusion polypeptide, the third domain comprises IgD1, IgD2, IgD3 and / or IgD4 of LAG3 or a functionally active fragment thereof.

[0187] In one embodiment of the fusion polypeptide, the third domain comprises IgD1, IgD1-IgD2, IgD1-IgD2-IgD3, and / or IgD1-IgD2-IgD3-IgD4 of LAG3 or a functionally active fragment thereof.

[0188] In one embodiment of the fusion polypeptide, the third domain comprises a LAG3 variant polypeptide.

[0189] In one embodiment of the fusion polypeptide, the third domain comprises a LAG3 variant polypeptide comprising truncations and / or mutations based on human LAG3.

[0190] In one embodiment of the fusion polypeptide, the third domain comprises a LAG3 variant polypeptide comprising a truncation of a wild-type human LAG3 extracellular domain polypeptide.

[0191] In one embodiment of the fusion polypeptide, the third domain comprises a LAG3 variant polypeptide comprising 0-124 amino acids truncated at the N-terminus based on a wild-type human LAG3 extracellular region polypeptide.

[0192] In one embodiment of the fusion polypeptide, the third domain comprises a LAG3 variant polypeptide comprising 0, 5, 21, 37, 45, 60, 71, 74, 79, 84, 89, 94, 99, 104, 109 or 114 amino acids truncated at the N-terminus based on the wild-type human LAG3 extracellular region polypeptide.

[0193] In one embodiment of the fusion polypeptide, the third domain comprises a LAG3 variant polypeptide, which comprises 0, 5, 21, 37, 45, 60, 71 or 74 amino acids truncated at the N-terminus based on the wild-type human LAG3 extracellular region polypeptide.

[0194] In one embodiment of the fusion polypeptide, the third domain comprises a LAG3 variant polypeptide, wherein the LAG3 variant polypeptide comprises a wild-type human LAG3 extracellular region polypeptide and terminates at amino acid positions 122-167 at the C-terminus.

[0195] In one embodiment of the fusion polypeptide, the third domain comprises a LAG3 variant polypeptide, which comprises a wild-type human LAG3 extracellular region polypeptide ending at amino acid position 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 or 167 at the C-terminus.

[0196] In one embodiment of the fusion polypeptide, the third domain comprises a LAG3 variant polypeptide, which comprises a wild-type human LAG3 extracellular region polypeptide and terminates at amino acid position 122, 129, 136, 146, 156, 161 or 167 at the C-terminus.

[0197] In one embodiment of the fusion polypeptide, the third domain comprises a LAG3 variant polypeptide, which comprises a wild-type human LAG3 extracellular region polypeptide and terminates at amino acid position 156, 161 or 167 at the C-terminus.

[0198] In one embodiment of the fusion polypeptide, the third domain comprises a LAG3 variant polypeptide, which comprises 0, 5, 21, 37, 45, 60, 71 or 74 amino acids truncated at the N-terminus based on the wild-type human LAG3 extracellular region polypeptide, and terminates at amino acid position 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 or 167 at the C-terminus based on the wild-type human LAG3 extracellular region polypeptide.

[0199] In one embodiment of the fusion polypeptide, the third domain comprises a LAG3 variant polypeptide comprising an amino acid site mutation.

[0200] In one embodiment of the fusion polypeptide, the third domain comprises a LAG3 variant polypeptide, which comprises an amino acid site mutation, wherein the amino acid mutation site is an Arg amino acid at position 97.

[0201] In one embodiment of the fusion polypeptide, the third domain comprises a LAG3 variant polypeptide, which comprises an amino acid site mutation, wherein the amino acid mutation site is a mutation of the Arg amino acid at position 97 to a Glu amino acid.

[0202] In one embodiment of the fusion polypeptide, the third domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 20 to SEQ ID NO: 28 and SEQ ID NO: 244 to SEQ ID NO: 349.

[0203] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly linked to the second domain.

[0204] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly linked to the third domain.

[0205] In one embodiment of the fusion polypeptide, the second domain is directly or indirectly linked to the third domain.

[0206] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly linked to the second domain, and the first domain is directly or indirectly linked to the third domain.

[0207] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly linked to the second domain, and the second domain is directly or indirectly linked to the third domain.

[0208] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly linked to the third domain, and the second domain is directly or indirectly linked to the third domain.

[0209] In one embodiment of the fusion polypeptide, the indirect linkage comprises linkage via a linker.

[0210] In one embodiment of the fusion polypeptide, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.

[0211] In one embodiment, the fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 751 to SEQ ID NO: 1024, 1249, 1251, 1253 and 1255.

[0212] In another aspect, the present application provides a LAG3 variant polypeptide comprising truncations and / or mutations based on human LAG3. The present application also provides a method for innovatively modifying LAG3 polypeptides to obtain functional LAG3 variant polypeptides, and methods for using such polypeptides to modulate immune responses in antigen-presenting cells, thereby treating or preventing diseases such as infections and tumors caused by suppressed immune cell function.

[0213] In one embodiment, the LAG3 variant polypeptide comprises a truncation of a wild-type human LAG3 extracellular region polypeptide.

[0214] In one embodiment, the LAG3 variant polypeptide comprises 0-124 amino acids truncated at the N-terminus based on the wild-type human LAG3 extracellular region polypeptide, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 118, 119, 120, 121, 122, 123, or 124 amino acids;and the C-terminus terminates at the following positions of the wild-type LAG3 extracellular region polypeptide: amino acid positions 122-167, such as amino acid positions 122-167, 123-167, 124-167, 125-167, 126-167, 127-167, 128-167, 129-167, 130-167, 131-167, 132-167, 133-167, 134-167, 135-167, 136-167, 137-167, 138-167, 139-167, 140-167, 141-167, 142-167, 143-167, 144-167, 145-167, 146-167, 147-167, 148-167, 149-167, 150-167, 151-167, 152-167, 153-167, 154-167, 155 -167, 156-167, 157-167, 158-167, 159-167, 160-167, 161-167, 162-167, 163-167, 164-167, 165-167, 166-167, for example, the C-terminus terminates at positions corresponding to amino acid positions 122, 123, 124, 125, 126, 127, 128 of the wild-type LAG3 extracellular region polypeptide. , 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, or 167. ;

[0215] In certain preferred embodiments, the LAG3 variant polypeptide comprises 0, 5, 21, 37, 45, 60, 71, 74, 79, 84, 89, 94, 99, 104, 109 or 114 amino acids truncated at the N-terminus based on the wild-type human LAG3 extracellular region polypeptide, and the C-terminus terminates at the following positions of the wild-type LAG3 extracellular region polypeptide: 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 or 167.

[0216] In one embodiment of the LAG3 variant, the LAG3 variant polypeptide comprises an amino acid mutation, wherein the amino acid mutation site is an Arg amino acid at position 97.

[0217] In one embodiment of the LAG3 variant, the LAG3 variant polypeptide comprises an amino acid mutation, wherein the amino acid mutation site is a mutation of the Arg amino acid at position 97 to a Glu amino acid.

[0218] In certain preferred embodiments, the truncated LAG3 variant polypeptide has at least one characteristic selected from the group consisting of:

[0219] (1) The N-terminus is truncated by 74 amino acids, and the C-terminus terminates at the following positions: positions corresponding to amino acid positions 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, or 167 of the wild-type LAG3 extracellular region polypeptide; wherein, preferably, the C-terminus terminates at the following positions: positions corresponding to amino acid positions 122, 129, 136, 146, 156, or 161 of the wild-type LAG3 extracellular region polypeptide.

[0220] (2) The N-terminus is truncated by 74 amino acids, the Arg amino acid at position 97 is mutated to a Glu amino acid, and the C-terminus terminates at the following positions: positions corresponding to amino acid positions 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, or 167 of the wild-type LAG3 extracellular region polypeptide; wherein, preferably, the C-terminus terminates at the following positions: positions corresponding to amino acid positions 122, 129, 136, 146, 156, or 161 of the wild-type LAG3 extracellular region polypeptide.

[0221] (3) The N-terminus is truncated by 5 amino acids, and the C-terminus terminates at the following positions: positions corresponding to amino acid positions 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0222] (4) The N-terminus was truncated by 5 amino acids, the Arg amino acid at position 97 was mutated to a Glu amino acid, and the C-terminus terminated at the following positions: the position corresponding to amino acid position 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0223] (5) The N-terminus is truncated by 21 amino acids, and the C-terminus terminates at the following positions: positions corresponding to amino acid positions 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0224] (6) The N-terminus was truncated by 21 amino acids, the Arg amino acid at position 97 was mutated to a Glu amino acid, and the C-terminus terminated at the following positions: the position corresponding to amino acid position 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0225] (7) The N-terminus is truncated by 37 amino acids, and the C-terminus terminates at the following positions: positions corresponding to amino acid positions 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0226] (8) The N-terminus was truncated by 37 amino acids, the Arg amino acid at position 97 was mutated to a Glu amino acid, and the C-terminus terminated at the following positions: the position corresponding to amino acid position 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0227] (9) The N-terminus is truncated by 45 amino acids, and the C-terminus terminates at the following positions: positions corresponding to amino acid positions 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0228] (10) The N-terminus was truncated by 45 amino acids, the Arg amino acid at position 97 was mutated to a Glu amino acid, and the C-terminus terminated at the following positions: the position corresponding to amino acid position 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0229] (11) The N-terminus is truncated by 60 amino acids, and the C-terminus terminates at the following positions: positions corresponding to amino acid positions 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0230] (12) The N-terminus was truncated by 60 amino acids, the Arg amino acid at position 97 was mutated to a Glu amino acid, and the C-terminus terminated at the following positions: positions corresponding to amino acid positions 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0231] (13) The N-terminus is truncated by 71 amino acids, and the C-terminus terminates at the following positions: positions corresponding to amino acid positions 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0232] (14) The N-terminus was truncated by 71 amino acids, the Arg amino acid at position 97 was mutated to a Glu amino acid, and the C-terminus terminated at the following positions: positions corresponding to amino acid positions 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0233] (15) The N-terminus is truncated by 79 amino acids, and the C-terminus terminates at the following positions: positions corresponding to amino acid positions 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0234] (16) The N-terminus is truncated by 84 amino acids, and the C-terminus terminates at the following positions: positions corresponding to amino acid positions 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0235] (17) The N-terminus is truncated by 89 amino acids, and the C-terminus terminates at the following positions: positions corresponding to amino acid positions 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0236] (18) The N-terminus is truncated by 94 amino acids, and the C-terminus terminates at the following positions: positions corresponding to amino acid positions 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0237] (19) The N-terminus is truncated by 99 amino acids, and the C-terminus terminates at the following positions: positions corresponding to amino acid positions 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0238] (20) The N-terminus is truncated by 104 amino acids, and the C-terminus terminates at the following positions: positions corresponding to amino acid positions 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0239] (21) The N-terminus is truncated by 109 amino acids, and the C-terminus terminates at the following positions: positions corresponding to amino acid positions 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0240] (22) The N-terminus is truncated by 114 amino acids, and the C-terminus terminates at the following positions: positions corresponding to amino acid positions 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0241] (23) The N-terminus is truncated by 0 amino acids, and the C-terminus terminates at the following positions: positions corresponding to amino acid positions 122, 129, 136, 146, 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0242] (24) The N-terminus was truncated by 0 amino acids, the Arg amino acid at position 97 was mutated to a Glu amino acid, and the C-terminus terminated at the following positions: positions corresponding to amino acid positions 122, 129, 136, 146, 156, 161, or 167 of the wild-type LAG3 extracellular domain polypeptide.

[0243] In one embodiment of the LAG3 variant, the LAG3 variant polypeptide is selected from the amino acid sequences shown in SEQ ID NO: 244 to SEQ ID NO: 349.

[0244] On the other hand, the present application provides a LAG3 variant polypeptide comprising truncations and / or mutations based on human LAG3.

[0245] In certain embodiments, the LAG3 variant polypeptide comprises a truncation based on a wild-type human LAG3 extracellular domain polypeptide.

[0246] In certain embodiments, the LAG3 variant polypeptide comprises 0-124 amino acids truncated at the N-terminus based on the wild-type human LAG3 extracellular region polypeptide and terminates at amino acid positions 121-167 based on the wild-type human LAG3 extracellular region polypeptide at the C-terminus.

[0247] In certain embodiments, the LAG3 variant polypeptide comprises 0, 5, 21, 37, 45, 60, 71, 74, 79, 81, 84, 89, 94, 99, 104, 109 or 121 amino acids truncated at the N-terminus based on the wild-type human LAG3 extracellular region polypeptide.

[0248] In certain embodiments, the LAG3 variant polypeptide comprises 0, 5, 21, 37, 45, 60, 71, 74, 81 or 99 amino acids truncated at the N-terminus based on the wild-type human LAG3 extracellular region polypeptide.

[0249] In certain embodiments, the LAG3 variant polypeptide comprises a wild-type human LAG3 extracellular region polypeptide ending at amino acid position 121, 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, or 167 at the C-terminus.

[0250] In certain embodiments, the LAG3 variant polypeptide comprises a wild-type human LAG3 extracellular region polypeptide ending at amino acid position 121, 122, 129, 136, 146, 156, 161, or 167 at the C-terminus.

[0251] In certain embodiments, the LAG3 variant polypeptide comprises a wild-type human LAG3 extracellular domain polypeptide ending at amino acid position 121, 146, 156, 161, or 167 at the C-terminus.

[0252] In certain embodiments, the LAG3 variant polypeptide comprises 0, 5, 21, 37, 45, 60, 71, 74, 81 or 99 amino acids truncated at the N-terminus based on the wild-type human LAG3 extracellular region polypeptide, and terminates at amino acid position 121, 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 or 167 at the C-terminus based on the wild-type human LAG3 extracellular region polypeptide.

[0253] In certain embodiments, the LAG3 variant polypeptide comprises an amino acid position mutation.

[0254] In certain embodiments, the LAG3 variant polypeptide comprises an amino acid mutation at R110, R113, R119, R129, G130, and / or R141.

[0255] In certain embodiments, the LAG3 variant polypeptide is truncated at 81 amino acids at the N-terminus and terminates at amino acid position 121 at the C-terminus, and mutations are introduced at positions R110, and / or R113, and / or R119.

[0256] In certain embodiments, the LAG3 variant polypeptide is truncated at 99 amino acids at the N-terminus and terminates at amino acid position 146 at the C-terminus, and mutations are introduced at R129, and / or G130, and / or R141.

[0257] In certain embodiments, the LAG3 variant polypeptide is truncated at the N-terminus by 81 amino acids and terminates at amino acid position 146 at the C-terminus, and mutations are introduced at R110, and / or R113, and / or R119, and / or R129, and / or G130, and / or R141.

[0258] In certain embodiments, the LAG3 variant polypeptide comprises an amino acid site mutation, wherein the R110 site is mutated to K110.

[0259] In certain embodiments, the LAG3 variant polypeptide comprises an amino acid site mutation, wherein the R113 site is mutated to K113.

[0260] In certain embodiments, the LAG3 variant polypeptide comprises an amino acid site mutation, wherein the R119 site is mutated to K119.

[0261] In certain embodiments, the LAG3 variant polypeptide comprises an amino acid site mutation, wherein the R129 site is mutated to K129.

[0262] In certain embodiments, the LAG3 variant polypeptide comprises an amino acid position mutation, wherein the G130 position is mutated to P130, or A130, or T130, or Y130, or S130.

[0263] In certain embodiments, the LAG3 variant polypeptide comprises an amino acid site mutation, wherein the R141 site is mutated to K141.

[0264] In certain embodiments, the amino acid sequence of the LAG3 variant polypeptide is selected from SEQ ID NO: 1285-SEQ ID NO: 1318.

[0265] On the other hand, the present application provides a fusion polypeptide, which comprises a first domain and a second domain, wherein the first domain comprises the LAG3 variant polypeptide described in the present application, and the second domain comprises an antibody or an antigen-binding fragment thereof, or an immunoglobulin domain.

[0266] In one embodiment of the fusion polypeptide, the second domain comprises an antibody or an antigen-binding fragment thereof.

[0267] In one embodiment of the fusion polypeptide, the second domain is capable of binding to one or more of the following targets: PD-L1, PD-L2, PD-1, OX40, 4-1BB, ICOS, TIGIT, CTLA4, LAG3, CD3, VEGF, VEGFR, CD47, HGF, Trop2, EpCAM, CCR8, CCR4, CCR5, GPRC5D, BCMA, CD19, CD20, HER-2neu, DLL1, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CD123, CD33, CD30, CD38, NKG2A, Nkp36 and Tim3.

[0268] In one embodiment of the fusion polypeptide, the second domain is capable of binding to PD-L1, PD-1 and / or PD-L2.

[0269] In one embodiment of the fusion polypeptide, the second domain comprises HCDR1, HCDR2 and / or HCDR3 of an antibody heavy chain, wherein the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17.

[0270] In one embodiment of the fusion polypeptide, the second domain comprises the heavy chain variable region VH of an antibody heavy chain, and the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 17.

[0271] In one embodiment of the fusion polypeptide, the second domain comprises an antibody heavy chain, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17.

[0272] In one embodiment of the fusion polypeptide, the second domain comprises LCDR1, LCDR2 and / or LCDR3 of a light chain of an antibody light chain, wherein the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 18.

[0273] In one embodiment of the fusion polypeptide, the second domain comprises a light chain variable region VL of an antibody light chain, and the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 18.

[0274] In one embodiment of the fusion polypeptide, the second domain comprises an antibody light chain, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 18.

[0275] In one embodiment of the fusion polypeptide, the second domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of Sugemalimab, Durvalumab, Atezolizumab, Avelumab, Envafolimab, Permbrolizumab and Nivolumab.

[0276] In one embodiment of the fusion polypeptide, the second domain comprises Sugemalimab or an antigen-binding fragment thereof. In one embodiment of the fusion polypeptide, the antigen-binding fragment is selected from one or more of the following groups: Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, VHH, and dAb.

[0277] In one embodiment of the fusion polypeptide, the second domain comprises an immunoglobulin domain.

[0278] In one embodiment of the fusion polypeptide, the second domain comprises an immunoglobulin IgG antibody, and the immunoglobulin IgG antibody comprises one or more selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b and mouse IgG3.

[0279] In one embodiment of the fusion polypeptide, the second domain comprises an immunoglobulin Fc domain.

[0280] In one embodiment of the fusion polypeptide, the second domain comprises the Fc domain of an immunoglobulin IgG antibody.

[0281] In one embodiment of the fusion polypeptide, the second domain comprises the Fc domain of an immunoglobulin IgG antibody, and the Fc domain of the immunoglobulin IgG antibody comprises a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgG1 Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain or a mouse IgG3 Fc domain.

[0282] In one embodiment of the fusion polypeptide, the second domain comprises the Fc domain of human immunoglobulin IgG4.

[0283] In one embodiment of the fusion polypeptide, the second domain is selected from the group consisting of amino acid sequences shown in SEQ ID NO: 4 to SEQ ID NO: 18, SEQ ID NO: 29 to SEQ ID NO: 30, and SEQ ID NO: 634 to SEQ ID NO: 639.

[0284] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly linked to the second domain.

[0285] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly connected to the N-terminus of the second domain.

[0286] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly connected to the C-terminus of the second domain.

[0287] In one embodiment of the fusion polypeptide, the indirect linkage comprises linkage via a linker.

[0288] In one embodiment of the fusion polypeptide, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.

[0289] In one embodiment, the fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 640 to SEQ ID NO: 745 and SEQ ID NO: 747 to SEQ ID NO: 750.

[0290] In certain embodiments, the fusion polypeptide comprises an antibody heavy chain, preferably the antibody heavy chain polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4; the amino acid sequence comprising the antibody heavy chain is selected from SEQ ID NO: 1353 to SEQ ID NO: 1386.

[0291] In certain embodiments, the fusion polypeptide comprises a light chain, and the light chain comprises an antibody light chain polypeptide, preferably an antibody light chain polypeptide comprising the amino acid sequence shown in SEQ ID NO:5.

[0292] In certain embodiments, the fusion polypeptide heavy chain and the fusion polypeptide light chain are combined into the fusion polypeptide.

[0293] In certain embodiments, the first domain is directly or indirectly linked to the second domain.

[0294] In certain embodiments, the first domain is directly or indirectly connected to the N-terminus of the second domain.

[0295] In certain embodiments, the first domain is directly or indirectly connected to the C-terminus of the second domain.

[0296] In certain embodiments, the second domain may comprise an antibody heavy chain, and the LAG3 variant polypeptide may be directly or indirectly linked to the antibody heavy chain of the second domain.

[0297] In certain embodiments, the second domain may comprise an antibody heavy chain, and the LAG3 variant polypeptide may be directly or indirectly linked to the C-terminus of the antibody heavy chain of the second domain.

[0298] In certain embodiments, the second domain may comprise an antibody heavy chain, and the N-terminus of the LAG3 variant polypeptide may be directly or indirectly linked to the C-terminus of the antibody heavy chain of the second domain.

[0299] In certain embodiments, the second domain may comprise an antibody heavy chain, and the LAG3 variant polypeptide may be directly or indirectly linked to the N-terminus of the antibody heavy chain of the second domain.

[0300] In certain embodiments, the second domain may comprise an antibody heavy chain, and the C-terminus of the LAG3 variant polypeptide may be directly or indirectly linked to the N-terminus of the antibody heavy chain of the second domain.

[0301] In certain embodiments, the second domain may comprise an antibody light chain, and the LAG3 variant polypeptide may be directly or indirectly linked to the antibody light chain of the second domain.

[0302] In certain embodiments, the second domain may comprise an antibody light chain, and the LAG3 variant polypeptide may be directly or indirectly linked to the C-terminus of the antibody light chain of the second domain.

[0303] In certain embodiments, the second domain may comprise an antibody light chain, and the N-terminus of the LAG3 variant polypeptide may be directly or indirectly linked to the C-terminus of the antibody light chain of the second domain.

[0304] In certain embodiments, the second domain may comprise an antibody light chain, and the LAG3 variant polypeptide may be directly or indirectly linked to the N-terminus of the antibody light chain of the second domain.

[0305] In certain embodiments, the second domain may comprise an antibody light chain, and the C-terminus of the LAG3 variant polypeptide may be directly or indirectly linked to the N-terminus of the antibody light chain of the second domain.

[0306] In certain embodiments, the indirect linkage comprises linkage through a linker.

[0307] In certain embodiments, the second domain comprises an immunoglobulin Fc domain.

[0308] In certain embodiments, the second domain comprises the Fc domain of an immunoglobulin IgG antibody.

[0309] In certain embodiments, the second domain comprises the Fc domain of an immunoglobulin IgG antibody, and the Fc domain of the immunoglobulin IgG antibody comprises a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgG1 Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain, or a mouse IgG3 Fc domain.

[0310] In certain embodiments, the second domain comprises the Fc domain of human immunoglobulin IgG4.

[0311] In certain embodiments, the LAG3 variant polypeptide is covalently linked to the second domain with or without a linker peptide.

[0312] In certain embodiments, the second domain is selected from the group consisting of amino acid sequences shown in SEQ ID NO: 1313 to SEQ ID NO: 1352.

[0313] In certain embodiments, the fusion polypeptide further comprises a third domain, and the third domain is an antibody or antigen-binding fragment or a functional protein or an active fragment thereof that is the same as or different from the second domain.

[0314] On the other hand, the present application provides a fusion polypeptide comprising a first domain, a second domain, and a third domain, wherein the first domain comprises the LAG3 variant polypeptide described herein, the second domain comprises an antibody or an antigen-binding fragment thereof, or an immunoglobulin domain, and the third domain is an antibody or antigen-binding fragment or a functional protein or an active fragment thereof that is the same as or different from the second domain.

[0315] In one embodiment of the fusion polypeptide, the second domain comprises an antibody or an antigen-binding fragment thereof.

[0316] In one embodiment of the fusion polypeptide, the second domain is capable of binding to one or more of the following targets: PD-L1, PD-L2, PD-1, OX40, 4-1BB, ICOS, TIGIT, CTLA4, LAG3, CD3, VEGF, VEGFR, CD47, HGF, Trop2, EpCAM, CCR8, CCR4, CCR5, GPRC5D, BCMA, CD19, CD20, HER-2neu, DLL1, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CD123, CD33, CD30, CD38, NKG2A, Nkp36 and Tim3.

[0317] In one embodiment of the fusion polypeptide, the second domain is capable of binding to PD-L1, PD-1, and / or PD-L2. In one embodiment of the fusion polypeptide, the second domain comprises HCDR1, HCDR2, and / or HCDR3 of an antibody heavy chain, wherein the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 17.

[0318] In one embodiment of the fusion polypeptide, the second domain comprises the heavy chain variable region VH of an antibody heavy chain, and the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 17.

[0319] In one embodiment of the fusion polypeptide, the second domain comprises an antibody heavy chain, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17.

[0320] In one embodiment of the fusion polypeptide, the second domain comprises LCDR1, LCDR2 and / or LCDR3 of a light chain of an antibody light chain, wherein the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 18.

[0321] In one embodiment of the fusion polypeptide, the second domain comprises a light chain variable region VL of an antibody light chain, and the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 18.

[0322] In one embodiment of the fusion polypeptide, the second domain comprises an antibody light chain, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO: 18.

[0323] In one embodiment of the fusion polypeptide, the second domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of Sugemalimab, Durvalumab, Atezolizumab, Avelumab, Envafolimab, Permbrolizumab and Nivolumab.

[0324] In one embodiment of the fusion polypeptide, the second domain comprises Sugemalimab or an antigen-binding fragment thereof.

[0325] In one embodiment of the fusion polypeptide, the antibody is selected from the group consisting of an immunoglobulin antibody, a recombinant antibody, a chimeric antibody, a heavy chain antibody, a single domain antibody, and a bispecific antibody.

[0326] In one embodiment of the fusion polypeptide, the antigen-binding fragment is selected from one or more of the following groups: Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, VHH and dAb.

[0327] In one embodiment of the fusion polypeptide, the second domain comprises an immunoglobulin domain.

[0328] In one embodiment of the fusion polypeptide, the second domain comprises an immunoglobulin IgG antibody, and the immunoglobulin IgG comprises one or more selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b and mouse IgG3.

[0329] In one embodiment of the fusion polypeptide, the second domain comprises an immunoglobulin Fc domain.

[0330] In one embodiment of the fusion polypeptide, the second domain comprises an immunoglobulin IgG Fc domain, and the immunoglobulin Fc domain comprises a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgG1 Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain or a mouse IgG3 Fc domain.

[0331] In one embodiment of the fusion polypeptide, the immunoglobulin Fc domain comprises the Fc domain of human immunoglobulin IgG4.

[0332] In one embodiment of the fusion polypeptide, the second domain is selected from the group consisting of amino acid sequences shown in SEQ ID NO: 4 to SEQ ID NO: 18, SEQ ID NO: 29 to SEQ ID NO: 30, and SEQ ID NO: 634 to SEQ ID NO: 639.

[0333] In one embodiment of the fusion polypeptide, the third domain is a functional protein or an active fragment thereof, and the functional protein or the active fragment thereof can activate the innate immune response.

[0334] In one embodiment of the fusion polypeptide, the third domain is capable of binding to CD28 and / or CTLA4.

[0335] In one embodiment of the fusion polypeptide, the third domain comprises CD80, CD86 or an active fragment thereof.

[0336] In one embodiment of the fusion polypeptide, the third domain is selected from the following group: CD86 derived from humans or a functionally active fragment thereof, CD86 derived from mice or a functionally active fragment thereof, CD80 derived from humans or a functionally active fragment thereof, and CD80 derived from mice or a functionally active fragment thereof.

[0337] In one embodiment of the fusion polypeptide, the third domain comprises the IgV domain of CD80, the IgV domain of CD86, or an active fragment thereof.

[0338] In one embodiment of the fusion polypeptide, the third domain comprises the extracellular domain of CD80, the extracellular domain of CD86, or an active fragment thereof.

[0339] In one embodiment of the fusion polypeptide, the third domain comprises a CD86 variant polypeptide.

[0340] In one embodiment of the fusion polypeptide, the third domain comprises a CD86 variant polypeptide, and the mutant comprises an amino acid substitution mutation of humanized CD86.

[0341] In one embodiment of the fusion polypeptide, the third domain comprises a CD86 variant polypeptide comprising an amino acid substitution mutant of the IgV domain of CD86 and / or an amino acid substitution mutant of the extracellular domain of humanized CD86.

[0342] In one embodiment of the fusion polypeptide, the third domain comprises a CD86 variant polypeptide, which comprises an amino acid substitution mutant of the IgV domain of CD86, and the mutation site of the amino acid substitution mutant of the IgV domain of CD86 comprises one or more amino acid site mutations selected from the group consisting of A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, and H90F.

[0343] In one embodiment of the fusion polypeptide, the third domain comprises a CD86 variant polypeptide, which comprises an amino acid substitution mutant of the extracellular domain of CD86, and the amino acid substitution mutant of the extracellular domain of CD86 comprises one or more amino acid site mutations selected from the group consisting of A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, and H90F.

[0344] In one embodiment of the fusion polypeptide, the third domain is selected from the group consisting of amino acid sequences shown in SEQ ID NO: 2 to SEQ ID NO: 3, and SEQ ID NO: 178 to SEQ ID NO: 243.

[0345] In certain embodiments, the amino acid substitution mutants of the IgV or extracellular domain of CD86 can be selected from the following groups of amino acid mutation sites: Q25I / F33L / H90I, Q25V / F33L / H90I, Q25I / F33V / H90I, Q25V / F33V / H90I, Q25I / F33L / H90V, Q25V / F33L / H90V, Q25I / F33 V / H90V, Q25V / F33V / H90V, A13L / Q25I / F33L / H90I, A13I / Q25I / F33L / H90I, A13L / Q25V / F3 3L / H90I, A13I / Q25V / F33L / H90I, Q25I / F33L / I89L / H90I, Q25I / F33L / M60R / H90I, Q25V / F3 3L / I89L / H90I, Q25V / F33L / M60R / H90I, Q25F / F33L / H90I, Q25I / F33L / H90F, Q25I / F33A / H 90I, Q25I / H90I, Q25I, H90I, Q25V / H90V, Q25V / H90I, Q25F / H90I, Q25F / H90V, A13F / Q25I / F 33L / H90I, A13M / Q25I / F33L / H90I, Q25A / F33L / H90I, Q25M / F33L / H90I, Q25I / F33M / H90I, Q25I / F33L / I89V / H90I, Q25I / F33L / I89F / H90I, Q25I / F33L / I89M / H90I, and Q25I / F33L / H90M.

[0346] In certain embodiments, the amino acid substitution mutant of the IgV or extracellular domain of CD86 comprises the following amino acid mutation sites: Q25I / F33L / H90I.

[0347] In one embodiment of the fusion polypeptide, the first domain and the second domain are directly or indirectly linked.

[0348] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly linked to the third domain.

[0349] In one embodiment of the fusion polypeptide, the second domain and the third domain are directly or indirectly linked.

[0350] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly linked to the second domain, and the first domain is directly or indirectly linked to the third domain.

[0351] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly linked to the second domain, and the second domain is directly or indirectly linked to the third domain.

[0352] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly linked to the third domain, and the second domain is directly or indirectly linked to the third domain.

[0353] In one embodiment of the fusion polypeptide, the indirect linkage comprises linkage via a linker.

[0354] In one embodiment of the fusion polypeptide, the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.

[0355] In one embodiment, the fusion polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 751 to SEQ ID NO: 1024, 1249, 1251, 1253 and 1255.

[0356] In certain embodiments, the fusion polypeptide comprises a multifunctional LAG3 variant fused to the heavy chain of the Sugemalimab-CD86 IgV Q25I / F33L / H90I antibody polypeptide complex, wherein the heavy chain amino acid sequence is selected from SEQ ID NO: 1387 to SEQ ID NO: 1420.

[0357] In certain embodiments, the fusion polypeptide comprises a multifunctional LAG3 variant fused to the heavy chain of the Sugemalimab-CD86 IgV Q25I / F33L / H90I antibody polypeptide complex, wherein the heavy chain amino acid sequence is selected from SEQ ID NO: 1421 and SEQ ID NO: 1426.

[0358] In certain embodiments, the nucleic acid encoding the fusion polypeptide comprises the nucleotide sequence shown in any one of SEQ ID NOs: 1422-1425, 1427-1433.

[0359] On the other hand, the present application provides an immunoconjugate comprising the fusion polypeptide described in the present application, the CD86 variant polypeptide described in the present application, and / or the LAG3 variant described in the present application.

[0360] On the other hand, the present application provides a nucleic acid molecule encoding the fusion polypeptide described in the present application, the CD86 variant polypeptide described in the present application and / or the LAG3 variant described in the present application.

[0361] In another aspect, the present application provides a vector comprising the nucleic acid molecule described in the present application.

[0362] On the other hand, the present application provides a cell comprising and / or expressing the fusion polypeptide described herein, the CD86 variant polypeptide described herein, the LAG3 variant described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, and / or the vector described herein.

[0363] On the other hand, the present application provides a composition comprising the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, and / or the cell described herein, and optionally a pharmaceutically acceptable carrier.

[0364] On the other hand, the present application provides a method for preparing the fusion polypeptide described herein, the CD86 variant polypeptide described herein, and the LAG3 variant described herein, comprising culturing the cell described herein under conditions that allow the expression of the fusion polypeptide.

[0365] On the other hand, the present application provides a method for blocking the interaction between PD-L1 protein and PD-1, comprising administering an effective amount of the fusion polypeptide described herein, the CD86 variant polypeptide described herein, the LAG3 variant described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the composition described herein.

[0366] On the other hand, the present application provides a method for stimulating antigen-presenting cells and / or activating T cells, which comprises administering an effective amount of the fusion polypeptide described herein, the CD86 variant polypeptide described herein, the LAG3 variant described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the composition described herein.

[0367] According to the method described in the present application, the stimulation of antigen-presenting cells comprises a method selected from the group consisting of increasing the expression of co-stimulatory molecules in antigen-presenting cells, causing morphological changes and maturation of antigen-presenting cells, increasing the secretion of chemokines in antigen-presenting cells, and enhancing the phagocytic ability of antigen-presenting cells.

[0368] On the other hand, the present application provides a method for inhibiting the growth and / or proliferation of tumors or tumor cells, which comprises administering an effective amount of the fusion polypeptide described herein, the CD86 variant polypeptide described herein, the LAG3 variant described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the composition described herein.

[0369] On the other hand, the present application provides the use of the fusion polypeptide described herein, the CD86 variant polypeptide described herein, the LAG3 variant described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the composition described herein in the preparation of a medicament, wherein the medicament is used to prevent, improve and / or treat tumors.

[0370] According to the use described in the present application, the tumor includes solid tumors and / or hematological tumors.

[0371] According to the use described in the present application, the tumor is selected from the following group: colon tumor, breast tumor, lung tumor, gastric tumor, melanoma, head and neck tumor, lymphoma, nasopharyngeal tumor, cervical tumor, esophageal tumor, kidney tumor, skin squamous cell carcinoma, endometrial tumor, liver tumor, bladder tumor, urothelial tumor and skin tumor.

[0372] On the other hand, the present application provides the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the composition described herein, which are used to prevent, improve and / or treat tumors.

[0373] On the other hand, the present application provides the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the composition described herein, which are used to prevent, improve and / or treat tumors, wherein the tumors include solid tumors and hematological tumors.

[0374] On the other hand, the present application provides the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the composition described herein, which are used to prevent, improve and / or treat tumors, wherein the tumor is selected from the following group: colon tumor, breast tumor, lung tumor, gastric tumor, melanoma, head and neck tumor, lymphoma, nasopharyngeal tumor, cervical tumor, esophageal tumor, kidney tumor, skin squamous cell carcinoma, endometrial tumor, liver tumor, bladder tumor, urothelial tumor and skin tumor.

[0375] On the other hand, the present application provides a method for preventing, improving and / or treating tumors, which may comprise administering the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the composition described herein to a subject in need thereof.

[0376] According to the method described in the present application, the tumor includes solid tumors and hematological tumors.

[0377] According to the method described in the present application, the tumor is selected from the following groups: colon tumor, breast tumor, lung tumor, gastric tumor, melanoma, head and neck tumor, lymphoma, nasopharyngeal tumor, cervical tumor, esophageal tumor, kidney tumor, skin squamous cell carcinoma, endometrial tumor, liver tumor, bladder tumor, urothelial tumor and skin tumor.

[0378] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0379] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0380] Figure 1 shows an exemplary structural diagram of the fusion polypeptide described in the present application. The present application provides exemplary structures that may include any one or more subunits in the first domain derived from any anti-PD-L1 and / or anti-PD-1 antibody antigen recognition region (i), an immunoglobulin Fc region (ii), the second domain (iii) and / or the third domain (iv) (herein, the second domain (iii) and / or the third domain (iv) having one subunit are shown, although two or more of the same subunits can be considered in series, and although it can be extended to any subunit mixed combination in series in (iv); the first domain is connected to the second domain via a linker or covalently through the immunoglobulin Fc region. Similarly, the subunits of the third domain and the second domain can be connected via a linker or covalently.

[0381] FIG2 shows the results of SDS-PAGE detection of the expression of the fusion polypeptide complex of the present application.

[0382] FIG3 shows the binding of the fusion polypeptide complex of the present application to the human PDL1 protein detected by ELISA.

[0383] FIG4 shows the results of ELISA detection of the binding between the fusion polypeptide complex of the present application and the human CTLA4 protein.

[0384] FIG5 shows the results of ELISA detection of the binding between the fusion polypeptide complex of the present application and human CD28 protein.

[0385] FIG6 shows the results of the regulation of Jurkat T cell activation by the fusion polypeptide complex of the present application.

[0386] FIG7 shows the result of the fusion polypeptide complex of the present application regulating the up-regulation of the expression of costimulatory molecules in antigen presenting cells.

[0387] FIG8 shows the results of the fusion polypeptide complex of the present application regulating the morphological differentiation of antigen-presenting cells.

[0388] FIG9 shows the results of the fusion polypeptide complex of the present application regulating antigen presentation and secretion of CCL4.

[0389] FIG10 shows the expression of CD86 mutant fusion polypeptide complexes detected by SDS-PAGE.

[0390] FIG11 shows the expression of CD86 variant polypeptide fusion polypeptide complex detected by AKTA.

[0391] FIG12 shows the results of the present application's fusion polypeptide complex regulating antigen presentation and secretion of CCL4.

[0392] FIG13 shows the effect of the fusion polypeptide complex of the present application on inhibiting tumor growth.

[0393] FIG14 shows the results of SDS-PAGE detection of the expression of the LAG3 variant polypeptide complex of the present application.

[0394] FIG15 shows the results of the fusion polypeptide complex of the present application regulating the secretion of CCL4 by antigen-presenting cells.

[0395] FIG16 shows the results of the fusion polypeptide complex of the present application regulating the secretion of CCL4 by primary PBMC cells.

[0396] FIG17 shows the results of the fusion polypeptide complex of the present application regulating the morphological differentiation of primary DCs.

[0397] FIG18 shows the results of the fusion polypeptide complex of the present application regulating the up-regulation of co-stimulatory molecule expression in primary DCs cells.

[0398] FIG19 shows the results of the fusion polypeptide complex of the present application regulating the secretion of TNFa by primary DCs cells.

[0399] FIG20 shows the results of the fusion polypeptide complex of the present application regulating T cell activation.

[0400] FIG21 shows the results of the LAG3 variant polypeptide complex of the present application regulating the morphological differentiation of primary DCs cells.

[0401] FIG22 shows the results of the LAG3 variant polypeptide complex of the present application regulating the up-regulation of co-stimulatory molecule expression in primary DCs cells.

[0402] FIG23 shows the results of the LAG3 variant polypeptide complex of the present application regulating the secretion of TNFa by primary DCs cells.

[0403] FIG24 shows the deglycosylated and reduced LC-MS mass analysis of the LAG3 variant polypeptide complex of the present application. DETAILED DESCRIPTION

[0404] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0405] Definition of terms

[0406] In this application, the term "regulating T cell immune response" generally refers to the regulation of lymphocyte T cell function by the dual and / or multifunctional fusion polypeptide disclosed in this application. For example, it can also be extended to any other related T cell regulators that affect the expression of T cell NFAT transcription factors, the secretion of cytokines such as IL-2, IFN-γ, TNFα and Granzyme B, cell proliferation and cell killing of target cells including tumor cells after treating T cells. The T cells can be expanded to T cell lines and / or primary T cells including but not limited to CD4 T cells and CD4 T cell subsets Th1, Th2, Th9, Th17, TFH and / or Treg cells; CD8 T cells also include but are not limited to tumor tissue infiltrating CD8 T cells, effector CD8 T cells, immune memory CD8 T cells, etc.

[0407] In the present application, the term "stimulating antigen-presenting cells" generally refers to regulating the activation of antigen-presenting cells by the dual and / or multifunctional fusion polypeptide disclosed in the present application. For example, it can also be expanded to any other antigen-presenting cell regulators that have the ability to affect antigen-presenting cells after treating antigen-presenting cells, including but not limited to the expression of co-stimulatory molecules ICOSL, CD40L, CD137L, OX40L, CD80, CD83 and / or CD86, antigen-presenting cell activation and differentiation and maturation, antigen phagocytosis and presentation, production of chemokines CCL4, CCL22, CCL17, etc., expression of cytokines IL-1beta, TNFa, IFNγ, IL-6, IL-12, etc., recruitment of T cells to migrate to secondary lymphoid organs, and enhancement of T cell immune responses; the antigen-presenting cells can be expanded to but not limited to B lymphocyte lines and primary B lymphocytes, monocyte lines and primary monocytes, macrophage lines and primary macrophages, dendritic cell (DC) cell lines and primary DC cells, etc., and the antigen phagocytosis includes but is not limited to the effect of phagocytosis of bacteria, viruses, proteins, polysaccharides, etc.

[0408] In the present application, the term "relieving the inhibition of PD-L1 / PD-1 on T cells" generally refers to the dual and / or multifunctional fusion polypeptide disclosed in the present application binding to the PDL1 protein and blocking the binding of PD-L1 to PD-1, thereby improving the inhibition of PD-L1 / PD-1 on T cells, including but not limited to the expression of NFAT transcription factors by T cells, the secretion of cytokines such as IL-2, IFN-γ, TNFα and Granzyme B, cell proliferation and cell killing of target cells.

[0409] In this application, the term "anti-tumor activity" generally refers to any biological activity that reduces or prevents the proliferation or viability of tumor cells in vivo and / or in vitro. In one embodiment, the anti-tumor activity is the anti-tumor effect of the dual and / or multifunctional fusion polypeptides described herein.

[0410] In this application, the term "CD86" generally refers to a class of molecules that activate cells. For example, in this application, CD86 includes its full length, variants, and / or functionally active fragments. In the present application, CD86 can refer to a polypeptide or fragment thereof that has at least about 85% amino acid identity with the protein encoded by the gene with NCBI accession number Gene ID: 942 and has CD28 (protein encoded by the gene with NCBI accession number Gene ID: 940) and / or CTLA4 (protein encoded by the gene with NCBI accession number Gene ID: 1493). An exemplary human CD86 amino acid sequence is provided below (SEQ ID NO: 1); the terms "CD86 extracellular region", "CD86 extracellular domain" and "CD86 ECD region" refer to the amino acid sequence of the domain in the extracellular region of the CD86 protein and have at least about 85% amino acid identity, and have CD28 and / or CTLA4 binding activity. An exemplary CD86 extracellular region amino acid sequence is provided below (SEQ ID NO: 2). The terms "CD86 extracellular domain IgV domain" and "CD86 ECD region" refer to polypeptides or fragments thereof that have CD28 and / or CTLA4 binding activity. "IgV domain" refers to an amino acid sequence having at least about 85% amino acid identity with an IgV-like domain in the extracellular region of a CD86 protein, and a polypeptide or fragment thereof having CD28 and / or CTLA4 binding activity. An exemplary amino acid sequence of an extracellular domain IgV domain of CD86 is provided below (SEQ ID NO: 3). The number of CD86 extracellular domains or tandem CD86 extracellular domain IgV domains disclosed in this invention is not limited to the exemplary number shown, and any tandem combination comprising this functional domain is considered within the scope of the present invention.

[0411] In this application, the term "anti-PD-L1 antibody" generally refers to an antibody that selectively binds to and has the activity of blocking PD-L1 polypeptide. For example, Chinese Patent No. CN102245640(B), U.S. Patent Application Publication No. 7,943,743, U.S. Patent Application Publication No. 8,779,108 and U.S. Patent Application Publication No. 7,943,743, which are incorporated herein by reference; including but not limited to anti-PD-L1 antibodies such as Durvalumab (MEDI4736) developed by AstraZeneca, Sugemalimab (CS1001) developed by CStone Pharmaceuticals, JS003 developed by Junshi Pharmaceuticals, Envafolimab (KN035) developed by Alphamab Oncology, Atezolizumab (MPDL3280A) developed by Roche and Avelumab (MSB0010718C) jointly developed by Merck KGaA / Pfizer, Germany. The amino acid sequence of the heavy chain polypeptide of exemplary anti-PD-L1 antibody Sugemalimab (CS1001) is provided below (SEQ ID NO: 4) and light chain polypeptide amino acid sequence (SEQ ID NO: 5), JS003 heavy chain polypeptide amino acid sequence (SEQ ID NO: 6) and light chain polypeptide amino acid sequence (SEQ ID NO: 7), durvalumab (MEDI4736) heavy chain polypeptide amino acid sequence (SEQ ID NO: 8) and light chain polypeptide amino acid sequence (SEQ ID NO: 9), atezolizumab (MPDL3280A) heavy chain polypeptide amino acid sequence (SEQ ID NO: 10) and light chain polypeptide amino acid sequence (SEQ ID NO: 11), avelumab (MSB0010718C) heavy chain polypeptide amino acid sequence (SEQ ID NO: 12) and light chain polypeptide amino acid sequence (SEQ ID NO: 13), and envafolimab (KN035) amino acid sequence (SEQ ID NO: 14).

[0412] As used herein, the term "anti-PD-1 antibody" refers to an antibody that selectively binds to and has the activity of blocking a PD-1 polypeptide. Examples include U.S. Patent Application Publication No. 8,354,509 and U.S. Patent Application Publication No. 7,488,802, which are incorporated herein by reference; and include, but are not limited to, anti-PD-1 antibodies such as Permbrolizumab (MK-3475) developed by Merck and Nivolumab (BMS-936558) developed by Bristol-Myers Squibb. Exemplary anti-PD-1 antibodies Permbrolizumab (MK-3475) heavy chain polypeptide amino acid sequence (SEQ ID NO: 15) and light chain polypeptide amino acid sequence (SEQ ID NO: 16), and Nivolumab (BMS-936558) heavy chain polypeptide amino acid sequence (SEQ ID NO: 17) and light chain polypeptide amino acid sequence (SEQ ID NO: 18) are provided below.

[0413] In this application, the term "LAG3" generally refers to a class of proteins or polypeptides. For example, in this application, LAG3 includes its full-length, variants, and / or functionally active fragments. For example, LAG3 can indicate a polypeptide or fragment thereof that has at least about 85% amino acid identity with the protein encoded by the NCBI accession number Gene ID: 3902 gene of LAG3 (Lymphocyte activation gene 3), and has binding activity with the complex protein MHCII (or HLA-DR) assembled by the α subunit (protein encoded by the NCBI accession number Gene ID: 3122 gene) and the β subunit (protein encoded by the NCBI accession number Gene ID: 3123 gene, or protein encoded by the NCBI accession number Gene ID: 3125 gene), and / or FGL1 (protein encoded by the NCBI accession number Gene ID: 2267 gene), and / or activates antigen-presenting cells. An exemplary human LAG3 amino acid sequence is provided below (SEQ ID NO: 19); the term "LAG3 extracellular region" refers to a polypeptide or fragment thereof having an amino acid sequence with at least about 85% amino acid identity to the extracellular region of the full-length LAG3 protein (a polypeptide comprising amino acids 23-450 of the LAG3 protein according to NCBI numbering; the extracellular region starts at amino acid position 23, so, for example, a truncation of 74 amino acids may start at position 97), and having MHCII and / or FGL1 binding activity and / or activating antigen-presenting cells. An exemplary human LAG3 extracellular domain amino acid sequence (SEQ ID NO: 20) and a tandem human LAG3 extracellular domain amino acid sequence covalently linked by a linker (SEQ ID NO: 21) are provided below; the number of tandem structures disclosed in this invention is not limited to the exemplary number shown; any tandem combination comprising this functional domain is considered within the scope of the present invention.

[0414] As used herein, the term "LAG3-IgD1" refers to a polypeptide or fragment thereof having an amino acid sequence of at least about 85% amino acid identity with the Ig-like V-type domain in the extracellular region of human LAG3. An exemplary human LAG3-IgD1 amino acid sequence is provided below (SEQ ID NO: 22). The term "LAG3-IgD2" refers to an amino acid sequence of at least about 85% amino acid identity with the Ig-like C2-type 1 domain in the extracellular region of human LAG3. An exemplary human LAG3-IgD2 amino acid sequence is provided below (SEQ ID NO: 23). The term "LAG3-IgD3" refers to an amino acid sequence of at least about 85% amino acid identity with the Ig-like C2-type 2 domain in the extracellular region of human LAG3. An exemplary human LAG3-IgD3 amino acid sequence is provided below (SEQ ID NO: NO: 24); the term "LAG3-IgD4" refers to the amino acid sequence of the Ig-like C2-type 3 domain of the extracellular region of human LAG3 and has at least about 85% amino acid identity. An exemplary human LAG3-IgD4 amino acid sequence is provided below (SEQ ID NO: 25); the term "LAG3-IgD1 / D2" refers to a polypeptide or fragment thereof comprising the amino acid sequences of human LAG3-IgD1 and human LAG3-IgD2 and having at least about 85% amino acid identity, and having MHCII and / or FGL1 binding activity, and / or activating antigen-presenting cells. An exemplary human LAG3-D1 / D2 amino acid sequence is provided below (SEQ ID NO: NO: 26); the number of tandems disclosed in this invention is not limited to the number shown as an example, and any tandem combination comprising the functional domain of LAG3-D1 / D2 is considered to be within the scope of the present invention; the term "LAG3-D1 / D2 / D3" refers to a polypeptide or fragment thereof comprising the amino acid sequence of human LAG3-IgD1, human LAG3-IgD2, and human LAG3-IgD3, and having at least about 85% amino acid identity, and having MHCII and / or FGL1 binding activity. An exemplary human LAG3-IgD1 / D2 / D3 amino acid sequence is provided below (SEQ ID NO: 27). The number of tandems disclosed in this invention is not limited to the number shown as an example, and any tandem combination comprising the functional domain of LAG3-D1 / D2 / D3 is considered to be within the scope of the present invention;The term "LAG3-D1 / D2 / D3 / D4" refers to a polypeptide or fragment thereof comprising the amino acid sequence of human LAG3-IgD1, human LAG3-IgD2, human LAG3-IgD3, and human LAG3-IgD4, sharing at least about 85% amino acid identity, and having MHCII and / or FGL1 binding activity and / or antigen-presenting cell activation. An exemplary human LAG3-D1 / D2 / D3 / D4 amino acid sequence is provided below (SEQ ID NO: 28). The number of tandem domains disclosed in this invention is not limited to the exemplary number shown; any tandem combination comprising the functional LAG3-D1 / D2 / D3 / D4 domains is considered within the scope of the present invention.

[0415] In this application, the term "fusion polypeptide" generally refers to a polypeptide obtained by the fusion of two or more proteins or polypeptides. In this application, "fusion polypeptide" and "fusion protein" can be used interchangeably. Fusion polypeptides can include fusion polypeptide complexes. Fusion polypeptides can be artificially prepared using recombinant DNA technology. For example, genes or nucleic acid molecules encoding the two or more proteins or polypeptides can be linked to form a fusion gene or fused nucleic acid molecule, which can encode the fusion polypeptide. Translation of the fusion gene can produce a single polypeptide that can have the properties of at least one, or even each, of the two or more proteins or polypeptides before fusion.

[0416] In this application, the term "dual and / or multifunctional fusion polypeptide" generally refers to a polypeptide or protein formed by the fusion of polypeptides or their domains from one or more sources. The anti-PD-L1 antibody and / or anti-PD-1 antibody is valently linked to a functional fusion polypeptide comprising at least the CD86 extracellular domain, or / and at least the LAG3 extracellular domain, or / and at least one LAG3 extracellular structure, through the linker, wherein the disclosed two or more LAG3 extracellular domains or / and at least one LAG3 extracellular domain functional domain are tandemly linked to the LAG3 extracellular domain or / and at least one LAG3 extracellular structure functional domain. The structural domain or / and the functional domain comprising at least one LAG3 extracellular structure are connected by the linker; the dual and / or multifunctional fusion polypeptides disclosed in the present application include but are not limited to the anti-PD-L1 antibody and / or the anti-PD-1 antibody; any form of anti-PD-L1 antibody and / or anti-PD-1 antibody covalently linked to the CD86 extracellular domain, and / or LAG3 extracellular domain, and / or at least one subunit functional domain in the LAG3 extracellular structure through a linker, or a functional fusion polypeptide or combination thereof should be considered within the scope of the present application.

[0417] In this application, the term "antibody-related antigen-binding fragment" generally refers to an amino acid fragment within an antibody that is responsible for specific antigen binding. It can be a fragment that determines the key differences in an antibody molecule, and can also be called an antigen-binding domain, or an "epitope" or "antigenic determinant." The antigen-binding domain is generally composed of an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), but it does not necessarily include both. The antigen-binding domain of the antibody disclosed in this application is not limited to the domain composed of traditional VH and VL, but also includes any other type of antibody, but not limited to, antigen-binding domains contained in recombinant antibodies, single-domain antibodies, heavy-chain antibodies, chimeric antibodies, bispecific antibodies, and other unconventional antibodies, and combinations thereof.

[0418] Furthermore, the anti-PD-1 antibodies and / or anti-PD-L1 antibodies disclosed in the present application are not limited to traditional natural antibodies, but should include any other type of antibodies with anti-PD-1 antibody and / or anti-PD-L1 properties, but are not limited to, for example, recombinant antibodies, single domain antibodies, heavy chain antibodies, chimeric antibodies, bispecific antibodies and other unconventional antibodies and their combinations.

[0419] In this application, the term "Fab" generally refers to an antibody fragment consisting of the VL, VH, CL and CH1 domains.

[0420] In this application, the term "Fab'" generally refers to an antibody fragment that has a few additional residues at the carboxyl terminus of the CH1 domain compared to the Fab fragment. For example, Fab' may include one or more cysteines from the antibody hinge region.

[0421] In this application, the term "F(ab)2" generally refers to an antigen-binding fragment obtained by a pair of cysteine-linked Fab fragments.

[0422] In this application, the term "dAb fragment" generally refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341: 544 546 (1989)).

[0423] In this application, the term "complementarity determining region CDR" generally refers to the three hypervariable regions (HVRs) of the light chain variable region (VL) and the heavy chain variable region (VH). Because this part can form precise complementarity with the antigenic determinant cluster in terms of spatial structure, the hypervariable region is also called the complementarity determining region.

[0424] In this application, the term "Fv fragment" generally refers to an antibody fragment consisting of the VL and VH domains of a single arm of an antibody.

[0425] In this application, the term "scFv" generally refers to a molecule composed of the heavy chain variable region and the light chain variable region of an antibody connected by a short peptide linker, also known as a single-chain antibody.

[0426] In this application, the term "immunoglobulin" refers to an antibody composed of an antigen-specific binding amino acid segment and a constant region segment. The antigen-specific binding region is the segment that determines the key difference in the immunoglobulin, which can also be called the antigen-binding domain, or also called the "epitope" or "antigenic determinant". The antigen-binding domain is generally composed of the antibody heavy chain variable region (VH) and the antibody light chain variable region (VL). However, it does not necessarily include both. The antigen-binding domain of the antibody disclosed in the present invention is not limited to the domain composed of traditional VH and VL, but also includes any other type of antibody, but not limited to, such as recombinant antibodies, single-domain antibodies, heavy chain antibodies, chimeric antibodies, bispecific antibodies and other unconventional antibodies and their combinations. The constant region refers to the common structural region of immunoglobulins, which includes the antibody light chain constant region and the heavy chain constant region.

[0427] In this application, the term "immunoglobulin Fc domain" generally refers to the Fc fragment of a traditional antibody IgG formed by papain hydrolysis to form an Fc fragment and two identical Fab fragments. The Fc domain can include antibody heavy chain CH2, CH3 and hinge region fragments. Traditional Fc fragments have the function of binding to Fc fragment receptors to mediate related biological effects, and site-specific mutations can change their ability to bind to corresponding target receptors, thereby affecting their biological functions. The immunoglobulin Fc domain disclosed in this application should include but is not limited to traditional Fc fragments and any other forms of Fc mutants. The biological functions that the immunoglobulin Fc domain can provide include but are not limited to extending half-life, improving molecular stability, facilitating the expression and detection of fusion proteins, mediating passage through the placenta and mucosal barriers, mediating antibody-dependent cell-mediated cytotoxicity (ADCC), mediating inflammatory responses, mediating antibody-dependent cell-mediated phagocytosis (ADCP), mediating complement-dependent cytotoxicity (CDC), mediating the promotion of dendritic cell (DC) maturation, regulating cytokine secretion, and regulating B cell proliferation and differentiation. Exemplary human immunoglobulin IgG1 Fc domain (SEQ ID NO: 20) and human immunoglobulin IgG4 Fc domain (SEQ ID NO: 21) are provided below.

[0428] In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0429] The proteins, polypeptides and / or amino acid sequences involved in this application should also be understood to include at least the following scope: variants or homologs that have the same or similar functions as the proteins or polypeptides.

[0430] In the present application, the "variant" generally refers to a peptide that is substantially similar to the peptide, and may be a protein or polypeptide that has been substituted, deleted or added with one or more amino acids in the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to the protein). For example, the functional variant may comprise a protein or polypeptide that has been subjected to amino acid changes by at least 1, such as 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4 or 5 amino acid substitutions, deletions and / or insertions. The functional variant may substantially retain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide before the change. For example, the substitution may be a conservative substitution.

[0431] In the present application, the homolog can be a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., an antibody or fragment thereof that specifically binds to the protein).

[0432] In the present application, described homology generally refers to the similarity, similarity or association between two or more sequences.Can calculate " sequence homology per-cent " in the following manner: two sequences to be compared are compared in comparison window, determine that there is identical nucleic acid base (for example, A, T, C, G, I) or identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) number in the position to obtain the number of matching positions, with the number of matching positions divided by the total number of positions (that is, window size) in the comparison window, and result is multiplied by 100, to produce sequence homology per-cent.Comparison carried out in order to determine the sequence homology per-cent, can realize by several ways known in the art, for example, use publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for comparing sequences, including any algorithm required to achieve maximum alignment within the full-length sequence being compared or within the target sequence region. The homology can also be determined by, but is not limited to, the following methods: FASTA and BLAST.

[0433] Typically, in a polypeptide chain, an amino group is linked to another carboxyl group in the polypeptide chain to form a single chain. However, at the two ends of a protein, there are amino acid residues that do not form a peptide bond: the end of the polypeptide chain with a free amino group and the end of the polypeptide chain with a carboxyl group. In this application, the term "N-terminus" generally refers to the end of the polypeptide chain where the amino acid residue carries a free amino group. In this application, the term "C-terminus" generally refers to the end of the polypeptide chain where the amino acid residue carries a free carboxyl group.

[0434] In this application, the term "nucleic acid molecule" generally refers to isolated forms of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or their analogs, isolated from their natural environment or artificially synthesized.

[0435] As used herein, the term "immunoconjugate" generally refers to a polypeptide molecule conjugated to one or more heterologous molecules, including but not limited to cytotoxins.

[0436] In the present invention, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed.

[0437] In this application, the term "linker" generally refers to a connecting molecule that connects one or more polypeptides or their domains. For example, the linker can have conformational flexibility and be a short peptide chain formed by a combination of amino acid Gly (G) and Ser (S) residues, wherein the ratio of the number of amino acid Gly to the number of amino acid Ser can be ≥1. The linkers disclosed in this application can be extended to any short peptide with this property. Exemplary linker amino acid sequences are provided below, including but not limited to GGGGS (SEQ ID NO: 22), GGGGSGGGGS (SEQ ID NO: 23), GGGGSGGGGSGGGGS (SEQ ID NO: 24), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 25), and GGGGSGGGS (SEQ ID NO: 26).

[0438] In this application, the term "CD3" generally refers to a protein complex and is composed of four unique chains. In mammals, the complex contains one CD3γ chain, one CD3δ chain, and two CD3ε chains. These chains associate with the T cell receptor (TCR) and the ζ-chain to generate activation signals in T lymphocytes. TCR, ζ-chain, and some of the CD3 molecules form the TCR complex. As used herein, the term "CD3" refers to any natural CD3 from any human source. The term also encompasses "full-length" and unprocessed proteins and any form of protein or one or more CD3 chains (polypeptides) (e.g., mature polypeptides) derived from processing in cells. The term also encompasses naturally occurring variants and isoforms of CD3, such as splice variants or allelic variants. For example, www.uniprot.org / uniprot / P04234, www.uniprot.org / uniprot / P07766, and www.uniprot.org / uniprot / P09693 provide descriptions of the CD3 gamma chain, CD3 delta chain, and CD3 epsilon chain and sequences.

[0439] In this application, the term "anti-CD3 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize a single CD3 subunit (e.g., B, δ, γ, or ζ), as well as antibodies and antigen-binding fragments thereof that specifically recognize a dimeric complex of two CD3 subunits (e.g., γ / ε, δ / ε, and ζ / ζ CD3 dimers). For example, monoclonal antibodies, including human, humanized, chimeric, or murine antibodies, are directed against the CD3 receptor in the T cell antigen receptor of mature T cells. For example, the anti-CD3 antibody can be the CD3B219 antibody or an antigen-binding fragment thereof.

[0440] In this application, the term "amino acid mutation" generally refers to amino acid replacement, deletion, insertion and modification. Any combination of substitution, deletion, insertion and modification can be performed to achieve the final construct, as long as the final construct has the desired properties. In one embodiment, the amino acid mutation is a replacement. The term "amino acid mutation at a certain position" refers to the replacement or deletion of a specified residue or the insertion of at least one amino acid residue adjacent to a specified residue. Amino acid replacement can be conservative or non-conservative. For example, "A13I" can represent that the 13th alanine (A, Ala) is replaced with isoleucine (I, Ile).

[0441] In this application, the term "active fragment" generally refers to a nucleic acid or amino acid fragment or variant having a certain biological activity or function. For example, a functionally active fragment can retain or partially retain the ability of the full-length protein to bind to another molecule.

[0442] In this application, "CD86 active fragment" refers to the CD86 IgV or CD86 ECD protein and its derivative variant polypeptides described in the present invention, which have the activity of binding to CD28 and / or CTLA4 or its fragments, including but not limited to the characteristics of regulating T cell activity, immune system activation and anti-tumor activity.

[0443] As used herein, "LAG3 active fragment" refers to a polypeptide or fragment thereof of the LAG3 protein and its derivative variant polypeptides described herein that has the ability to activate antigen-presenting cells, including but not limited to the ability to modulate antigen-presenting cell activity, activate the immune system, and exhibit anti-tumor activity.

[0444] The term "CD86 variant polypeptide" refers to a protein or polypeptide in which one or more amino acids have been substituted, deleted, or added to the amino acid sequence of wild-type CD86. For example, the functional variant may comprise a protein or polypeptide having an amino acid alteration by at least one, such as 1-30, 1-20, or 1-10, or further such as 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and / or insertions. The functional variant may substantially retain the biological properties of CD86 prior to the alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide prior to the alteration. For example, the substitution may be a conservative substitution. "CD86 variant polypeptide" means an active fragment derived from a CD86 protein that possesses the functional properties of the CD86 protein, including but not limited to CD28 and / or CTLA4 binding activity.

[0445] The term "fusion polypeptide complex of variant polypeptide" is also referred to as "variant polypeptide complex", "polypeptide complex", "fusion polypeptide complex" or "fusion complex" in the present invention. The term "CD86 mutant fusion polypeptide complex" refers to a functional fusion polypeptide complex in which a CD86 ECD mutant or a CD86 IgV mutant is linked to an antibody or an antigen-binding fragment thereof, or an immunoglobulin Fc domain or a functional polypeptide fragment, with or without a linker peptide. The antibodies in the functional fusion polypeptide complex in which the disclosed CD86 mutant is linked to an antibody or an antigen-binding fragment thereof, with or without a linker peptide, include but are not limited to anti-PD-L1 antibodies, anti-PD-1 antibodies, anti-TIGIT antibodies, anti-CTLA4 antibodies, anti-CEA antibodies, anti-BCMA antibodies, anti-LAG3 antibodies, anti-CD3 antibodies, anti-Her2 antibodies, anti-Her3 antibodies, anti-VEGF antibodies, anti-VEGFR antibodies, anti-EGFR antibodies, anti-c-Met antibodies, anti-CD19 antibodies, anti-CD20 antibodies, anti-CD38 antibodies, anti-TROP-2 antibodies, anti-CD40 antibodies, anti-4-1BB antibodies, anti-CD30 antibodies, etc.; any form of antibody is linked to the CD86 ECD mutant or CD86 disclosed in the present invention, with or without a linker peptide. Functional fusion proteins covalently linked to IgV mutants or combinations thereof should be considered within the scope of the present invention.

[0446] The term "LAG3 variant polypeptide" refers to a protein or polypeptide in which one or more amino acids have been substituted, deleted, or added to the amino acid sequence of wild-type LAG3. For example, the functional variant may comprise a protein or polypeptide having an amino acid alteration through at least one, e.g., 1-30, 1-20, or 1-10, or further e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and / or insertions. The functional variant may substantially retain the biological properties of LAG3 prior to the alteration (e.g., substitution, deletion, or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding ability) of the protein or polypeptide prior to the alteration. For example, the substitution may be a conservative substitution. A "LAG3 variant polypeptide" refers to an active fragment derived from a LAG3 protein that possesses the functional properties of the LAG3 protein, including, but not limited to, promoting the activation and maturation of antigen-presenting cells. The term "LAG3 variant polypeptide" refers to an active fragment derived from a LAG3 protein that possesses the functional properties of the LAG3 protein. These functional properties include, but are not limited to, binding to MHCII (or HLA-DR) and / or FGL1, and promoting activation and maturation of antigen-presenting cells.

[0447] In this application, the term "LAG3 variant fusion polypeptide complex" generally refers to a polypeptide or protein formed by the fusion of polypeptides or domains from one or more sources. The term "variant polypeptide fusion polypeptide complex" is also referred to as "variant polypeptide complex," "polypeptide complex," "fusion polypeptide complex," or "fusion complex" in the present invention. The LAG3 variant polypeptide, with or without the linker peptide, is covalently linked to at least the Fc domain, or / and to at least the antibody or antigen-binding fragment thereof. The LAG3 variant fusion polypeptide complexes disclosed herein include, but are not limited to, functional fusion polypeptides covalently linked to any form of antibody, functional protein fragment, and / or at least one subunit functional domain of the Fc domain, or any combination thereof, and are considered within the scope of this application.

[0448] The activation and maturation of antigen-presenting cells include, but are not limited to, up-regulation of the expression of antigen-presenting cell co-stimulatory receptors, enhanced antigen phagocytosis, enhanced cytokine secretion, and enhanced chemokine secretion.

[0449] The co-stimulatory receptors include but are not limited to ICOSL, CD40L, CD137L, OX40L, CD80, CD83 and CD86.

[0450] The antigen phagocytosis includes but is not limited to the phagocytosis of bacteria, viruses, proteins, polysaccharides and the like.

[0451] The cytokines include but are not limited to IL-1beta, TNFa, IFNγ, IL-6, IL-12 and the like.

[0452] The chemokines include but are not limited to CCL1, CCL2, CCL3, CCL4, CCL5, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, etc.

[0453] In this application, the expression "N-terminally truncated by X amino acids" refers to the deletion of amino acid residues 1-X at the N-terminus of the extracellular region of a protein. For example, the expression "LAG3 extracellular region polypeptide with an N-terminal truncation of 74 amino acids" may refer to a variant polypeptide obtained by deleting amino acid residues 23-96 of the N-terminus of the wild-type LAG3 extracellular region polypeptide according to NCBI numbering. For example, the expression "LAG3 extracellular region polypeptide with an N-terminal truncation of 74 amino acids" may refer to a polypeptide derived from a wild-type human LAG3 polypeptide starting at amino acid position 97. For example, the expression "LAG3 extracellular region polypeptide with an N-terminal truncation of 74 amino acids" may be expressed as "LAG3 97-450."

[0454] In this application, the expression "the C-terminus ends at amino acid position X" means that all amino acid residues after amino acid position X (i.e., starting from amino acid position X+1) are deleted. For example, the expression "the C-terminus ends at amino acid position 156 of the LAG3 extracellular region polypeptide" can refer to a variant polypeptide obtained by deleting all amino acid residues after amino acid position NCBI number 156 of the wild-type LAG3 extracellular region polypeptide (i.e., starting from amino acid position 157). For example, the expression "the C-terminus ends at amino acid position 156 of the LAG3 extracellular region polypeptide" can refer to a variant polypeptide obtained by truncating 294 amino acids from the C-terminus of the wild-type LAG3 extracellular region polypeptide. For example, the expression "the C-terminus ends at amino acid position 156 of the LAG3 extracellular region polypeptide" can be expressed as "LAG3 23-156". For example, the expression "the N-terminus is truncated by 74 amino acids and the C-terminus ends at amino acid position 156" can be expressed as "LAG3 97-156".

[0455] In this application, the term "comprising" generally means including the features specifically stated, but not excluding other elements.

[0456] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0457] Detailed Description of the Invention

[0458] In one aspect, the present application provides a fusion polypeptide, which may comprise a first domain and a second domain, wherein the first domain is capable of blocking PD-1 / PD-L1 signaling, and the second domain comprises CD86 or a functionally active fragment thereof. For example, the fusion polypeptide may comprise one or more of the first domains and one or more of the second domains.

[0459] For example, the first domain is capable of binding to PD-L1 and / or PD-1.

[0460] For example, the first domain may comprise HCDR3 of an antibody heavy chain, which may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 17.

[0461] For example, the first domain may comprise HCDR2 of an antibody heavy chain, and the antibody heavy chain may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 17.

[0462] For example, the first domain may comprise HCDR1 of an antibody heavy chain, and the antibody heavy chain may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17.

[0463] For example, the first domain may comprise the heavy chain variable region VH of an antibody heavy chain, and the antibody heavy chain variable region may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17.

[0464] For example, the first domain may comprise an antibody heavy chain, which may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 17.

[0465] For example, the first domain may comprise LCDR3 of an antibody light chain, which may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO: 18.

[0466] For example, the first domain may comprise LCDR2 of an antibody light chain, which may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO: 18.

[0467] For example, the first domain may comprise LCDR1 of an antibody light chain, which may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO: 18.

[0468] For example, the first domain may comprise a light chain variable region VL of an antibody light chain, and the antibody light chain variable region may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO: 18.

[0469] For example, the first domain may comprise an antibody light chain, which may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO: 18.

[0470] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 4. For example, the antibody may be Sugemalimab.

[0471] For example, the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 5. For example, the antibody may be Sugemalimab.

[0472] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 4, and the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence set forth in SEQ ID NO: 5. For example, the antibody may be Sugemalimab.

[0473] For example, the first domain may comprise the heavy chain variable region VH of an antibody heavy chain, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 4, and the first domain may comprise the light chain variable region VL of an antibody light chain, the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 5. For example, the antibody may be Sugemalimab.

[0474] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 6. For example, the antibody may be JS003.

[0475] For example, the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 7. For example, the antibody may be JS003.

[0476] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 6, and the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 7. For example, the antibody may be JS003.

[0477] For example, the first domain may comprise the heavy chain variable region VH of an antibody heavy chain, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 6, and the first domain may comprise the light chain variable region VL of an antibody light chain, the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 7. For example, the antibody may be JS003.

[0478] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 8. For example, the antibody may be Durvalumab.

[0479] For example, the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 9. For example, the antibody may be Durvalumab.

[0480] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 8, and the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence set forth in SEQ ID NO: 9. For example, the antibody may be Durvalumab.

[0481] For example, the first domain may comprise the heavy chain variable region VH of an antibody heavy chain, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 8, and the first domain may comprise the light chain variable region VL of an antibody light chain, the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 9. For example, the antibody may be Durvalumab.

[0482] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 10. For example, the antibody may be Atezolizumab.

[0483] For example, the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 11. For example, the antibody may be Atezolizumab.

[0484] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 10, and the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence set forth in SEQ ID NO: 11. For example, the antibody may be atezolizumab.

[0485] For example, the first domain may comprise a heavy chain variable region VH of an antibody heavy chain, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 10, and the first domain may comprise a light chain variable region VL of an antibody light chain, the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 11. For example, the antibody may be atezolizumab.

[0486] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 12. For example, the antibody may be Avelumab.

[0487] For example, the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 13. For example, the antibody may be Avelumab.

[0488] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 12, and the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence set forth in SEQ ID NO: 13. For example, the antibody may be Avelumab.

[0489] For example, the first domain may comprise a heavy chain variable region VH of an antibody heavy chain, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 12, and the first domain may comprise a light chain variable region VL of an antibody light chain, the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 13. For example, the antibody may be avelumab.

[0490] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody, wherein the antibody may comprise the amino acid sequence shown in SEQ ID NO: 14. For example, the antibody may be Envafolimab.

[0491] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 15. For example, the antibody may be Permbrolizumab.

[0492] For example, the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 16. For example, the antibody may be Permbrolizumab.

[0493] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 15, and the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 16. For example, the antibody may be Permbrolizumab.

[0494] For example, the first domain may comprise the heavy chain variable region VH of an antibody heavy chain, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 15, and the first domain may comprise the light chain variable region VL of an antibody light chain, the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 16. For example, the antibody may be Permbrolizumab.

[0495] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 17. For example, the antibody may be Nivolumab.

[0496] For example, the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 18. For example, the antibody may be Nivolumab.

[0497] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 17, and the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence set forth in SEQ ID NO: 18. For example, the antibody may be Nivolumab.

[0498] For example, the first domain may comprise a heavy chain variable region VH of an antibody heavy chain, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 17, and the first domain may comprise a light chain variable region VL of an antibody light chain, the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 18. For example, the antibody may be nivolumab.

[0499] For example, the first domain may comprise an antibody or an antigen-binding fragment thereof.

[0500] For example, the antibody can be selected from the group consisting of a recombinant antibody, a single domain antibody, a heavy chain antibody, a chimeric antibody, and a bispecific antibody.

[0501] For example, the antigen-binding fragment can be selected from one or more of the following groups: Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, VHH and dAb.

[0502] For example, the first domain may comprise an antibody or an antigen thereof selected from the group consisting of

[0503] Binding fragments: Sugemalimab, Durvalumab, Atezolizumab, Avelumab, Envafolimab, Permbrolizumab and Nivolumab.

[0504] For example, the first domain can comprise Sugemalimab or an antigen-binding fragment thereof.

[0505] For example, the second domain may comprise CD86 or a functionally active fragment thereof.

[0506] For example, the second domain can be selected from the group consisting of human-derived CD86 or a functionally active fragment thereof and mouse-derived CD86 or a functionally active fragment thereof.

[0507] For example, the second domain can bind CD28, and / or CTLA4.

[0508] For example, the second domain may comprise the IgV domain of CD86 or a functionally active fragment thereof.

[0509] For example, the second domain may comprise the extracellular domain of CD86 or a functionally active fragment thereof.

[0510] For example, the second domain may comprise the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0511] For example, the first domain and the second domain may be linked directly or indirectly.

[0512] For example, the first domain and the N-terminus of the second domain may be directly or indirectly connected.

[0513] For example, the C-termini of the first domain and the second domain may be directly or indirectly connected.

[0514] For example, the first domain may comprise an antibody heavy chain, the antibody heavy chain of the first domain being directly or indirectly linked to the second domain.

[0515] For example, the first domain may comprise an antibody heavy chain, and the C-terminus of the first domain antibody heavy chain is directly or indirectly connected to the N-terminus of the second domain.

[0516] For example, the first domain may comprise an antibody heavy chain, and the N-terminus of the antibody heavy chain of the first domain is directly or indirectly connected to the C-terminus of the second domain.

[0517] For example, the first domain may comprise an antibody light chain, and the first domain antibody light chain and the second domain may be directly or indirectly connected.

[0518] For example, the first domain may comprise an antibody light chain, and the C-terminus of the antibody light chain of the first domain is directly or indirectly connected to the N-terminus of the second domain.

[0519] For example, the first domain may comprise an antibody light chain, and the N-terminus of the antibody light chain of the first domain is directly or indirectly connected to the C-terminus of the second domain.

[0520] For example, the indirect connection may comprise connection via a linker.

[0521] For example, the linker may comprise an amino acid sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35.

[0522] :58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99 NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86 and SEQ ID NO:87.

[0523] For example, the fusion polypeptide can comprise an amino acid sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and SEQ ID NO:77.

[0524] In one aspect, the present application further provides a fusion polypeptide, which may comprise a first domain, a second domain, and a third domain, wherein the first domain is capable of blocking PD-1 / PD-L1 signals, the second domain comprises CD86 or a functionally active fragment thereof, and the third domain is capable of activating an innate immune response. For example, the fusion polypeptide may comprise one or more of the first domains, one or more of the second domains, and one or more of the third domains.

[0525] For example, the first domain is capable of binding to PD-L1 and / or PD-1.

[0526] For example, the first domain may comprise HCDR3 of an antibody heavy chain, which may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 17.

[0527] For example, the first domain may comprise HCDR2 of an antibody heavy chain, and the antibody heavy chain may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 17.

[0528] For example, the first domain may comprise HCDR1 of an antibody heavy chain, and the antibody heavy chain may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17.

[0529] For example, the first domain may comprise the heavy chain variable region VH of an antibody heavy chain, and the antibody heavy chain variable region may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 17.

[0530] For example, the first domain may comprise an antibody heavy chain, which may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 17.

[0531] For example, the first domain may comprise LCDR3 of an antibody light chain, which may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO: 18.

[0532] For example, the first domain may comprise LCDR2 of an antibody light chain, which may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO: 18.

[0533] For example, the first domain may comprise LCDR1 of an antibody light chain, which may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO: 18.

[0534] For example, the first domain may comprise a light chain variable region VL of an antibody light chain, and the antibody light chain variable region may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO: 18.

[0535] For example, the first domain may comprise an antibody light chain, which may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO: 18.

[0536] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 4. For example, the antibody may be Sugemalimab.

[0537] For example, the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 5. For example, the antibody may be Sugemalimab.

[0538] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 4, and the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence set forth in SEQ ID NO: 5. For example, the antibody may be Sugemalimab.

[0539] For example, the first domain may comprise the heavy chain variable region VH of an antibody heavy chain, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 4, and the first domain may comprise the light chain variable region VL of an antibody light chain, the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 5. For example, the antibody may be Sugemalimab.

[0540] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 6. For example, the antibody may be JS003.

[0541] For example, the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 7. For example, the antibody may be JS003.

[0542] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 6, and the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 7. For example, the antibody may be JS003.

[0543] For example, the first domain may comprise the heavy chain variable region VH of an antibody heavy chain, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 6, and the first domain may comprise the light chain variable region VL of an antibody light chain, the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 7. For example, the antibody may be JS003.

[0544] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 8. For example, the antibody may be Durvalumab.

[0545] For example, the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 9. For example, the antibody may be Durvalumab.

[0546] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 8, and the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence set forth in SEQ ID NO: 9. For example, the antibody may be Durvalumab.

[0547] For example, the first domain may comprise the heavy chain variable region VH of an antibody heavy chain, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 8, and the first domain may comprise the light chain variable region VL of an antibody light chain, the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 9. For example, the antibody may be Durvalumab.

[0548] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 10. For example, the antibody may be Atezolizumab.

[0549] For example, the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 11. For example, the antibody may be Atezolizumab.

[0550] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 10, and the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence set forth in SEQ ID NO: 11. For example, the antibody may be atezolizumab.

[0551] For example, the first domain may comprise a heavy chain variable region VH of an antibody heavy chain, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 10, and the first domain may comprise a light chain variable region VL of an antibody light chain, the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 11. For example, the antibody may be atezolizumab.

[0552] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 12. For example, the antibody may be Avelumab.

[0553] For example, the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 13. For example, the antibody may be Avelumab.

[0554] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 12, and the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence set forth in SEQ ID NO: 13. For example, the antibody may be Avelumab.

[0555] For example, the first domain may comprise a heavy chain variable region VH of an antibody heavy chain, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 12, and the first domain may comprise a light chain variable region VL of an antibody light chain, the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 13. For example, the antibody may be avelumab.

[0556] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody, wherein the antibody may comprise the amino acid sequence shown in SEQ ID NO: 14. For example, the antibody may be Envafolimab.

[0557] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 15. For example, the antibody may be Permbrolizumab.

[0558] For example, the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 16. For example, the antibody may be Permbrolizumab.

[0559] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 15, and the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 16. For example, the antibody may be Permbrolizumab.

[0560] For example, the first domain may comprise the heavy chain variable region VH of an antibody heavy chain, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 15, and the first domain may comprise the light chain variable region VL of an antibody light chain, the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 16. For example, the antibody may be Permbrolizumab.

[0561] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 17. For example, the antibody may be Nivolumab.

[0562] For example, the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 18. For example, the antibody may be Nivolumab.

[0563] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence set forth in SEQ ID NO: 17, and the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise the amino acid sequence set forth in SEQ ID NO: 18. For example, the antibody may be Nivolumab.

[0564] For example, the first domain may comprise a heavy chain variable region VH of an antibody heavy chain, the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 17, and the first domain may comprise a light chain variable region VL of an antibody light chain, the antibody light chain may comprise the amino acid sequence shown in SEQ ID NO: 18. For example, the antibody may be nivolumab.

[0565] For example, the first domain may comprise an antibody or an antigen-binding fragment thereof.

[0566] For example, the antibody can be selected from the group consisting of a recombinant antibody, a single domain antibody, a heavy chain antibody, a chimeric antibody, and a bispecific antibody.

[0567] For example, the antigen-binding fragment can be selected from one or more of the following groups: Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, VHH and dAb.

[0568] For example, the first domain may comprise an antibody or antigen-binding fragment thereof selected from the group consisting of Sugemalimab, Durvalumab, Atezolizumab, Avelumab, Envafolimab, Permbrolizumab and Nivolumab.

[0569] For example, the first domain can comprise Sugemalimab or an antigen-binding fragment thereof.

[0570] For example, the second domain may comprise CD86 or a functionally active fragment thereof.

[0571] For example, the second domain can be selected from the group consisting of human-derived CD86 or a functionally active fragment thereof and mouse-derived CD86 or a functionally active fragment thereof.

[0572] For example, the second domain can bind CD28, and / or CTLA4.

[0573] For example, the second domain may comprise the IgV domain of CD86 or a functionally active fragment thereof.

[0574] For example, the second domain may comprise the extracellular domain of CD86 or a functionally active fragment thereof.

[0575] For example, the second domain may comprise the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3.

[0576] For example, the third domain is capable of binding to an MHC II molecule on an antigen presenting cell and / or activating antigen presentation.

[0577] For example, the third domain may comprise LAG3 or a functionally active fragment thereof.

[0578] For example, the third domain can be selected from the group consisting of human-derived LAG3 or a functionally active fragment thereof and mouse-derived LAG3 or a functionally active fragment thereof.

[0579] For example, the third domain may comprise the extracellular domain of LAG3 or a functionally active fragment thereof.

[0580] For example, the third domain may comprise IgD1, IgD2, IgD3 and / or IgD4 of LAG3 or a functionally active fragment thereof.

[0581] For example, the third domain may comprise IgD1, IgD1-IgD2, IgD1-IgD2-IgD3, and / or IgD1-IgD2-IgD3-IgD4 of LAG3 or a functionally active fragment thereof.

[0582] For example, the third domain may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28.

[0583] For example, the first domain and the second domain may be linked directly or indirectly.

[0584] For example, the first domain comprises an antibody heavy chain, and the first domain antibody heavy chain and the second domain may be directly or indirectly connected.

[0585] For example, the first domain comprises an antibody heavy chain, and the C-terminus of the antibody heavy chain of the first domain and the N-terminus of the second domain can be directly or indirectly connected.

[0586] For example, the first domain comprises an antibody heavy chain, and the N-terminus of the antibody heavy chain of the first domain and the C-terminus of the second domain can be directly or indirectly connected.

[0587] For example, the first domain comprises an antibody light chain, and the first domain antibody light chain and the second domain may be directly or indirectly connected.

[0588] For example, the first domain comprises an antibody light chain, and the C-terminus of the antibody light chain of the first domain and the N-terminus of the second domain can be directly or indirectly connected.

[0589] For example, the first domain comprises an antibody light chain, and the N-terminus of the antibody light chain of the first domain and the C-terminus of the second domain may be directly or indirectly connected.

[0590] For example, the first domain comprises an antibody heavy chain and an antibody light chain, the C-terminus of the antibody heavy chain of the first domain may be directly or indirectly connected to the N-terminus of the second domain, and the C-terminus of the antibody light chain of the first domain may be directly or indirectly connected to the N-terminus of the second domain.

[0591] For example, the first domain comprises an antibody heavy chain and an antibody light chain, the N-terminus of the antibody heavy chain of the first domain can be directly or indirectly connected to the C-terminus of the second domain, and the N-terminus of the antibody light chain of the first domain can be directly or indirectly connected to the C-terminus of the second domain.

[0592] For example, the first domain and the third domain may be linked directly or indirectly.

[0593] For example, the first domain comprises an antibody heavy chain, and the first domain antibody heavy chain and the third domain may be directly or indirectly connected.

[0594] For example, the first domain comprises an antibody heavy chain, and the C-terminus of the antibody heavy chain of the first domain can be directly or indirectly connected to the N-terminus of the third domain.

[0595] For example, the first domain comprises an antibody heavy chain, and the N-terminus of the antibody heavy chain of the first domain can be directly or indirectly connected to the C-terminus of the third domain.

[0596] For example, the first domain comprises an antibody light chain, and the first domain light chain and the third domain may be directly or indirectly connected.

[0597] For example, the first domain comprises an antibody light chain, and the C-terminus of the light chain of the first domain and the N-terminus of the third domain may be directly or indirectly connected.

[0598] For example, the first domain comprises an antibody light chain, and the N-terminus of the light chain of the first domain and the C-terminus of the third domain may be directly or indirectly connected.

[0599] For example, the second domain and the third domain may be linked directly or indirectly.

[0600] For example, the C-terminus of the second domain and the N-terminus of the third domain may be directly or indirectly connected.

[0601] For example, the N-terminus of the second domain and the C-terminus of the third domain may be directly or indirectly connected.

[0602] For example, the first domain and the second domain may be directly or indirectly connected, and the second domain and the third domain may be directly or indirectly connected.

[0603] For example, the C-terminus of the first domain and the N-terminus of the second domain may be directly or indirectly connected, and the C-terminus of the second domain and the N-terminus of the third domain may be directly or indirectly connected.

[0604] For example, the first domain comprises an antibody heavy chain, the C-terminus of the antibody heavy chain of the first domain may be directly or indirectly connected to the N-terminus of the second domain, and the C-terminus of the second domain may be directly or indirectly connected to the N-terminus of the third domain.

[0605] For example, the first domain comprises an antibody light chain, the C-terminus of the antibody light chain of the first domain may be directly or indirectly connected to the N-terminus of the second domain, and the C-terminus of the second domain may be directly or indirectly connected to the N-terminus of the third domain.

[0606] For example, the N-terminus of the first domain and the C-terminus of the second domain may be directly or indirectly connected, and the N-terminus of the second domain and the C-terminus of the third domain may be directly or indirectly connected.

[0607] For example, the first domain comprises an antibody heavy chain, the N-terminus of the antibody heavy chain of the first domain may be directly or indirectly connected to the C-terminus of the second domain, and the N-terminus of the second domain may be directly or indirectly connected to the C-terminus of the third domain.

[0608] For example, the first domain comprises an antibody light chain, the N-terminus of the antibody light chain of the first domain may be directly or indirectly connected to the C-terminus of the second domain, and the N-terminus of the second domain may be directly or indirectly connected to the C-terminus of the third domain.

[0609] For example, the first domain and the third domain may be directly or indirectly connected, and the third domain and the second domain may be directly or indirectly connected.

[0610] For example, the C-terminus of the first domain may be directly or indirectly connected to the N-terminus of the third domain, and the C-terminus of the third domain may be directly or indirectly connected to the N-terminus of the second domain.

[0611] For example, the first domain comprises an antibody heavy chain, the C-terminus of the antibody heavy chain of the first domain may be directly or indirectly connected to the N-terminus of the third domain, and the C-terminus of the third domain may be directly or indirectly connected to the N-terminus of the second domain.

[0612] For example, the first domain comprises an antibody light chain, the C-terminus of the antibody light chain of the first domain may be directly or indirectly connected to the N-terminus of the third domain, and the C-terminus of the third domain may be directly or indirectly connected to the N-terminus of the second domain.

[0613] For example, the N-terminus of the first domain may be directly or indirectly connected to the C-terminus of the third domain, and the N-terminus of the third domain may be directly or indirectly connected to the C-terminus of the second domain.

[0614] For example, the first domain comprises an antibody heavy chain, the N-terminus of the antibody heavy chain of the first domain may be directly or indirectly connected to the C-terminus of the third domain, and the N-terminus of the third domain may be directly or indirectly connected to the C-terminus of the second domain.

[0615] For example, the first domain comprises an antibody light chain, the N-terminus of the antibody light chain of the first domain may be directly or indirectly connected to the C-terminus of the third domain, and the N-terminus of the third domain may be directly or indirectly connected to the C-terminus of the second domain.

[0616] For example, the first domain and the second domain may be directly or indirectly connected, and the first domain and the third domain may be directly or indirectly connected.

[0617] For example, the C-terminus of the first domain and the N-terminus of the second domain may be directly or indirectly connected, and the N-terminus of the first domain and the C-terminus of the third domain may be directly or indirectly connected.

[0618] For example, the first domain comprises an antibody heavy chain, the C-terminus of the first domain antibody heavy chain and the N-terminus of the second domain can be directly or indirectly connected, and the N-terminus of the first domain heavy chain and the C-terminus of the third domain can be directly or indirectly connected.

[0619] For example, the first domain comprises an antibody light chain, the C-terminus of the first domain antibody light chain and the N-terminus of the second domain may be directly or indirectly connected, and the N-terminus of the first domain light chain and the C-terminus of the third domain may be directly or indirectly connected.

[0620] For example, the first domain comprises an antibody heavy chain, the C-terminus of the antibody heavy chain of the first domain may be directly or indirectly connected to the N-terminus of the second domain, and the N-terminus of the light chain of the first domain may be directly or indirectly connected to the C-terminus of the third domain.

[0621] For example, the first domain comprises an antibody light chain, the C-terminus of the first domain antibody light chain and the N-terminus of the second domain can be directly or indirectly connected, and the N-terminus of the first domain heavy chain and the C-terminus of the third domain can be directly or indirectly connected.

[0622] For example, the N-terminus of the first domain may be directly or indirectly connected to the C-terminus of the second domain, and the C-terminus of the first domain may be directly or indirectly connected to the N-terminus of the third domain.

[0623] For example, the first domain comprises an antibody heavy chain, the N-terminus of the first domain antibody heavy chain may be directly or indirectly connected to the C-terminus of the second domain, and the C-terminus of the first domain heavy chain may be directly or indirectly connected to the N-terminus of the third domain.

[0624] For example, the first domain comprises an antibody light chain, the N-terminus of the first domain antibody light chain and the C-terminus of the second domain may be directly or indirectly connected, and the C-terminus of the first domain light chain and the N-terminus of the third domain may be directly or indirectly connected.

[0625] For example, the first domain comprises an antibody heavy chain and an antibody light chain, the N-terminus of the antibody heavy chain of the first domain may be directly or indirectly connected to the C-terminus of the second domain, and the C-terminus of the antibody light chain of the first domain may be directly or indirectly connected to the N-terminus of the third domain.

[0626] For example, the first domain comprises an antibody heavy chain and an antibody light chain, the N-terminus of the antibody light chain of the first domain can be directly or indirectly connected to the C-terminus of the second domain, and the C-terminus of the antibody heavy chain of the first domain can be directly or indirectly connected to the N-terminus of the third domain.

[0627] For example, the indirect connection may comprise connection via a linker.

[0628] For example, the linker may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35.

[0629] : 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135 NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 171, SEQ ID NO: 173.

[0630] For example, the fusion polypeptide may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 104, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 171, and SEQ ID NO: 173.

[0631] In one aspect, the present application discloses a dual and / or multifunctional fusion polypeptide comprising a two-chain fusion polypeptide, wherein the fusion polypeptide includes (i) an antigen-binding fragment with specific binding to a first cellular target, the presence or absence of (ii) an Fc domain, and / or (iii) a second domain with specific binding to a second cellular target, and / or (iv) a third domain with specific binding to a third cellular target, wherein the second cellular target binding second domain is covalently linked through the C-terminus of the first domain polypeptide, or through the N-terminus of the first domain polypeptide fragment, with or without a linker, and wherein the third cellular target binding third domain is covalently linked through the C-terminus of the first domain polypeptide, or through the C-terminus of the second domain, or through the N-terminus of the first domain polypeptide, or through the N-terminus of the second domain, with or without a linker; it should be understood that the first domain disclosed in the present invention can be extended to certain anti-PD-L1 and / or anti-PD-1 antibodies without an Fc domain, including but not limited to single-domain antibodies, recombinant antibodies, and single-chain antibodies. For example, if the C-terminus of the heavy chain polypeptide or light chain polypeptide of the first domain is covalently linked to the second domain and / or the third domain via a linker, it should be considered within the scope of the claims of the present invention; similarly, if the N-terminus of the heavy chain polypeptide or light chain polypeptide of the first domain is covalently linked to the second domain and / or the third domain via a linker, it should also be considered within the scope of the claims of the present invention. Thus, the dual- and / or multifunctional fusion polypeptide can simultaneously bind to the first cell target, the second cell target, and / or the third cell target.

[0632] In one aspect, the present invention provides a specific polypeptide complex constituting a dual and / or multifunctional fusion polypeptide, wherein the specific polypeptide complex comprises a first polypeptide and a second polypeptide constituting the polypeptide complex. It should be understood that the second polypeptide may not be present in certain specific antibodies.

[0633] On the other hand, the second polypeptide of the polypeptide complex of the present invention is the light chain of the first domain; the first polypeptide of the polypeptide complex of the present invention includes, from the N-terminus to the C-terminus of the polypeptide, the third domain and / or the second domain, a linker, the heavy chain of the first domain, a linker, and the third domain and / or the second domain covalently connected, and the first polypeptide and the second polypeptide constitute a specific polypeptide complex of a dual and / or multifunctional fusion polypeptide; wherein the linker can exist or not exist independently of each other; in some cases, only one of the second domain or the third domain exists.

[0634] On the other hand, the second polypeptide of the polypeptide complex of the present invention is the light chain of the first domain; the first polypeptide of the polypeptide complex of the present invention includes, from the N-terminus to the C-terminus of the polypeptide, the heavy chain of the first domain, a linker, the third domain and / or the second domain, and the linker and the third domain and / or the second domain are covalently linked, and the first polypeptide and the second polypeptide constitute a specific polypeptide complex of a dual and / or multifunctional fusion polypeptide; wherein the linker can exist or not exist independently of each other; in some cases, only one of the second domain or the third domain exists.

[0635] On the other hand, the second polypeptide of the polypeptide complex of the present invention is the light chain of the first domain; the first polypeptide of the polypeptide complex of the present invention includes, from the N-terminus to the C-terminus of the polypeptide, the third domain and / or the second domain, a linker, the third domain and / or the second domain, a linker and the first domain heavy chain covalently connected, and the first polypeptide and the second polypeptide constitute a specific polypeptide complex of a dual and / or multifunctional fusion polypeptide; wherein the linker can exist or not exist independently of each other; in some cases, only one of the second domain or the third domain exists.

[0636] On the other hand, the first polypeptide of the polypeptide complex of the present invention is the heavy chain of the first domain; the second polypeptide of the polypeptide complex of the present invention includes, from the N-terminus to the C-terminus of the polypeptide, the light chain of the first domain, a linker, the third domain and / or the second domain, and the linker and the third domain and / or the second domain are covalently linked. The first polypeptide and the second polypeptide constitute a specific polypeptide complex of a dual and / or multifunctional fusion polypeptide; wherein the linker can exist or not exist independently of each other; in some cases, only one of the second domain or the third domain exists.

[0637] On the other hand, the first polypeptide of the polypeptide complex of the present invention is the heavy chain of the first domain; the second polypeptide of the polypeptide complex of the present invention includes, from the N-terminus to the C-terminus of the polypeptide, the light chain of the first domain, a linker, the third domain and / or the second domain, and the linker and the third domain and / or the second domain are covalently linked. The first polypeptide and the second polypeptide constitute a specific polypeptide complex of a dual and / or multifunctional fusion polypeptide; wherein the linker can exist or not exist independently of each other; in some cases, only one of the second domain or the third domain exists.

[0638] On the other hand, the first polypeptide of the polypeptide complex of the present invention is the heavy chain of the first domain; the second polypeptide of the polypeptide complex of the present invention includes, from the N-terminus to the C-terminus of the polypeptide, the third domain and / or the second domain, a linker, the third domain and / or the second domain, a linker and the light chain of the first domain covalently connected, and the first polypeptide and the second polypeptide constitute a specific polypeptide complex of a dual and / or multifunctional fusion polypeptide; wherein the linker can exist or not exist independently of each other; in some cases, only one of the second domain or the third domain exists.

[0639] In one aspect, the first polypeptide of the polypeptide complex of the present invention comprises, from the N-terminus to the C-terminus of the polypeptide, a third domain and / or a second domain, a linker, and a covalently linked heavy chain of the first domain; the second polypeptide of the polypeptide complex of the present invention comprises, from the N-terminus to the C-terminus of the polypeptide, a light chain of the first domain, a linker, and a covalently linked third domain and / or a second domain; wherein the linker may be independently present or absent; in some cases, only one of the second domain or the third domain is present.

[0640] In one aspect, the first polypeptide of the polypeptide complex of the present invention comprises, from the N-terminus to the C-terminus of the polypeptide, a third domain and / or a second domain, a linker, and a covalently linked heavy chain of the first domain; the second polypeptide of the polypeptide complex of the present invention comprises, from the N-terminus to the C-terminus of the polypeptide, a third domain and / or a second domain, a linker, and a covalently linked light chain of the first domain; wherein the linker may be independently present or absent; in some cases, only one of the second domain or the third domain is present.

[0641] In one aspect, the first polypeptide of the polypeptide complex of the present invention comprises, from the N-terminus to the C-terminus of the polypeptide, a first domain heavy chain, a linker, and a third domain and / or a second domain covalently linked together; the second polypeptide of the polypeptide complex of the present invention comprises, from the N-terminus to the C-terminus of the polypeptide, a third domain and / or a second domain, a linker, and a first domain light chain covalently linked together; wherein the linker may be independently present or absent; in some cases, only one of the second domain or the third domain is present.

[0642] In one aspect, the first polypeptide of the polypeptide complex of the present invention comprises, from the N-terminus to the C-terminus of the polypeptide, a first domain heavy chain, a linker, and a covalently linked third domain and / or second domain; the second polypeptide of the polypeptide complex of the present invention comprises, from the N-terminus to the C-terminus of the polypeptide, a first domain light chain, a linker, and a covalently linked third domain and / or second domain; wherein the linker may be independently present or absent; in some cases, only one of the second domain or the third domain is present.

[0643] On the other hand, the polypeptide complex of the present invention only comprises the structure of the first polypeptide, which includes, from the N-terminus to the C-terminus of the polypeptide, the first domain heavy chain, the linker, the third domain and / or the second domain, and the linker and the third domain and / or the second domain are covalently linked to form a specific polypeptide complex of a dual and / or multifunctional fusion polypeptide; wherein the linker can be independently present or absent; in some cases, only one of the second domain or the third domain exists.

[0644] On the other hand, the polypeptide complex of the present invention only comprises the structure of the first polypeptide, which includes, from the N-terminus to the C-terminus of the polypeptide, the first domain heavy chain, the linker, the third domain and / or the second domain, and the linker and the third domain and / or the second domain are covalently linked to form a specific polypeptide complex of a dual and / or multifunctional fusion polypeptide; wherein the linker can be independently present or absent; in some cases, only one of the second domain or the third domain exists.

[0645] On the other hand, the polypeptide complex of the present invention only comprises the structure of the first polypeptide, and its structure includes, from the N-terminus to the C-terminus of the polypeptide, the third domain and / or the second domain, the linker, the third domain and / or the second domain, the linker and the first domain heavy chain covalently linked to form a specific polypeptide complex of a dual and / or multifunctional fusion polypeptide; wherein the linker can exist independently or not; in some cases, only one of the second domain or the third domain exists.

[0646] The (a) antigen-binding fragment comprising the first domain in the dual- and / or multifunctional fusion polypeptide of the present invention can be derived from any anti-PD-L1 and / or anti-PD-1 antibody, including any antibody that can block or reduce the binding of PD-L1 to its receptor and thereby relieve the PD-L1-mediated immunosuppressive effect, including existing published anti-PD-L1 and / or anti-PD-1 antibodies and anti-PD-L1 antibodies and / or anti-PD-1 antibodies developed in the future.

[0647] The dual and / or multifunctional fusion polypeptide of the present invention comprises an extracellular region in which the second domain comprises one and / or multiple ligands, including at least a polypeptide consisting of the extracellular domain of CD86 or the IgV region of the extracellular domain of CD86. For example, CD86 is a member protein of the B7 family of proteins that can co-stimulate and activate T cells. Mature CD86 molecules are composed of an extracellular domain (ECD), a transmembrane domain, and an intracellular domain, wherein the extracellular domain (ECD) is the key region for these molecules to bind to the corresponding receptors on T cells. The extracellular domain of CD86 contains an immunoglobulin-like V (IgV) region and an immunoglobulin-like C2 (IgC2) region, and the IgV domain is the key domain directly involved in its receptor binding; both the extracellular domain and the IgV domain of CD86 can bind to CD28 and participate in inducing T cell activation, proliferation, and effector function. CTLA4 can also bind to the IgV region of CD86 and participate in immunosuppressive regulation.

[0648] The dual- and / or multifunctional fusion polypeptides of the present invention comprise a third domain comprising an extracellular region of one or more ligands, including at least the LAG3 extracellular domain or one or more subunits of a polypeptide composed of IgD1, IgD2, IgD3, and IgD4 within the LAG3 extracellular domain. For example, LAG3 can be a type 1 transmembrane protein at immune checkpoints, primarily expressed on activated NK cells and T cells. LAG3 is expressed on CD8+ T cells in tumor-depleted cells and may be an effective target for tumor immunotherapy. For example, the function of LAG3 can be two-sided. On the one hand, LAG3 can play a negative regulatory role in T cell proliferation and activation. After binding to its ligand, histocompatibility complex II (MHCII) or ligand, fibrinogen-like protein 1 (FGL1), LAG3 can participate in inhibiting T cell activation and function. On the other hand, LAG3, by binding to MHCII molecules on antigen-presenting cells, can participate in inducing the activation of antigen-presenting cells, such as upregulating the expression of CD80 / CD83 / CD86, enhancing antigen presentation, and secreting chemokines to recruit T cells, etc., to assist T cell activation. While antibodies that antagonize LAG3 have the ability to directly relieve LAG3-mediated T cell inhibition, they also block LAG3-mediated antigen-presenting cell activation, and therefore cannot achieve a comprehensive enhancement of LAG3-related immune activation effects. The LAG3 extracellular domain chimeric protein provided in this application can simultaneously take into account the effects of T cell immunosuppression relief and antigen-presenting cell activation, and may have potential prospects for tumor treatment.

[0649] The following provides an exemplary anti-PD-L1 antibody Sugemalimab bifunctional fusion polypeptide complex, wherein the first polypeptide is covalently linked to the extracellular domain of CD86 via an Fc domain linker (SEQ ID NO: 36), and the first polypeptide and the second polypeptide of the Sugemalimab light chain (SEQ ID NO: 5) constitute the bifunctional fusion polypeptide complex; Sugemalimab bifunctional fusion polypeptide complex, wherein the first polypeptide is covalently linked to the IgV region of the extracellular domain of CD86 via an Fc domain linker (SEQ ID NO: 37), and the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the bifunctional antibody polypeptide complex; Sugemalimab bifunctional fusion polypeptide complex, wherein the first polypeptide is covalently linked to the extracellular domain of CD86 via an antigen binding region heavy chain linker (SEQ ID NO: 38). NO: 38), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the bifunctional fusion polypeptide complex; Sugemalimab is covalently linked to the CD86 extracellular domain IgV region bifunctional fusion polypeptide complex first polypeptide amino acid sequence (SEQ ID NO: 39) via a linker connected to the N-terminus of the heavy chain of the antigen binding region, the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the bifunctional fusion polypeptide complex; Sugemalimab is covalently linked to the CD86 extracellular domain bifunctional fusion polypeptide complex second polypeptide amino acid sequence (SEQ ID NO: 40) via a linker connected to the C-terminus of the light chain of the antigen binding region, the second polypeptide and the first polypeptide of the Sugemalimab heavy chain (SEQ ID NO: 4) constitute the bifunctional fusion polypeptide complex; Sugemalimab is covalently linked to the CD86 extracellular domain IgV region bifunctional fusion polypeptide complex second polypeptide amino acid sequence (SEQ ID NO: 50) via a linker connected to the C-terminus of the light chain of the antigen binding region NO: 41), the second polypeptide and the first polypeptide of the Sugemalimab heavy chain constitute the bifunctional fusion polypeptide complex; the second polypeptide and the first polypeptide of the Sugemalimab heavy chain constitute the bifunctional antibody polypeptide complex; Sugemalimab is covalently linked to the second polypeptide amino acid sequence of the bifunctional fusion polypeptide complex of the CD86 extracellular domain through the antigen-binding region light chain N-linker (SEQ ID NO: 42), the second polypeptide and the first polypeptide of the Sugemalimab heavy chain constitute the bifunctional fusion polypeptide complex;Sugemalimab is covalently linked to the second polypeptide amino acid sequence (SEQ ID NO: 43) of the bifunctional fusion polypeptide complex of the CD86 extracellular domain IgV region via an N-linker in the antigen-binding region of the light chain. The second polypeptide and the first polypeptide of the Sugemalimab heavy chain form the bifunctional fusion polypeptide complex.

[0650] Provided below are the amino acid sequences of the first polypeptide of a bifunctional fusion polypeptide complex in which the exemplary anti-PD-L1 antibody Durvalumab is covalently linked to the extracellular domain of CD86 via an Fc domain linker (SEQ ID NO: 44), wherein the first polypeptide and the second Durvalumab light chain polypeptide (SEQ ID NO: 9) constitute the bifunctional fusion polypeptide complex; the amino acid sequence of the first polypeptide of a bifunctional fusion polypeptide complex in which Durvalumab is covalently linked to the IgV region of the extracellular domain of CD86 via an Fc domain linker (SEQ ID NO: 45), wherein the first polypeptide and the second Durvalumab light chain polypeptide constitute the bifunctional antibody polypeptide complex; and the amino acid sequence of the first polypeptide of a bifunctional fusion polypeptide complex in which Durvalumab is covalently linked to the extracellular domain of CD86 via a linker at the N-terminus of the antigen-binding region of the heavy chain (SEQ ID NO: 46). The first polypeptide and the second polypeptide of the Durvalumab light chain constitute the bifunctional fusion polypeptide complex; Durvalumab is covalently linked to the first polypeptide amino acid sequence of the bifunctional fusion polypeptide complex of the IgV region of the CD86 extracellular domain via a linker at the N-terminus of the heavy chain of the antigen binding region, and the first polypeptide and the second polypeptide of the Durvalumab light chain constitute the bifunctional fusion polypeptide complex; Durvalumab is covalently linked to the second polypeptide amino acid sequence of the bifunctional fusion polypeptide complex of the CD86 extracellular domain via a linker at the C-terminus of the light chain of the antigen binding region, and the second polypeptide and the first polypeptide of the Durvalumab heavy chain (SEQ ID NO: 8) constitute the bifunctional fusion polypeptide complex; Durvalumab is covalently linked to the second polypeptide amino acid sequence of the bifunctional fusion polypeptide complex of the IgV region of the CD86 extracellular domain via a linker at the C-terminus of the light chain of the antigen binding region, and the second polypeptide and the first polypeptide of the Durvalumab heavy chain (SEQ ID NO: 8) constitute the bifunctional fusion polypeptide complex; Durvalumab is covalently linked to the second polypeptide amino acid sequence of the bifunctional fusion polypeptide complex of the IgV region of the CD86 extracellular domain via a linker at the C-terminus of the light chain of the antigen binding region NO: 49), the second polypeptide and the first polypeptide of the Durvalumab heavy chain constitute the bifunctional fusion polypeptide complex; the second polypeptide and the first polypeptide of the Durvalumab heavy chain constitute the bifunctional antibody polypeptide complex; Durvalumab is covalently linked to the second polypeptide amino acid sequence of the bifunctional fusion polypeptide complex of the CD86 extracellular domain through the antigen-binding region light chain N-linked linker (SEQ ID NO: 50), the second polypeptide and the first polypeptide of the Durvalumab heavy chain constitute the bifunctional fusion polypeptide complex; Durvalumab is covalently linked to the second polypeptide amino acid sequence of the bifunctional fusion polypeptide complex of the CD86 extracellular domain IgV region through the antigen-binding region light chain N-linked linker (SEQ ID NO: 51), the second polypeptide and the first polypeptide of the Durvalumab heavy chain constitute the bifunctional fusion polypeptide complex.

[0651] The following provides an exemplary anti-PD-L1 antibody Atezolizumab bifunctional fusion polypeptide complex, wherein the first polypeptide is covalently linked to the CD86 extracellular domain via an Fc domain linker (SEQ ID NO: 52), wherein the first polypeptide and the Atezolizumab light chain second polypeptide (SEQ ID NO: 11) constitute the bifunctional fusion polypeptide complex; an exemplary anti-PD-L1 antibody Atezolizumab bifunctional fusion polypeptide complex, wherein the first polypeptide is covalently linked to the CD86 extracellular domain IgV region via an Fc domain linker (SEQ ID NO: 53), wherein the first polypeptide and the Atezolizumab light chain second polypeptide constitute the bifunctional antibody polypeptide complex; an exemplary anti-PD-L1 antibody bifunctional fusion polypeptide complex, wherein the first polypeptide is covalently linked to the CD86 extracellular domain via an antigen binding region heavy chain N-terminus linker (SEQ ID NO: 54). NO: 54), the first polypeptide and the second polypeptide of the atezolizumab light chain constitute the bifunctional fusion polypeptide complex; atezolizumab is covalently linked to the CD86 extracellular domain IgV region bifunctional fusion polypeptide complex first polypeptide amino acid sequence (SEQ ID NO: 55) via a linker connected to the N-terminus of the antigen-binding region heavy chain, the first polypeptide and the second polypeptide of the atezolizumab light chain constitute the bifunctional fusion polypeptide complex; atezolizumab is covalently linked to the CD86 extracellular domain second polypeptide amino acid sequence (SEQ ID NO: 56) via a linker connected to the C-terminus of the antigen-binding region light chain, the second polypeptide and the first polypeptide of the atezolizumab heavy chain (SEQ ID NO: 10) constitute the bifunctional fusion polypeptide complex; atezolizumab is covalently linked to the CD86 extracellular domain IgV region bifunctional fusion polypeptide complex second polypeptide amino acid sequence (SEQ ID NO: 10) via a linker connected to the C-terminus of the antigen-binding region light chain. NO: 57), the second polypeptide and the first polypeptide of the atezolizumab heavy chain constitute the bifunctional fusion polypeptide complex; the second polypeptide and the first polypeptide of the atezolizumab heavy chain constitute the bifunctional antibody polypeptide complex; atezolizumab is covalently linked to the CD86 extracellular domain by an N-linker in the antigen-binding region light chain, and the second polypeptide and the first polypeptide of the atezolizumab heavy chain constitute the bifunctional fusion polypeptide complex;Atezolizumab is covalently linked to a second polypeptide amino acid sequence (SEQ ID NO: 59) of the bifunctional fusion polypeptide complex of the CD86 extracellular domain IgV region via an N-linker in the antigen-binding region of the light chain. The second polypeptide and the first polypeptide of the atezolizumab heavy chain form the bifunctional fusion polypeptide complex.

[0652] The following provides an exemplary anti-PD-L1 antibody Avelumab, wherein the first polypeptide amino acid sequence of the bifunctional fusion polypeptide complex is covalently linked to the extracellular domain of CD86 via an Fc domain linker (SEQ ID NO: 60), wherein the first polypeptide and the second Avelumab light chain polypeptide (SEQ ID NO: 13) constitute the bifunctional fusion polypeptide complex; a bifunctional fusion polypeptide complex wherein Avelumab is covalently linked to the IgV region of the extracellular domain of CD86 via an Fc domain linker (SEQ ID NO: 61), wherein the first polypeptide and the second Avelumab light chain polypeptide constitute the bifunctional antibody polypeptide complex; a bifunctional fusion polypeptide complex wherein Avelumab is covalently linked to the extracellular domain of CD86 via an antigen binding region heavy chain N-terminus linker (SEQ ID NO: 62). NO: 62), the first polypeptide and the avelumab light chain second polypeptide constitute the bifunctional fusion polypeptide complex; avelumab is covalently linked to the CD86 extracellular domain IgV region bifunctional fusion polypeptide complex first polypeptide amino acid sequence (SEQ ID NO: 63) via a linker connected to the N-terminus of the antigen-binding region heavy chain, the first polypeptide and the avelumab light chain second polypeptide constitute the bifunctional fusion polypeptide complex; avelumab is covalently linked to the CD86 extracellular domain second polypeptide amino acid sequence (SEQ ID NO: 64) via a linker connected to the C-terminus of the antigen-binding region light chain, the second polypeptide and the avelumab heavy chain first polypeptide (SEQ ID NO: 12) constitute the bifunctional fusion polypeptide complex; avelumab is covalently linked to the CD86 extracellular domain IgV region bifunctional fusion polypeptide complex second polypeptide amino acid sequence (SEQ ID NO: 13) via a linker connected to the C-terminus of the antigen-binding region light chain. NO: 65), the second polypeptide and the first avelumab heavy chain polypeptide constitute the bifunctional fusion polypeptide complex; the second polypeptide and the first avelumab heavy chain polypeptide constitute the bifunctional antibody polypeptide complex; avelumab is covalently linked to the second polypeptide amino acid sequence of the bifunctional fusion polypeptide complex of the CD86 extracellular domain through an antigen-binding region light chain N-linked linker (SEQ ID NO: 66), the second polypeptide and the first avelumab heavy chain polypeptide constitute the bifunctional fusion polypeptide complex; avelumab is covalently linked to the second polypeptide amino acid sequence of the bifunctional fusion polypeptide complex of the CD86 extracellular domain IgV region through an antigen-binding region light chain N-linked linker (SEQ ID NO: 67), the second polypeptide and the first avelumab heavy chain polypeptide constitute the bifunctional fusion polypeptide complex.

[0653] Provided below are the amino acid sequence of the first polypeptide of a bifunctional fusion polypeptide complex in which the exemplary anti-PD-L1 antibody Envafolimab is covalently linked to the extracellular domain of CD86 via an Fc domain linker (SEQ ID NO: 68), the first polypeptide constituting the bifunctional fusion polypeptide complex; the amino acid sequence of the first polypeptide of a bifunctional fusion polypeptide complex in which Envafolimab is covalently linked to the IgV region of the extracellular domain of CD86 via an Fc domain linker (SEQ ID NO: 69), the first polypeptide constituting the bifunctional antibody polypeptide complex; the amino acid sequence of the first polypeptide of a bifunctional fusion polypeptide complex in which Envafolimab is covalently linked to the extracellular domain of CD86 via an antigen-binding region heavy chain N-terminus linker (SEQ ID NO: 70), the first polypeptide constituting the bifunctional fusion polypeptide complex; the amino acid sequence of the first polypeptide of a bifunctional fusion polypeptide complex in which Envafolimab is covalently linked to the IgV region of the extracellular domain of CD86 via an antigen-binding region heavy chain N-terminus linker (SEQ ID NO: 71), the first polypeptide constituting the bifunctional fusion polypeptide complex;

[0654] The following provides an exemplary anti-PD-1 antibody Permbrolizumab bifunctional fusion polypeptide complex, wherein the first polypeptide is covalently linked to the CD86 extracellular domain via an Fc domain linker (SEQ ID NO: 72), wherein the first polypeptide and the Permbrolizumab light chain second polypeptide (SEQ ID NO: 16) constitute the bifunctional fusion polypeptide complex; the first polypeptide is covalently linked to the CD86 extracellular domain IgV region via an Fc domain linker (SEQ ID NO: 73), wherein the first polypeptide and the Permbrolizumab light chain second polypeptide constitute the bifunctional antibody polypeptide complex; the first polypeptide is covalently linked to the CD86 extracellular domain via an antigen binding region heavy chain N-terminus linker (SEQ ID NO: 74). NO: 74), the first polypeptide and the second polypeptide of the light chain of Permbrolizumab constitute the bifunctional fusion polypeptide complex; Permbrolizumab is covalently linked to the CD86 extracellular domain IgV region bifunctional fusion polypeptide complex first polypeptide amino acid sequence (SEQ ID NO: 75) via a linker at the N-terminus of the heavy chain of the antigen binding region, the first polypeptide and the second polypeptide of the light chain of Permbrolizumab constitute the bifunctional fusion polypeptide complex; Permbrolizumab is covalently linked to the CD86 extracellular domain bifunctional fusion polypeptide complex second polypeptide amino acid sequence (SEQ ID NO: 76) via a linker at the C-terminus of the light chain of the antigen binding region, the second polypeptide and the first polypeptide of the heavy chain of Permbrolizumab (SEQ ID NO: 15) constitute the bifunctional fusion polypeptide complex; Permbrolizumab is covalently linked to the CD86 extracellular domain IgV region bifunctional fusion polypeptide complex second polypeptide amino acid sequence (SEQ ID NO: 16) via a linker at the C-terminus of the light chain of the antigen binding region NO: 77), the second polypeptide and the first polypeptide of the heavy chain of Permbrolizumab constitute the bifunctional fusion polypeptide complex; the second polypeptide and the first polypeptide of the heavy chain of Permbrolizumab constitute the bifunctional antibody polypeptide complex; the second polypeptide amino acid sequence of the bifunctional fusion polypeptide complex in which Permbrolizumab is covalently linked to the extracellular domain of CD86 via an N-linked linker of the antigen-binding region light chain (SEQ ID NO: 78), the second polypeptide and the first polypeptide of the heavy chain of Permbrolizumab constitute the bifunctional fusion polypeptide complex;Permbrolizumab is covalently linked to a second polypeptide amino acid sequence (SEQ ID NO: 79) of a bifunctional fusion polypeptide complex of the CD86 extracellular domain IgV region via an N-linked linker in the antigen-binding region of the light chain. The second polypeptide and the first polypeptide of the heavy chain of Permbrolizumab form the bifunctional fusion polypeptide complex.

[0655] The following provides an exemplary anti-PD-1 antibody Nivolumab bifunctional fusion polypeptide complex, wherein the first polypeptide is covalently linked to the CD86 extracellular domain via an Fc domain linker (SEQ ID NO: 80), wherein the first polypeptide and the Nivolumab light chain second polypeptide (SEQ ID NO: 18) constitute the bifunctional fusion polypeptide complex; a bifunctional fusion polypeptide complex, wherein Nivolumab is covalently linked to the IgV region of the CD86 extracellular domain via an Fc domain linker (SEQ ID NO: 81), wherein the first polypeptide and the Nivolumab light chain second polypeptide constitute the bifunctional antibody polypeptide complex; a bifunctional fusion polypeptide complex, wherein the first polypeptide is covalently linked to the CD86 extracellular domain via an antigen binding region heavy chain N-terminus linker (SEQ ID NO: 82). NO: 82), the first polypeptide and the second polypeptide of the nivolumab light chain constitute the bifunctional fusion polypeptide complex; nivolumab is covalently linked to the first polypeptide amino acid sequence of the bifunctional fusion polypeptide complex of the IgV region of the CD86 extracellular domain via a linker at the N-terminus of the antigen-binding region heavy chain (SEQ ID NO: 83), the first polypeptide and the second polypeptide of the nivolumab light chain constitute the bifunctional fusion polypeptide complex; nivolumab is covalently linked to the second polypeptide amino acid sequence of the bifunctional fusion polypeptide complex of the CD86 extracellular domain via a linker at the C-terminus of the antigen-binding region light chain (SEQ ID NO: 84), the second polypeptide and the first polypeptide of the nivolumab heavy chain (SEQ ID NO: 17) constitute the bifunctional fusion polypeptide complex; nivolumab is covalently linked to the second polypeptide amino acid sequence of the bifunctional fusion polypeptide complex of the IgV region of the CD86 extracellular domain via a linker at the C-terminus of the antigen-binding region light chain (SEQ ID NO: NO: 85), the second polypeptide and the first polypeptide of the nivolumab heavy chain constitute the bifunctional fusion polypeptide complex; the second polypeptide and the first polypeptide of the nivolumab heavy chain constitute the bifunctional antibody polypeptide complex; nivolumab is covalently linked to the second polypeptide amino acid sequence of the bifunctional fusion polypeptide complex of the CD86 extracellular domain through the antigen-binding region light chain N-linked linker (SEQ ID NO: 86), the second polypeptide and the first polypeptide of the nivolumab heavy chain constitute the bifunctional fusion polypeptide complex; nivolumab is covalently linked to the second polypeptide amino acid sequence of the bifunctional fusion polypeptide complex of the CD86 extracellular domain IgV region through the antigen-binding region light chain N-linked linker (SEQ ID NO: 87), the second polypeptide and the first polypeptide of the nivolumab heavy chain constitute the bifunctional fusion polypeptide complex.

[0656] Provided below is an exemplary anti-PD-L1 antibody Sugemalimab heavy chain covalently linked to the CD86 extracellular domain via a linker at the C-terminus of the Fc domain, which in turn is covalently linked to the LAG3 extracellular domain via a linker at the C-terminus of the CD86 extracellular domain. The first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the first polypeptide amino acid sequence of the Sugemalimab heavy chain covalently linked to the CD86 extracellular domain via a linker at the C-terminus of the Fc domain, which in turn is covalently linked to the LAG3 extracellular domain D1 / D2 / D3 via a linker at the C-terminus of the CD86 extracellular domain is covalently linked to the LAG3 extracellular domain D1 / D2 / D3 via a linker at the C-terminus of the Fc domain. NO: 89), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the Sugemalimab heavy chain is covalently linked to the CD86 extracellular domain via the Fc domain C-terminus connecting linker, and the CD86 extracellular domain C-terminus connecting linker is covalently linked to the LAG3 extracellular domain D1 / D2 first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex (SEQ ID NO: 90), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the Sugemalimab heavy chain is covalently linked to the CD86 extracellular domain via the Fc domain C-terminus connecting linker, and the CD86 extracellular domain C-terminus connecting linker is covalently linked to the LAG3 extracellular domain D1 first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex (SEQ ID NO: 91), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the Sugemalimab heavy chain is covalently linked to the LAG3 extracellular domain via a linker at the C-terminus of the Fc domain, and the LAG3 extracellular domain is covalently linked to the CD86 extracellular domain via a linker at the C-terminus of the Fc domain, and the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the Sugemalimab heavy chain is covalently linked to the LAG3 extracellular domain D1 / D2 / D3 via a linker at the C-terminus of the Fc domain, and the LAG3 extracellular domain D1 / D2 / D3 is covalently linked to the CD86 extracellular domain via a linker at the C-terminus of the Fc domain, and the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex;The first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the Sugemalimab heavy chain is covalently linked to the LAG3 extracellular domains D1 / D2 via the Fc domain C-terminus connecting linker, and the LAG3 extracellular domains D1 / D2 are covalently linked to the CD86 extracellular domain via the C-terminus connecting linker, and the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the Sugemalimab heavy chain is covalently linked to the LAG3 extracellular domain D1 via the Fc domain C-terminus connecting linker, and the LAG3 extracellular domain D1 is covalently linked to the CD86 extracellular domain via the C-terminus connecting linker, and the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex;

[0657] Provided below is an exemplary anti-PD-L1 antibody Sugemalimab heavy chain covalently linked to the CD86 extracellular domain IgV region via a linker at the C-terminus of the Fc domain, which in turn is covalently linked to the LAG3 extracellular domain via a linker at the C-terminus of the CD86 extracellular domain IgV region. The first polypeptide and the Sugemalimab light chain second polypeptide constitute the multifunctional fusion polypeptide complex; the Sugemalimab heavy chain covalently linked to the CD86 extracellular domain IgV region via a linker at the C-terminus of the Fc domain, which in turn is covalently linked to the LAG3 extracellular domain D1 / D2 / D3 via a linker at the C-terminus of the CD86 extracellular domain IgV region. NO: 97), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the Sugemalimab heavy chain is covalently linked to the CD86 extracellular domain IgV region via the Fc domain C-terminus connected to a linker, and the CD86 extracellular domain IgV region C-terminus connected to a linker is covalently linked to the LAG3 extracellular domain D1 / D2 first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex (SEQ ID NO: 98), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the Sugemalimab heavy chain is covalently linked to the CD86 extracellular domain IgV region via the Fc domain C-terminus connected to a linker, and the CD86 extracellular domain IgV region C-terminus connected to a linker is covalently linked to the LAG3 extracellular domain D1 first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex (SEQ ID NO: NO: 99), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the Sugemalimab heavy chain is covalently linked to the LAG3 extracellular domain via a linker at the C-terminus of the Fc domain, and the LAG3 extracellular domain is covalently linked to the IgV region of the CD86 extracellular domain via a linker, the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex;The heavy chain of Sugemalimab is covalently linked to the extracellular domains D1 / D2 / D3 of LAG3 via a linker at the C-terminus of the Fc domain, and the C-terminus of the extracellular domains D1 / D2 / D3 of LAG3 are covalently linked to the IgV region of the extracellular domain of CD86 via a linker at the C-terminus of the Fc domain, wherein the first polypeptide and the second polypeptide of the light chain of Sugemalimab constitute the multifunctional fusion polypeptide complex; the first polypeptide and the second polypeptide of the light chain of Sugemalimab constitute the multifunctional fusion polypeptide complex; the heavy chain of Sugemalimab is covalently linked to the extracellular domains D1 / D2 of LAG3 via a linker at the C-terminus of the Fc domain, and the C-terminus of the extracellular domains D1 / D2 of LAG3 are covalently linked to the IgV region of the extracellular domain of CD86 via a linker at the C-terminus of the Fc domain, wherein the first polypeptide and the second polypeptide of the light chain of Sugemalimab constitute the multifunctional fusion polypeptide complex. NO: 102), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the Sugemalimab heavy chain is covalently linked to the LAG3 extracellular domain D1 via a linker connected to the C-terminus of the Fc domain, and the LAG3 extracellular domain D1 C-terminus is covalently linked to the CD86 extracellular domain IgV region via a linker, and the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex.

[0658] The following provides an exemplary first polypeptide amino acid sequence of a multifunctional fusion polypeptide complex in which the C-terminus of the CD86 extracellular domain is covalently linked to the heavy chain of the anti-PD-L1 antibody Sugemalimab, which is covalently linked to the C-terminus of Sugemalimab (SEQ ID NO: 104), wherein the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the first polypeptide amino acid sequence of a multifunctional fusion polypeptide complex in which the C-terminus of the CD86 extracellular domain is covalently linked to the heavy chain of the anti-PD-L1 antibody Sugemalimab, which is covalently linked to the C-terminus of the Sugemalimab heavy chain (SEQ ID NO: 105), wherein the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; NO: 105), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the CD86 extracellular domain C-terminus is connected to a linker to be covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain, the C-terminus of the Sugemalimab heavy chain is connected to a linker to be covalently linked to the LAG3 extracellular domain D1 / D2 first polypeptide amino acid sequence (SEQ ID NO: 106), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the CD86 extracellular domain C-terminus is connected to a linker to be covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain, the C-terminus of the Sugemalimab heavy chain is connected to a linker to be covalently linked to the LAG3 extracellular domain D1 first polypeptide amino acid sequence (SEQ ID NO: NO: 107), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the LAG3 extracellular domain C-terminus is connected to a linker to be covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain, and the Sugemalimab C-terminus is connected to a linker to be covalently linked to the CD86 extracellular domain, a first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex (SEQ ID NO: 108), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the LAG3 extracellular domain D1 / D2 / D3 C-terminus is connected to a linker to be covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain, and the Sugemalimab C-terminus is connected to a linker to be covalently linked to the CD86 extracellular domain, a first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex (SEQ ID NO: 109), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex;The LAG3 extracellular domain D1 / D2 C-terminus is connected to a linker covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain, the Sugemalimab C-terminus is connected to a linker covalently linked to a CD86 extracellular domain first polypeptide amino acid sequence (SEQ ID NO: 110), the first polypeptide and the Sugemalimab light chain second polypeptide constitute the multifunctional fusion polypeptide complex; the first polypeptide and the Sugemalimab light chain second polypeptide constitute the multifunctional fusion polypeptide complex; the LAG3 extracellular domain D1 C-terminus is connected to a linker covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain, the Sugemalimab C-terminus is connected to a linker covalently linked to the CD86 extracellular domain first polypeptide amino acid sequence (SEQ ID NO: 111), the first polypeptide and the Sugemalimab light chain second polypeptide constitute the multifunctional fusion polypeptide complex;

[0659] The following provides an exemplary first polypeptide amino acid sequence of a multifunctional fusion polypeptide complex in which the C-terminus of the IgV region of the extracellular domain of CD86 is linked to a linker to covalently link the heavy chain of the anti-PD-L1 antibody Sugemalimab, which is linked to the C-terminus of Sugemalimab to covalently link the LAG3 extracellular domain (SEQ ID NO: 112), wherein the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the first polypeptide amino acid sequence of a multifunctional fusion polypeptide complex in which the C-terminus of the IgV region of the extracellular domain of CD86 is linked to a linker to covalently link the heavy chain of the anti-PD-L1 antibody Sugemalimab, which is linked to the C-terminus of the Sugemalimab heavy chain to covalently link the LAG3 extracellular domain D1 / D2 / D3 (SEQ ID NO: 113). NO: 113), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the C-terminus of the IgV region of the CD86 extracellular domain is connected to a linker to be covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain, the C-terminus of the Sugemalimab heavy chain is connected to a linker to be covalently linked to the LAG3 extracellular domain D1 / D2 first polypeptide amino acid sequence (SEQ ID NO: 114), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the C-terminus of the IgV region of the CD86 extracellular domain is connected to a linker to be covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain, the C-terminus of the Sugemalimab heavy chain is connected to a linker to be covalently linked to the LAG3 extracellular domain D1 first polypeptide amino acid sequence (SEQ ID NO: 115), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the LAG3 extracellular domain C-terminus is connected to a linker to be covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain, and the Sugemalimab C-terminus is connected to a linker to be covalently linked to the CD86 extracellular domain IgV region, a first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex (SEQ ID NO: 116), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the LAG3 extracellular domain D1 / D2 / D3 C-terminus is connected to a linker to be covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain, and the Sugemalimab C-terminus is connected to a linker to be covalently linked to the CD86 extracellular domain IgV region, a first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex (SEQ ID NO: 117), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex;The LAG3 extracellular domain D1 / D2 C-terminus is covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain through a linker, the Sugemalimab C-terminus is covalently linked to the CD86 extracellular domain IgV region through a linker, and the first polypeptide of the multifunctional fusion polypeptide complex (SEQ ID NO: 118) and the Sugemalimab light chain second polypeptide constitute the multifunctional fusion polypeptide complex; the first polypeptide and the Sugemalimab light chain second polypeptide constitute the multifunctional fusion polypeptide complex; the LAG3 extracellular domain D1 C-terminus is covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain through a linker, and the Sugemalimab C-terminus is covalently linked to the CD86 extracellular domain IgV region through a linker, and the first polypeptide and the Sugemalimab light chain second polypeptide constitute the multifunctional fusion polypeptide complex;

[0660] The following provides an exemplary first polypeptide amino acid sequence (SEQ ID NO: 120) of a multifunctional fusion polypeptide complex in which the C-terminus of the CD86 extracellular domain is covalently linked to the LAG3 extracellular domain, which is covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain by a linker at the C-terminus of the LAG3 extracellular domain. The first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the first polypeptide amino acid sequence (SEQ ID NO: 121) of a multifunctional fusion polypeptide complex in which the C-terminus of the CD86 extracellular domain is covalently linked to the LAG3 extracellular domain D1 / D2 / D3, which is covalently linked to the C-terminus of the LAG3 extracellular domain D1 / D2 / D3 by a linker at the C-terminus of the LAG3 extracellular domain D1 / D2 / D3; the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the C-terminus of the CD86 extracellular domain is covalently linked to the LAG3 extracellular domain D1 / D2, which is covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain by a linker at the C-terminus of the LAG3 extracellular domain D1 / D2 The multifunctional fusion polypeptide complex comprises a first polypeptide amino acid sequence (SEQ ID NO: 122) in which a C-terminal linker is covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain, and the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the CD86 extracellular domain C-terminal linker is covalently linked to the LAG3 extracellular domain D1, and the LAG3 extracellular domain D1 is covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain, and the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the LAG3 extracellular domain C-terminal linker is covalently linked to the CD86 extracellular domain, and the CD86 extracellular domain C-terminal linker is covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain, and the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex. NO: 124), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the LAG3 extracellular domain D1 / D2 / D3 C-terminus is connected to a linker to covalently link to the CD86 extracellular domain, the CD86 extracellular domain C-terminus is connected to a linker to covalently link to the anti-PD-L1 antibody Sugemalimab heavy chain first polypeptide amino acid sequence (SEQ ID NO: 125), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex;The LAG3 extracellular domain D1 / D2 C-terminus is linked to a linker covalently linked to the CD86 extracellular domain, the CD86 extracellular domain C-terminus is linked to a linker covalently linked to a first polypeptide amino acid sequence (SEQ ID NO: 126) of a multifunctional fusion polypeptide complex of the anti-PD-L1 antibody Sugemalimab heavy chain, and the first polypeptide and the Sugemalimab light chain second polypeptide constitute the multifunctional fusion polypeptide complex; the LAG3 extracellular domain D1 C-terminus is linked to a linker covalently linked to the CD86 extracellular domain, the CD86 extracellular domain C-terminus is linked to a linker covalently linked to a first polypeptide amino acid sequence (SEQ ID NO: 127) of a multifunctional fusion polypeptide complex of the anti-PD-L1 antibody Sugemalimab heavy chain, and the first polypeptide and the Sugemalimab light chain second polypeptide constitute the multifunctional fusion polypeptide complex;

[0661] The following provides an exemplary first polypeptide amino acid sequence of a multifunctional fusion polypeptide complex in which the C-terminus of the IgV region of the CD86 extracellular domain is linked to a linker covalently linked to the LAG3 extracellular domain, which is in turn linked to the C-terminus of the LAG3 extracellular domain. The amino acid sequence of the first polypeptide of the multifunctional fusion polypeptide complex is provided (SEQ ID NO: 128), wherein the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the amino acid sequence of the first polypeptide amino acid sequence of the IgV region of the CD86 extracellular domain is linked to a linker covalently linked to the LAG3 extracellular domain D1 / D2 / D3, which is linked to the C-terminus of the LAG3 extracellular domain D1 / D2 / D3. NO: 129), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the C-terminus of the IgV region of the CD86 extracellular domain is connected to a linker to covalently link the extracellular domains D1 / D2 of LAG3, and the C-terminus of the LAG3 extracellular domains D1 / D2 is connected to a linker to covalently link the anti-PD-L1 antibody Sugemalimab heavy chain first polypeptide amino acid sequence (SEQ ID NO: 130), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the C-terminus of the IgV region of the CD86 extracellular domain is connected to a linker to covalently link the extracellular domain D1 of LAG3, and the C-terminus of the LAG3 extracellular domain D1 is connected to a linker to covalently link the anti-PD-L1 antibody Sugemalimab heavy chain first polypeptide amino acid sequence (SEQ ID NO: 131), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the LAG3 extracellular domain C-terminus is connected to a linker and covalently linked to the CD86 extracellular domain IgV region, which is in turn covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain by a first polypeptide amino acid sequence (SEQ ID NO: 132), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex; the LAG3 extracellular domain D1 / D2 / D3 C-terminus is connected to a linker and covalently linked to the CD86 extracellular domain IgV region, which is in turn covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain by a first polypeptide amino acid sequence (SEQ ID NO: 133), the first polypeptide and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex;The LAG3 extracellular domain D1 / D2 C-terminus is linked to a linker covalently linked to the CD86 extracellular domain IgV region, the CD86 extracellular domain IgV region C-terminus is linked to a linker covalently linked to a first polypeptide amino acid sequence (SEQ ID NO: 134) of a multifunctional fusion polypeptide complex of the anti-PD-L1 antibody Sugemalimab heavy chain, and the first polypeptide and the Sugemalimab light chain second polypeptide constitute the multifunctional fusion polypeptide complex; the LAG3 extracellular domain D1 C-terminus is linked to a linker covalently linked to the CD86 extracellular domain IgV region, the CD86 extracellular domain IgV region C-terminus is linked to a linker covalently linked to a first polypeptide amino acid sequence (SEQ ID NO: 135) of a multifunctional fusion polypeptide complex of the anti-PD-L1 antibody Sugemalimab heavy chain, and the first polypeptide and the Sugemalimab light chain second polypeptide constitute the multifunctional fusion polypeptide complex;

[0662] The following provides an exemplary first polypeptide amino acid sequence of a multifunctional fusion polypeptide complex in which the C-terminus of the CD86 extracellular domain is covalently linked to the heavy chain of the anti-PD-L1 antibody Sugemalimab by a linker (SEQ ID NO: 38), the first polypeptide and the LAG3 extracellular domain C-terminus are covalently linked to the second polypeptide of the Sugemalimab light chain by a linker to form the multifunctional fusion polypeptide complex; a first polypeptide amino acid sequence of a multifunctional fusion polypeptide complex in which the IgV region of the CD86 extracellular domain is covalently linked to the heavy chain of the anti-PD-L1 antibody Sugemalimab by a linker (SEQ ID NO: 39), the first polypeptide and the LAG3 extracellular domain C-terminus are covalently linked to the second polypeptide of the Sugemalimab light chain by a linker to form the multifunctional fusion polypeptide complex; a first polypeptide amino acid sequence of a multifunctional fusion polypeptide complex in which the C-terminus of the PD-L1 antibody Sugemalimab heavy chain is covalently linked to the CD86 extracellular domain by a linker (SEQ ID NO: NO: 40), the first polypeptide is connected to the C-terminus of the LAG3 extracellular domain by a linker and is covalently linked to the second polypeptide of the Sugemalimab light chain to form the multifunctional fusion polypeptide complex; the PD-L1 antibody Sugemalimab heavy chain C-terminus is connected to a linker and is covalently linked to the first polypeptide amino acid sequence of the CD86 extracellular domain IgV region (SEQ ID NO: 41), the first polypeptide is connected to the C-terminus of the LAG3 extracellular domain by a linker and is covalently linked to the second polypeptide of the Sugemalimab light chain to form the multifunctional fusion polypeptide complex.

[0663] The following provides exemplary amino acid sequences of a first polypeptide of a multifunctional fusion polypeptide complex in which a CD86 extracellular domain is covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain by a linker at the C-terminus, the first polypeptide is covalently linked to a second polypeptide of the Sugemalimab light chain by a linker at the C-terminus of the LAG3 extracellular domain D1 / D2 / D3 to form the multifunctional fusion polypeptide complex; an amino acid sequence of a first polypeptide of a multifunctional fusion polypeptide complex in which a CD86 extracellular domain IgV region is covalently linked to the anti-PD-L1 antibody Sugemalimab heavy chain by a linker at the C-terminus, the first polypeptide is covalently linked to a second polypeptide of the Sugemalimab light chain by a linker at the C-terminus of the LAG3 extracellular domain D1 / D2 / D3 to form the multifunctional fusion polypeptide complex; an amino acid sequence of a first polypeptide of a multifunctional fusion polypeptide complex in which a PD-L1 antibody Sugemalimab heavy chain is covalently linked to the CD86 extracellular domain by a linker, the first polypeptide is covalently linked to the LAG3 extracellular domain D1 / D2 / D3 The C-terminal connecting linker is covalently connected to the second polypeptide of the Sugemalimab light chain to form the multifunctional fusion polypeptide complex; the C-terminal connecting linker of the PD-L1 antibody Sugemalimab heavy chain is covalently connected to the first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the IgV region of the CD86 extracellular domain, and the first polypeptide and the LAG3 extracellular domain D1 / D2 / D3 C-terminal connecting linker are covalently connected to the second polypeptide of the Sugemalimab light chain to form the multifunctional fusion polypeptide complex.

[0664] The following provides an exemplary first polypeptide amino acid sequence of a multifunctional fusion polypeptide complex in which the C-terminus of the extracellular domain of CD86 is connected to a linker and covalently linked to the heavy chain of the anti-PD-L1 antibody Sugemalimab. The first polypeptide is covalently linked to the C-terminus of the LAG3 extracellular domain D1 / D2 by a linker and covalently linked to the second polypeptide (SEQ ID NO: 138) of the Sugemalimab light chain to form the multifunctional fusion polypeptide complex; the first polypeptide amino acid sequence of a multifunctional fusion polypeptide complex in which the C-terminus of the IgV region of the CD86 extracellular domain is covalently linked to the heavy chain of the anti-PD-L1 antibody Sugemalimab by a linker, the first polypeptide is covalently linked to the LAG3 extracellular domain D1 / D2 by a linker The C-terminal connecting linker is covalently connected to the second polypeptide of the Sugemalimab light chain to form the multifunctional fusion polypeptide complex; the C-terminal connecting linker of the PD-L1 antibody Sugemalimab heavy chain is covalently connected to the first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the CD86 extracellular domain, and the first polypeptide and the LAG3 extracellular domain D1 / D2 C-terminal connecting linker are covalently connected to the second polypeptide of the Sugemalimab light chain to form the multifunctional fusion polypeptide complex; the C-terminal connecting linker of the PD-L1 antibody Sugemalimab heavy chain is covalently connected to the first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the IgV region of the CD86 extracellular domain, and the first polypeptide and the LAG3 extracellular domain D1 / D2 C-terminal connecting linker are covalently connected to the second polypeptide of the Sugemalimab light chain to form the multifunctional fusion polypeptide complex.

[0665] The following provides exemplary amino acid sequences of a first polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the extracellular domain of CD86 is covalently linked to the heavy chain of the anti-PD-L1 antibody Sugemalimab by a linker, the first polypeptide being covalently linked to the C-terminus of the LAG3 extracellular domain D1 by a linker, and the second polypeptide of the Sugemalimab light chain being covalently linked to the C-terminus of the LAG3 extracellular domain D1 by a linker to form the multifunctional fusion polypeptide complex; amino acid sequences of a first polypeptide of a multifunctional fusion polypeptide complex in which the IgV region of the extracellular domain of CD86 is covalently linked to the heavy chain of the anti-PD-L1 antibody Sugemalimab by a linker, the first polypeptide being covalently linked to the C-terminus of the LAG3 extracellular domain D1 by a linker to form the second polypeptide of the Sugemalimab light chain; amino acid sequences of a first polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the PD-L1 antibody Sugemalimab heavy chain is covalently linked to the CD86 extracellular domain by a linker, the first polypeptide being covalently linked to the LAG3 extracellular domain D1 by a linker to form the multifunctional fusion polypeptide complex. The C-terminal connecting linker is covalently connected to the second polypeptide of the Sugemalimab light chain to form the multifunctional fusion polypeptide complex; the C-terminal connecting linker of the PD-L1 antibody Sugemalimab heavy chain is covalently connected to the first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the CD86 extracellular domain IgV region, and the first polypeptide and the LAG3 extracellular domain D1 C-terminal connecting linker are covalently connected to the second polypeptide of the Sugemalimab light chain to form the multifunctional fusion polypeptide complex.

[0666] The following provides an exemplary first polypeptide amino acid sequence of a multifunctional fusion polypeptide complex in which a linker is covalently linked to the heavy chain of the anti-PD-L1 antibody Sugemalimab at the C-terminus of the CD86 extracellular domain, and the first polypeptide is covalently linked to the second polypeptide of the LAG3 extracellular domain by a linker at the C-terminus of the Sugemalimab light chain (SEQ ID NO: NO: 140) to form the multifunctional fusion polypeptide complex; the C-terminus of the IgV region of the CD86 extracellular domain is connected to a linker and covalently linked to a first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the heavy chain of the anti-PD-L1 antibody Sugemalimab, the first polypeptide and the C-terminus of the Sugemalimab light chain are covalently linked to a second polypeptide of the LAG3 extracellular domain to form the multifunctional fusion polypeptide complex; the C-terminus of the PD-L1 antibody Sugemalimab heavy chain is connected to a linker and covalently linked to a first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the CD86 extracellular domain, the first polypeptide and the C-terminus of the Sugemalimab light chain are covalently linked to a second polypeptide of the LAG3 extracellular domain to form the multifunctional fusion polypeptide complex; the C-terminus of the PD-L1 antibody Sugemalimab heavy chain is connected to a linker and covalently linked to a first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the IgV region of the CD86 extracellular domain, the first polypeptide and the C-terminus of the Sugemalimab light chain are covalently linked to a second polypeptide of the LAG3 extracellular domain to form the multifunctional fusion polypeptide complex.

[0667] The following provides exemplary amino acid sequences of a first polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the extracellular domain of CD86 is linked to a linker and covalently linked to the heavy chain of the anti-PD-L1 antibody Sugemalimab. The first polypeptide and a second polypeptide (SEQ ID NO: 141) of the extracellular domain of LAG3 D1 / D2 / D3 are linked to the C-terminus of the light chain of Sugemalimab and covalently linked to form the multifunctional fusion polypeptide complex; an amino acid sequence of a first polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the IgV region of the extracellular domain of CD86 is linked to a linker and covalently linked to the heavy chain of the anti-PD-L1 antibody Sugemalimab. The first polypeptide and a second polypeptide of the extracellular domain of LAG3 D1 / D2 / D3 are linked to the C-terminus of the light chain of Sugemalimab and covalently linked to form the multifunctional fusion polypeptide complex; and a multifunctional fusion polypeptide complex in which the C-terminus of the heavy chain of the PD-L1 antibody Sugemalimab is linked to the extracellular domain of CD86 and covalently linked to form the multifunctional fusion polypeptide complex. A polypeptide amino acid sequence, wherein the first polypeptide is connected to the C-terminus of the Sugemalimab light chain and the second polypeptide of the LAG3 extracellular domain D1 / D2 / D3 is covalently linked to a linker to form the multifunctional fusion polypeptide complex; a PD-L1 antibody Sugemalimab heavy chain C-terminus is connected to a linker and covalently linked to the CD86 extracellular domain IgV region. The first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex, wherein the first polypeptide is connected to the C-terminus of the Sugemalimab light chain and the second polypeptide of the LAG3 extracellular domain D1 / D2 / D3 is covalently linked to a linker to form the multifunctional fusion polypeptide complex.

[0668] The following provides exemplary amino acid sequences of a first polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the extracellular domain of CD86 is linked to a linker and covalently linked to the heavy chain of the anti-PD-L1 antibody Sugemalimab. The first polypeptide and a second polypeptide (SEQ ID NO: 142) of the LAG3 extracellular domain D1 / D2 are linked to the C-terminus of the Sugemalimab light chain and covalently linked to form the multifunctional fusion polypeptide complex; an amino acid sequence of a first polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the IgV region of the extracellular domain of CD86 is linked to a linker and covalently linked to the heavy chain of the anti-PD-L1 antibody Sugemalimab. The first polypeptide and a second polypeptide of the LAG3 extracellular domain D1 / D2 are linked to the C-terminus of the Sugemalimab light chain and covalently linked to form the multifunctional fusion polypeptide complex; and a multifunctional fusion polypeptide complex in which the C-terminus of the PD-L1 antibody Sugemalimab heavy chain is linked to a linker and covalently linked to the CD86 extracellular domain. A first polypeptide amino acid sequence, wherein the first polypeptide and the second polypeptide of the LAG3 extracellular domain D1 / D2 are covalently linked to the C-terminus of the Sugemalimab light chain through a linker to form the multifunctional fusion polypeptide complex; a first polypeptide amino acid sequence of a multifunctional fusion polypeptide complex of the IgV region of the CD86 extracellular domain is covalently linked to the C-terminus of the PD-L1 antibody Sugemalimab heavy chain through a linker to form the multifunctional fusion polypeptide complex.

[0669] The following provides exemplary amino acid sequences of a first polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the extracellular domain of CD86 is linked to a linker and covalently linked to the heavy chain of the anti-PD-L1 antibody Sugemalimab. The first polypeptide and a second polypeptide (SEQ ID NO: 143) of the extracellular domain D1 of LAG3 are linked to a linker at the C-terminus of the light chain of Sugemalimab to form the multifunctional fusion polypeptide complex; amino acid sequences of a first polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the IgV region of the extracellular domain of CD86 is linked to a linker and covalently linked to the heavy chain of the anti-PD-L1 antibody Sugemalimab. The first polypeptide and a second polypeptide of the extracellular domain D1 of LAG3 are linked to a linker at the C-terminus of the light chain of Sugemalimab to form the multifunctional fusion polypeptide complex; and a multifunctional fusion polypeptide complex in which the C-terminus of the PD-L1 antibody Sugemalimab heavy chain is linked to a linker and covalently linked to the CD86 extracellular domain. The multifunctional fusion polypeptide complex comprises a first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex, wherein the first polypeptide and the second polypeptide of the LAG3 extracellular domain D1 are covalently linked to the C-terminus of the Sugemalimab light chain through a linker to form the multifunctional fusion polypeptide complex; the first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the IgV region of the CD86 extracellular domain is covalently linked to the C-terminus of the Sugemalimab heavy chain through a linker to form the multifunctional fusion polypeptide complex.

[0670] The following provides an exemplary second polypeptide amino acid sequence of a multifunctional fusion polypeptide complex in which the C-terminus of the CD86 extracellular domain is covalently linked to the anti-PD-L1 antibody Sugemalimab light chain by a linker (SEQ ID NO: 42), the second polypeptide and the LAG3 extracellular domain C-terminus are covalently linked to the first Sugemalimab heavy chain polypeptide (SEQ ID NO: 144) to form the multifunctional fusion polypeptide complex; a second polypeptide amino acid sequence of a multifunctional fusion polypeptide complex in which the IgV region of the CD86 extracellular domain is covalently linked to the anti-PD-L1 antibody Sugemalimab light chain by a linker (SEQ ID NO: 43), the second polypeptide and the LAG3 extracellular domain C-terminus are covalently linked to the first Sugemalimab heavy chain polypeptide to form the multifunctional fusion polypeptide complex; a second polypeptide amino acid sequence of a multifunctional fusion polypeptide complex in which the C-terminus of the anti-PD-L1 antibody Sugemalimab light chain is covalently linked to the CD86 extracellular domain by a linker (SEQ ID NO: NO: 40), the second polypeptide is covalently linked to the first polypeptide of the Sugemalimab heavy chain by a linker at the C-terminus of the LAG3 extracellular domain to form the multifunctional fusion polypeptide complex; the anti-PD-L1 antibody Sugemalimab light chain is covalently linked to the second polypeptide amino acid sequence of the CD86 extracellular domain IgV region by a linker (SEQ ID NO: 41), and the second polypeptide is covalently linked to the first polypeptide of the Sugemalimab heavy chain by a linker at the C-terminus of the LAG3 extracellular domain to form the multifunctional fusion polypeptide complex.

[0671] The following provides exemplary amino acid sequences of a second polypeptide of a multifunctional fusion polypeptide complex in which a CD86 extracellular domain C-terminus is covalently linked to the anti-PD-L1 antibody Sugemalimab light chain by a linker, the second polypeptide being covalently linked to a first polypeptide of the Sugemalimab heavy chain by a linker at the C-terminus of the LAG3 extracellular domain D1 / D2 / D3 to form the multifunctional fusion polypeptide complex; amino acid sequences of a second polypeptide of a multifunctional fusion polypeptide complex in which a CD86 extracellular domain IgV region is covalently linked to the anti-PD-L1 antibody Sugemalimab light chain by a linker at the C-terminus of the LAG3 extracellular domain D1 / D2 / D3 to form the multifunctional fusion polypeptide complex; amino acid sequences of a second polypeptide of a multifunctional fusion polypeptide complex in which a CD86 extracellular domain IgV region is covalently linked to the anti-PD-L1 antibody Sugemalimab light chain by a linker at the C-terminus of the LAG3 extracellular domain D1 / D2 / D3 to form the multifunctional fusion polypeptide complex. The C-terminal connecting linker is covalently connected to the first polypeptide of the Sugemalimab heavy chain to form the multifunctional fusion polypeptide complex; the C-terminal connecting linker of the anti-PD-L1 antibody Sugemalimab light chain is covalently connected to the second polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the IgV region of the CD86 extracellular domain, and the second polypeptide is covalently connected to the first polypeptide of the Sugemalimab heavy chain with the C-terminal connecting linker of the LAG3 extracellular domain D1 / D2 / D3 to form the multifunctional fusion polypeptide complex.

[0672] The following provides exemplary amino acid sequences of a second polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the CD86 extracellular domain is covalently linked to the anti-PD-L1 antibody Sugemalimab light chain by a linker, the second polypeptide being covalently linked to the LAG3 extracellular domain D1 / D2 C-terminus of the Sugemalimab heavy chain first polypeptide (SEQ ID NO: 146) by a linker to form the multifunctional fusion polypeptide complex; amino acid sequences of a second polypeptide of a multifunctional fusion polypeptide complex in which the IgV region of the CD86 extracellular domain is covalently linked to the anti-PD-L1 antibody Sugemalimab light chain by a linker, the second polypeptide being covalently linked to the LAG3 extracellular domain D1 / D2 C-terminus of the Sugemalimab heavy chain first polypeptide to form the multifunctional fusion polypeptide complex; amino acid sequences of a second polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the anti-PD-L1 antibody Sugemalimab light chain is covalently linked to the CD86 extracellular domain by a linker, the second polypeptide being covalently linked to the LAG3 extracellular domain D1 / D2 The C-terminal connecting linker is covalently connected to the first polypeptide of the Sugemalimab heavy chain to form the multifunctional fusion polypeptide complex; the C-terminal connecting linker of the anti-PD-L1 antibody Sugemalimab light chain is covalently connected to the second polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the IgV region of the CD86 extracellular domain, and the second polypeptide is covalently connected to the first polypeptide of the Sugemalimab heavy chain with the C-terminal connecting linker of the LAG3 extracellular domain D1 / D2 to form the multifunctional fusion polypeptide complex.

[0673] The following provides exemplary amino acid sequences of a second polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the extracellular domain of CD86 is covalently linked to the light chain of the anti-PD-L1 antibody Sugemalimab by a linker, the second polypeptide being covalently linked to the first polypeptide of the heavy chain of Sugemalimab (SEQ ID NO: 147) by a linker at the C-terminus of the extracellular domain D1 of LAG3 to form the multifunctional fusion polypeptide complex; amino acid sequences of a second polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the IgV region of the extracellular domain of CD86 is covalently linked to the light chain of the anti-PD-L1 antibody Sugemalimab by a linker at the C-terminus of the extracellular domain D1 of LAG3 to form the multifunctional fusion polypeptide complex; amino acid sequences of a second polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the light chain of Sugemalimab of the anti-PD-L1 antibody Sugemalimab is covalently linked to the light chain of the CD86 extracellular domain by a linker, the second polypeptide being covalently linked to the first polypeptide of the heavy chain of Sugemalimab by a linker at the C-terminus of the extracellular domain D1 of LAG3 to form the multifunctional fusion polypeptide complex. The C-terminal connecting linker is covalently connected to the first polypeptide of the Sugemalimab heavy chain to form the multifunctional fusion polypeptide complex; the C-terminal connecting linker of the anti-PD-L1 antibody Sugemalimab light chain is covalently connected to the second polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the IgV region of the CD86 extracellular domain, and the second polypeptide is covalently connected to the first polypeptide of the Sugemalimab heavy chain with the C-terminal connecting linker of the LAG3 extracellular domain D1 to form the multifunctional fusion polypeptide complex.

[0674] The following provides an exemplary second polypeptide amino acid sequence of a multifunctional fusion polypeptide complex in which a C-terminal linker of the CD86 extracellular domain is covalently linked to the anti-PD-L1 antibody Sugemalimab light chain, and the second polypeptide is covalently linked to the C-terminal linker of the Sugemalimab heavy chain to the LAG3 extracellular domain first polypeptide (SEQ ID NO: 148) to form the multifunctional fusion polypeptide complex; the C-terminus of the IgV region of the CD86 extracellular domain is connected to a linker covalently linked to a second polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the anti-PD-L1 antibody Sugemalimab light chain, the second polypeptide and the Sugemalimab heavy chain C-terminus are covalently linked to a first polypeptide of the LAG3 extracellular domain to form the multifunctional fusion polypeptide complex; the C-terminus of the anti-PD-L1 antibody Sugemalimab light chain is connected to a linker covalently linked to a second polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the CD86 extracellular domain, the second polypeptide and the Sugemalimab heavy chain C-terminus are covalently linked to a first polypeptide of the LAG3 extracellular domain to form the multifunctional fusion polypeptide complex; the C-terminus of the anti-PD-L1 antibody Sugemalimab light chain is connected to a linker covalently linked to a second polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the IgV region of the CD86 extracellular domain, the second polypeptide and the Sugemalimab heavy chain C-terminus are covalently linked to a first polypeptide of the LAG3 extracellular domain to form the multifunctional fusion polypeptide complex.

[0675] The following provides exemplary amino acid sequences of a second polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the extracellular domain of CD86 is linked to a linker and covalently linked to the light chain of the anti-PD-L1 antibody Sugemalimab, wherein the second polypeptide and the Sugemalimab heavy chain C-terminus are linked to a linker and covalently linked to the first polypeptide of the extracellular domain D1 / D2 / D3 of LAG3 to form the multifunctional fusion polypeptide complex; amino acid sequences of a second polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the IgV region of the extracellular domain of CD86 is linked to a linker and covalently linked to the light chain of the anti-PD-L1 antibody Sugemalimab, wherein the second polypeptide and the Sugemalimab heavy chain C-terminus are linked to a linker and covalently linked to the first polypeptide of the extracellular domain D1 / D2 / D3 of LAG3 to form the multifunctional fusion polypeptide complex; amino acid sequences of a second polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the extracellular domain of CD86 is linked to a linker and covalently linked to the light chain of the anti-PD-L1 antibody Sugemalimab Two polypeptide amino acid sequences, the second polypeptide and the Sugemalimab heavy chain C-terminal connecting linker are covalently linked to the LAG3 extracellular domain D1 / D2 / D3 first polypeptide to form the multifunctional fusion polypeptide complex; the anti-PD-L1 antibody Sugemalimab light chain C-terminal connecting linker is covalently linked to the CD86 extracellular domain IgV region second polypeptide amino acid sequence, the second polypeptide and the Sugemalimab heavy chain C-terminal connecting linker are covalently linked to the LAG3 extracellular domain D1 / D2 / D3 first polypeptide to form the multifunctional fusion polypeptide complex.

[0676] The following provides exemplary amino acid sequences of a second polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the extracellular domain of CD86 is covalently linked to the light chain of the anti-PD-L1 antibody Sugemalimab via a linker, wherein the second polypeptide and the Sugemalimab heavy chain C-terminus are covalently linked to the first polypeptide of the LAG3 extracellular domain D1 / D2 (SEQ ID NO: 150) via a linker to form the multifunctional fusion polypeptide complex; amino acid sequences of a second polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the IgV region of the extracellular domain of CD86 is covalently linked to the light chain of the anti-PD-L1 antibody Sugemalimab via a linker to form the multifunctional fusion polypeptide complex; and amino acid sequences of a multifunctional fusion polypeptide complex in which the C-terminus of the IgV region of the extracellular domain of CD86 is covalently linked to the light chain of the anti-PD-L1 antibody Sugemalimab via a linker to form the multifunctional fusion polypeptide complex. The present invention relates to a multifunctional fusion polypeptide complex comprising a second polypeptide amino acid sequence of a compound, wherein the second polypeptide and the Sugemalimab heavy chain C-terminus are connected to a linker and covalently linked to the LAG3 extracellular domain D1 / D2 first polypeptide to form the multifunctional fusion polypeptide complex; the anti-PD-L1 antibody Sugemalimab light chain C-terminus is connected to a linker and covalently linked to the CD86 extracellular domain IgV region second polypeptide amino acid sequence of a multifunctional fusion polypeptide complex, wherein the second polypeptide and the Sugemalimab heavy chain C-terminus are connected to a linker and covalently linked to the LAG3 extracellular domain D1 / D2 first polypeptide to form the multifunctional fusion polypeptide complex.

[0677] The following provides exemplary amino acid sequences of a second polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the extracellular domain of CD86 is connected to a linker and covalently linked to the light chain of the anti-PD-L1 antibody Sugemalimab. The second polypeptide and the Sugemalimab heavy chain C-terminus are covalently linked to the first polypeptide of the extracellular domain D1 of LAG3 (SEQ ID NO: 151) to form the multifunctional fusion polypeptide complex; amino acid sequences of a second polypeptide of a multifunctional fusion polypeptide complex in which the C-terminus of the IgV region of the extracellular domain of CD86 is connected to a linker and covalently linked to the light chain of the anti-PD-L1 antibody Sugemalimab. The second polypeptide and the Sugemalimab heavy chain C-terminus are covalently linked to the first polypeptide of the extracellular domain D1 of LAG3 to form the multifunctional fusion polypeptide complex; and a multifunctional fusion polypeptide complex in which the C-terminus of the anti-PD-L1 antibody Sugemalimab light chain is covalently linked to the CD86 extracellular domain. The second polypeptide amino acid sequence of the multifunctional fusion polypeptide complex is covalently linked to the first polypeptide of the LAG3 extracellular domain D1 by a linker connected to the C-terminus of the Sugemalimab heavy chain to form the multifunctional fusion polypeptide complex; the second polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the IgV region of the CD86 extracellular domain is covalently linked to the first polypeptide of the LAG3 extracellular domain D1 by a linker connected to the C-terminus of the anti-PD-L1 antibody Sugemalimab light chain to form the multifunctional fusion polypeptide complex.

[0678] For example, the antibody AN_Bif_Abs_0016 can be composed of the Sugemalimab heavy chain antibody AN_Bif_Abs_0016 first polypeptide (SEQ ID NO: 4) and the Sugemalimab light chain second polypeptide (SEQ ID NO: 5), and the assembly into the AN_Bif_Abs_0016 antibody is achieved by introducing a vector expressing the AN_Bif_Abs_0016 first polypeptide polynucleotide sequence (SEQ ID NO: 152) and a vector expressing the AN_Bif_Abs_0016 second polypeptide polynucleotide sequence (SEQ ID NO: 153) into host cells for expression.

[0679] For example, the bifunctional antibody AN_Bif_Abs_0220 can be composed of a CD86 extracellular domain C-terminal linker peptide (SEQ ID NO: 34) covalently linked to the Sugemalimab heavy chain N-terminus to form a chimeric antibody AN_Bif_Abs_0220 first polypeptide (SEQ ID NO: 38) and a Sugemalimab light chain second polypeptide (SEQ ID NO: 5), and the assembly into the AN_Bif_Abs_0220 bifunctional antibody is achieved by introducing a vector expressing the AN_Bif_Abs_0220 first polypeptide polynucleotide sequence (SEQ ID NO: 154) and a vector expressing the AN_Bif_Abs_0220 second polypeptide polynucleotide sequence (SEQ ID NO: 153) into host cells for expression.

[0680] For example, the bifunctional antibody AN_Bif_Abs_0222 can be composed of a Sugemalimab heavy chain Fc domain connecting linker peptide (SEQ ID NO: 34) covalently linked to the N-terminus of the CD86 extracellular domain to form a chimeric antibody AN_Bif_Abs_0222 first polypeptide (SEQ ID NO: 36) and a Sugemalimab light chain second polypeptide (SEQ ID NO: 5), and the assembly into the AN_Bif_Abs_0222 bifunctional antibody is achieved by introducing a vector expressing the AN_Bif_Abs_0222 first polypeptide polynucleotide sequence (SEQ ID NO: 155) and a vector expressing the AN_Bif_Abs_0222 second polypeptide polynucleotide sequence (SEQ ID NO: 153) into host cells for expression.

[0681] For example, the bifunctional antibody AN_Bif_Abs_0703 can be composed of a Sugemalimab heavy chain Fc domain connecting linker peptide (SEQ ID NO: 34) covalently linked to the CD86 extracellular domain IgV region to form a chimeric antibody AN_Bif_Abs_0703 first polypeptide (SEQ ID NO: 37) and a Sugemalimab light chain second polypeptide (SEQ ID NO: 5), and the assembly into the AN_Bif_Abs_0703 bifunctional antibody is achieved by introducing a vector expressing the AN_Bif_Abs_0703 first polypeptide polynucleotide sequence (SEQ ID NO: 156) and a vector expressing the AN_Bif_Abs_0703 second polypeptide polynucleotide sequence (SEQ ID NO: 153) into host cells for expression.

[0682] For example, the bifunctional antibody AN_Bif_Abs_0158 can be composed of a linker peptide (SEQ ID NO: 34) at the C-terminus of the IgV region of the CD86 extracellular domain covalently linked to the N-terminus of the Sugemalimab heavy chain to form a chimeric antibody AN_Bif_Abs_0158 first polypeptide (SEQ ID NO: 39) and a Sugemalimab light chain second polypeptide (SEQ ID NO: 5). The assembly into the AN_Bif_Abs_0158 bifunctional antibody is achieved by introducing a vector expressing the AN_Bif_Abs_0158 first polypeptide polynucleotide sequence (SEQ ID NO: 157) and a vector expressing the AN_Bif_Abs_0158 second polypeptide polynucleotide sequence (SEQ ID NO: 153) into host cells for expression.

[0683] For example, the bifunctional antibody AN_Bif_Abs_0675 can be composed of a Sugemalimab heavy chain first polypeptide (SEQ ID NO: 4) and a CD86 extracellular domain IgV region connecting linker peptide (SEQ ID NO: 34) covalently linked to a Sugemalimab light chain to form a chimeric antibody AN_Bif_Abs_0675 second polypeptide (SEQ ID NO: 43), and the assembly into the AN_Bif_Abs_0675 bifunctional antibody is achieved by introducing a vector expressing the AN_Bif_Abs_0675 first polypeptide polynucleotide sequence (SEQ ID NO: 152) and a vector expressing the AN_Bif_Abs_0675 second polypeptide polynucleotide sequence (SEQ ID NO: 158) into a host cell for expression.

[0684] For example, the bifunctional antibody AN_Bif_Abs_0676 can be composed of a Sugemalimab heavy chain first polypeptide (SEQ ID NO: 4) and a Sugemalimab light chain connecting linker peptide (SEQ ID NO: 34) covalently linked to the CD86 extracellular domain IgV region to form a chimeric antibody AN_Bif_Abs_0676 second polypeptide (SEQ ID NO: 41), and the assembly into the AN_Bif_Abs_0676 bifunctional antibody is achieved by introducing a vector expressing the AN_Bif_Abs_0676 first polypeptide polynucleotide sequence (SEQ ID NO: 152) and a vector expressing the AN_Bif_Abs_0676 second polypeptide polynucleotide sequence (SEQ ID NO: 159) into host cells for expression.

[0685] For example, the multifunctional antibody AN_Bif_Abs_0159 can be composed of the CD86 extracellular domain IgV region C-terminal connecting linker peptide (SEQ ID NO: 34) covalently linked to the Sugemalimab heavy chain N-terminus to form a chimeric antibody AN_Bif_Abs_0159 first polypeptide (SEQ ID NO: 39) and the LAG3 D1 / D2 C-terminal connecting linker peptide (SEQ ID NO: 34) covalently linked to the Sugemalimab light chain second polypeptide (SEQ ID NO: 138). The assembly into the AN_Bif_Abs_0159 multifunctional antibody is achieved by introducing a vector expressing the AN_Bif_Abs_0159 first polypeptide polynucleotide sequence (SEQ ID NO: 157) and a vector expressing the AN_Bif_Abs_0159 second polypeptide polynucleotide sequence (SEQ ID NO: 160) into host cells for expression.

[0686] For example, the multifunctional antibody AN_Bif_Abs_0160 can be composed of a first polypeptide (SEQ ID NO: 39) of the CD86 extracellular domain IgV region connected to a linker peptide (SEQ ID NO: 34) covalently linked to the N-terminus of the Sugemalimab heavy chain to form a chimeric antibody AN_Bif_Abs_0160 and a second polypeptide (SEQ ID NO: 142) of the Sugemalimab light chain connected to a linker peptide (SEQ ID NO: 34) covalently linked to the LAG3 D1 / D2. The multifunctional antibody AN_Bif_Abs_0160 is assembled by introducing a vector expressing the AN_Bif_Abs_0160 first polypeptide polynucleotide sequence (SEQ ID NO: 157) and a vector expressing the AN_Bif_Abs_0160 second polypeptide polynucleotide sequence (SEQ ID NO: 161) into host cells for expression.

[0687] For example, the multifunctional antibody AN_Bif_Abs_0161 can be composed of a CD86 extracellular domain IgV region C-terminal connecting linker peptide (SEQ ID NO: 34) covalently linked to the N-terminus of the Sugemalimab heavy chain, and a Sugemalimab heavy chain C-terminal connecting linker peptide (SEQ ID NO: 34) covalently linked to LAG3 D1 / D2 to form a chimeric antibody AN_Bif_Abs_0161 first polypeptide (SEQ ID NO: 114) and a Sugemalimab light chain second polypeptide (SEQ ID NO: 5). The AN_Bif_Abs_0161 multifunctional antibody is assembled by introducing a vector expressing the AN_Bif_Abs_0161 first polypeptide polynucleotide sequence (SEQ ID NO: 162) and a vector expressing the AN_Bif_Abs_0161 second polypeptide polynucleotide sequence (SEQ ID NO: 153) into host cells for expression.

[0688] For example, the multifunctional antibody AN_Bif_Abs_0644 can be composed of a CD86 extracellular domain C-terminal linker peptide (SEQ ID NO: 34) covalently linked to the Sugemalimab heavy chain N-terminus, and a Sugemalimab heavy chain C-terminal linker peptide (SEQ ID NO: 34) covalently linked to LAG3 D1 to form a chimeric antibody AN_Bif_Abs_0644 first polypeptide (SEQ ID NO: 107) and a Sugemalimab light chain second polypeptide (SEQ ID NO: 5). The AN_Bif_Abs_0644 multifunctional antibody is assembled by introducing a vector expressing the AN_Bif_Abs_0644 first polypeptide polynucleotide sequence (SEQ ID NO: 163) and a vector expressing the AN_Bif_Abs_0644 second polypeptide polynucleotide sequence (SEQ ID NO: 153) into a host cell for expression.

[0689] For example, the multifunctional antibody AN_Bif_Abs_0646 can be composed of a linker peptide (SEQ ID NO: 34) covalently linked to the N-terminus of the Sugemalimab heavy chain at the C-terminus of the CD86 extracellular domain IgV region, and a linker peptide (SEQ ID NO: 34) covalently linked to the LAG3 D1 to form a chimeric antibody AN_Bif_Abs_0646 first polypeptide (SEQ ID NO: 115) and a Sugemalimab light chain second polypeptide (SEQ ID NO: 5). The AN_Bif_Abs_0646 multifunctional antibody is assembled by introducing a vector expressing the AN_Bif_Abs_0646 first polypeptide polynucleotide sequence (SEQ ID NO: 164) and a vector expressing the AN_Bif_Abs_0646 second polypeptide polynucleotide sequence (SEQ ID NO: 153) into a host cell for expression.

[0690] For example, the multifunctional antibody AN_Bif_Abs_0162 can be composed of a linker peptide (SEQ ID NO: 34) at the C-terminus of the IgV region of the CD86 extracellular domain covalently linked to the N-terminus of the Sugemalimab heavy chain, and a linker peptide (SEQ ID NO: 34) at the C-terminus of the Sugemalimab heavy chain covalently linked to the LAG3 extracellular domain to form a chimeric antibody AN_Bif_Abs_0162 first polypeptide (SEQ ID NO: 112) and a Sugemalimab light chain second polypeptide (SEQ ID NO: 5). The assembly of the AN_Bif_Abs_0162 multifunctional antibody is achieved by introducing a vector expressing the AN_Bif_Abs_0162 first polypeptide polynucleotide (SEQ ID NO: 165) and a vector expressing the AN_Bif_Abs_0162 second polypeptide polynucleotide (SEQ ID NO: 153) into host cells for expression.

[0691] For example, the multifunctional antibody AN_Bif_Abs_0163 can be composed of a CD86 extracellular domain C-terminal linker peptide (SEQ ID NO: 34) covalently linked to the Sugemalimab heavy chain N-terminus, and a Sugemalimab heavy chain C-terminal linker peptide (SEQ ID NO: 34) covalently linked to the LAG3 extracellular domain to form a chimeric antibody AN_Bif_Abs_0163 first polypeptide (SEQ ID NO: 104) and a Sugemalimab light chain second polypeptide (SEQ ID NO: 5). The assembly of the AN_Bif_Abs_0163 multifunctional antibody is achieved by introducing a vector expressing the AN_Bif_Abs_0163 first polypeptide polynucleotide sequence (SEQ ID NO: 166) and a vector expressing the AN_Bif_Abs_0163 second polypeptide polynucleotide sequence (SEQ ID NO: 153) into host cells for expression.

[0692] For example, the multifunctional antibody AN_Bif_Abs_0164 can be composed of a LAG3 extracellular domain C-terminal linker peptide (SEQ ID NO: 34) covalently linked to the Sugemalimab heavy chain N-terminus, and a Sugemalimab heavy chain C-terminal linker peptide (SEQ ID NO: 34) covalently linked to the CD86 extracellular domain to form a chimeric antibody AN_Bif_Abs_0164 first polypeptide (SEQ ID NO: 108) and a Sugemalimab light chain second polypeptide (SEQ ID NO: 5). The AN_Bif_Abs_0164 multifunctional antibody is assembled by introducing a vector expressing the AN_Bif_Abs_0164 first polypeptide polynucleotide sequence (SEQ ID NO: 167) and a vector expressing the AN_Bif_Abs_0164 second polypeptide polynucleotide sequence (SEQ ID NO: 153) into a host cell for expression.

[0693] For example, the multifunctional antibody AN_Bif_Abs_0166 can be composed of a LAG3 extracellular domain C-terminal linker peptide (SEQ ID NO: 34) covalently linked to the Sugemalimab heavy chain N-terminus, and a Sugemalimab heavy chain C-terminal linker peptide (SEQ ID NO: 34) covalently linked to the CD86 extracellular domain IgV region to form a chimeric antibody AN_Bif_Abs_0166 first polypeptide (SEQ ID NO: 116) and a Sugemalimab light chain second polypeptide (SEQ ID NO: 5). The AN_Bif_Abs_0166 multifunctional antibody is assembled by introducing a vector expressing the AN_Bif_Abs_0166 first polypeptide polynucleotide (SEQ ID NO: 168) and a vector expressing the AN_Bif_Abs_0166 second polypeptide polynucleotide (SEQ ID NO: 153) into a host cell for expression.

[0694] For example, the antibody AN_Bif_Abs_0031 can be assembled into the antibody AN_Bif_Abs_0031 first polypeptide (SEQ ID NO: 14) by Envafolimab, and the AN_Bif_Abs_0031 antibody can be assembled by introducing a vector expressing the AN_Bif_Abs_0031 first polypeptide polynucleotide (SEQ ID NO: 169) into a host cell for expression.

[0695] For example, the bifunctional antibody AN_Bif_Abs_0129 can be covalently linked to the N-terminus of Envafolimab by connecting a linker peptide (SEQ ID NO: 34) at the C-terminus of the IgV region of the CD86 extracellular domain to form the chimeric antibody AN_Bif_Abs_0129 first polypeptide (SEQ ID NO: 71), and the assembly into the AN_Bif_Abs_0129 bifunctional antibody can be achieved by introducing a vector expressing the AN_Bif_Abs_0129 first polypeptide polynucleotide (SEQ ID NO: 170) into a host cell for expression.

[0696] For example, the multifunctional antibody AN_Bif_Abs_0130 can be covalently linked to the LAG3 D1 / D2 N-terminus by connecting the C-terminus linker peptide (SEQ ID NO: 34) of the IgV region of the CD86 extracellular domain, and the LAG3 D1 / D2 C-terminus linker peptide (SEQ ID NO: 34) is covalently linked to the N-terminus of Envafolimab to form the chimeric antibody AN_Bif_Abs_0130 first polypeptide (SEQ ID NO: 171). The AN_Bif_Abs_0130 multifunctional antibody is assembled by introducing a vector expressing the AN_Bif_Abs_0130 first polypeptide polynucleotide (SEQ ID NO: 172) into a host cell for expression.

[0697] For example, the multifunctional antibody AN_Bif_Abs_0131 can be covalently linked to the N-terminus of Envafolimab by connecting the C-terminus of the IgV region of the CD86 extracellular domain to a linker peptide (SEQ ID NO: 34), and the C-terminus of Envafolimab to a linker peptide (SEQ ID NO: 34) is covalently linked to the LAG3 D1 / D2 end to form the chimeric antibody AN_Bif_Abs_0131 first polypeptide (SEQ ID NO: 173). The AN_Bif_Abs_0131 multifunctional antibody is assembled by introducing a vector expressing the AN_Bif_Abs_0131 first polypeptide polynucleotide (SEQ ID NO: 174) into a host cell for expression.

[0698] For example, the bifunctional antibody AN_Bif_Abs_0012 can be composed of a Durvalumab heavy chain C-terminal connecting linker peptide (SEQ ID NO: 34) covalently linked to the CD86 extracellular domain to form a chimeric antibody AN_Bif_Abs_0012 first polypeptide (SEQ ID NO: 44) and a Durvalumab light chain second polypeptide (SEQ ID NO: 9), and the assembly into the AN_Bif_Abs_0012 multifunctional antibody is achieved by introducing a vector expressing the AN_Bif_Abs_0164 first polypeptide polynucleotide sequence (SEQ ID NO: 175) and a vector expressing the AN_Bif_Abs_0012 second polypeptide polynucleotide sequence (SEQ ID NO: 176) into host cells for expression.

[0699] For example, the antibody AN_Bif_Abs_0001 can be composed of the Durvalumab heavy chain antibody AN_Bif_Abs_0001 first polypeptide (SEQ ID NO: 8) and the Durvalumab light chain second polypeptide (SEQ ID NO: 9), and the assembly into the AN_Bif_Abs_0001 antibody is achieved by introducing a vector expressing the AN_Bif_Abs_0001 first polypeptide polynucleotide sequence (SEQ ID NO: 177) and a vector expressing the AN_Bif_Abs_0001 second polypeptide polynucleotide sequence (SEQ ID NO: 176) into host cells for expression.

[0700] In certain embodiments, the present application also provides a method for separating a polypeptide complex for separating the polypeptide complex.

[0701] In one aspect, the present invention provides a method for producing the polypeptide complex: this method comprises introducing the polynucleotide encoding the antibody polypeptide complex of the present invention into a host cell, expressing the first polypeptide, and / or the second polypeptide, wherein the first polypeptide and the second polypeptide, and the first polypeptide itself are capable of forming a stable dimer, and the stable dimer can be maintained by a natural bond or / and a stable dimerization comprising at least one non-natural interchain bond, the first polypeptide and the second polypeptide assemble into a stable polymer in the host cell, and form the stable complex having the ability to bind to the first cell target, the second cell target and / or the third cell target.

[0702] In other aspects, the content disclosed in this application provides a method for relieving the inhibitory effect of PD-L1 / PD-1 on T cells and stimulating the activation of antigen-presenting cells and / or T cells. The method includes using the dual and / or multifunctional fusion polypeptide disclosed in this application to stimulate the activation of T cells, but is not limited to the scheme used in the examples, and can be expanded to currently published or future developed schemes that can be used to evaluate the activation of antigen-presenting cells and / or T cells.

[0703] On the other hand, the dual and / or multifunctional fusion polypeptides disclosed in the present application have the effect of blocking tumor growth, including but not limited to colon tumors, breast tumors, lung tumors, gastric tumors, melanoma, head and neck tumors, lymphomas, nasopharyngeal tumors, cervical tumors, esophageal tumors, kidney tumors, skin squamous cell carcinoma, endometrial tumors, liver tumors, bladder tumors, urothelial tumors and / or skin tumors.

[0704] In another aspect, the present application provides an immunoconjugate, which may comprise the fusion polypeptide described herein.

[0705] On the other hand, the present application provides a nucleic acid molecule that can encode the fusion polypeptide described in the present application.

[0706] On the other hand, the present application provides a vector, which may contain the nucleic acid molecule described in the present application.

[0707] On the other hand, the present application provides a cell, which may contain and / or express the fusion polypeptide described in the present application, the immunoconjugate described in the present application, the nucleic acid molecule described in the present application, and / or the vector described in the present application.

[0708] On the other hand, the present application provides a composition, which may include the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, and / or the cell described herein, and optionally a pharmaceutically acceptable carrier.

[0709] On the other hand, the present application provides a method for preparing the fusion polypeptide described in the present application, which comprises culturing the cell described in the present application under conditions that allow the expression of the fusion polypeptide.

[0710] On the other hand, the present application provides a method for blocking the interaction between PD-L1 protein and PD-1, which may include administering an effective amount of the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the composition described herein.

[0711] On the other hand, the present application provides a method for stimulating antigen-presenting cells and / or activating T cells, which may include administering an effective amount of the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the composition described herein.

[0712] According to the method described in the present application, the stimulation of antigen-presenting cells may include a method selected from the following groups: increasing the expression of co-stimulatory molecules in antigen-presenting cells, causing morphological changes and maturation of antigen-presenting cells, increasing the secretion of chemokines in antigen-presenting cells, and enhancing the phagocytic ability of antigen-presenting cells.

[0713] On the other hand, the present application provides a method for inhibiting the growth and / or proliferation of tumors or tumor cells, which may include administering an effective amount of the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the composition described herein.

[0714] On the other hand, the present application provides the use of the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the composition described herein in the preparation of a medicament, wherein the medicament can be used to prevent, improve and / or treat tumors.

[0715] According to the use described in the present application, the tumor may include solid tumors and hematological tumors.

[0716] According to the use described in the present application, the tumor can be selected from the group consisting of colon tumors, breast tumors, lung tumors, stomach tumors, melanomas, head and neck tumors, lymphomas, nasopharyngeal tumors, cervical tumors, esophageal tumors, kidney tumors, endometrial tumors, liver tumors, bladder tumors, urothelial tumors, and skin tumors. For example, the skin tumor can comprise squamous cell carcinoma of the skin.

[0717] On the other hand, the present application provides the fusion polypeptide described in the present application, the immunoconjugate described in the present application, the nucleic acid molecule described in the present application, the vector described in the present application, the cell described in the present application, and / or the composition described in the present application, which can be used to prevent, improve and / or treat tumors.

[0718] On the other hand, the present application provides the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the composition described herein, which can be used to prevent, improve and / or treat tumors, wherein the tumors include solid tumors and hematological tumors.

[0719] On the other hand, the present application provides the fusion polypeptide described herein, the immunoconjugate described herein, the nucleic acid molecule described herein, the vector described herein, the cell described herein, and / or the composition described herein, which can be used to prevent, improve and / or treat tumors, wherein the tumor can be selected from the following group: colon tumor, breast tumor, lung tumor, gastric tumor, melano...

Claims

1. A fusion polypeptide comprising a first domain and a second domain, wherein the first domain is capable of blocking PD-1 / PD-L1 signaling, and the second domain comprises CD86 or a functionally active fragment thereof.

2. The fusion polypeptide according to claim 1, wherein the first domain is capable of binding to PD-L1 and / or PD-1.

3. The fusion polypeptide according to any one of claims 1 to 2, wherein the first domain comprises an antibody or an antigen-binding fragment thereof.

4. The fusion polypeptide according to claim 3, wherein the antibody is selected from the group consisting of a recombinant antibody, a single domain antibody, a heavy chain antibody, a chimeric antibody and a bispecific antibody.

5. The fusion polypeptide of claim 3, wherein the antigen-binding fragment is selected from one or more of the following groups: Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, VHH and dAb.

6. The fusion polypeptide according to any one of claims 1 to 5, wherein the first domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of Sugemalimab, Durvalumab, Atezolizumab, Avelumab, Envafolimab, Permbrolizumab and Nivolumab.

7. The fusion polypeptide of any one of claims 1 to 6, wherein the first domain comprises HCDR1, HCDR2 and / or HCDR3 of an antibody heavy chain, wherein the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO:

17.

8. The fusion polypeptide of any one of claims 1 to 7, wherein the first domain comprises a heavy chain variable region VH of an antibody heavy chain, and the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO:

17.

9. The fusion polypeptide of any one of claims 1 to 8, wherein the first domain comprises an antibody heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO:

17.

10. The fusion polypeptide of any one of claims 1 to 9, wherein the first domain comprises LCDR1, LCDR2 and / or LCDR3 of an antibody light chain, wherein the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO:

18.

11. The fusion polypeptide according to any one of claims 1 to 10, wherein the first domain comprises a light chain variable region (VL) of an antibody light chain, and the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO:

18.

12. The fusion polypeptide of any one of claims 1 to 11, wherein the first domain comprises an antibody light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO:

18.

13. The fusion polypeptide according to any one of claims 1 to 12, wherein the first domain comprises Sugemalimab or an antigen-binding fragment thereof.

14. The fusion polypeptide of any one of claims 1 to 13, wherein the second domain is capable of binding to CD28 and / or CTLA4. 15 . The fusion polypeptide according to claim 1 , wherein the second domain is selected from the group consisting of human-derived CD86 or a functionally active fragment thereof and mouse-derived CD86 or a functionally active fragment thereof.

16. The fusion polypeptide according to any one of claims 1 to 15, wherein the second domain comprises the IgV domain of CD86 or a functionally active fragment thereof.

17. The fusion polypeptide according to any one of claims 1 to 16, wherein the second domain comprises the extracellular domain of CD86 or a functionally active fragment thereof.

18. The fusion polypeptide according to any one of claims 1 to 17, wherein the second domain comprises the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO:

3.

19. The fusion polypeptide of any one of claims 1-15, wherein the CD86 or functionally active fragment thereof comprises a CD86 variant polypeptide.

20. The fusion polypeptide of claim 19, wherein the CD86 variant polypeptide comprises an amino acid substitution mutation of humanized CD86.

21. The CD86 variant polypeptide according to claim 20, comprising an amino acid substitution mutant of the humanized CD86 extracellular domain IgV domain.

22. The fusion polypeptide according to any one of claims 19 to 21, wherein the CD86 variant polypeptide comprises an amino acid substitution mutant of the IgV domain of CD86, and the mutation site of the amino acid substitution mutant of the IgV domain of CD86 comprises one or more amino acid site mutations selected from the group consisting of A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, and H90F.

23. The fusion polypeptide according to any one of claims 19 to 22, wherein: The CD86 variant polypeptide includes 1, 2, 3, 4, 5 or more combinations of amino acid position mutations selected from the following group: A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, H90F.

24. The fusion polypeptide according to any one of claims 19 to 23, wherein the CD86 variant polypeptide comprises an amino acid substitution mutant of the IgV domain of CD86, wherein the amino acid substitution mutant comprises one or more groups selected from the combination shown in the following group: Q25I / F33L / H90I, Q25V / F33L / H90I, Q25I / F33V / H90I, Q25V / F33V / H90I, Q25I / F33L / H90 V, Q25V / F33L / H90V, Q25I / F33V / H90V, Q25V / F33V / H90V, A13L / Q25I / F33L / H90I, A13I / Q25I / F 33L / H90I, A13L / Q25V / F33L / H90I, A13I / Q25V / F33L / H90I, Q25I / F33L / I89L / H90I, Q25I / F33L / M60R / H90I, Q25V / F33L / I89L / H90I, Q25V / F33L / M60R / H90I, Q25F / F33L / H90I, Q25I / F33L / H90 F, Q25I / F33A / H90I, Q25I / H90I, Q25I, H90I, Q25V / H90V, Q25V / H90I, Q25F / H90I, Q25F / H90V, A1 3F / Q25I / F33L / H90I, A13M / Q25I / F33L / H90I, Q25A / F33L / H90I, Q25M / F33L / H90I, Q25I / F33M / H90I, Q25I / F33L / I89V / H90I, Q25I / F33L / I89F / H90I, Q25I / F33L / I89M / H90I, and Q25I / F33L / H90M.

25. The fusion polypeptide according to any one of claims 19 to 24, wherein the CD86 variant polypeptide comprises the IgV domain amino acid substitution mutant Q25I / F33L / H90I of CD86.

26. The fusion polypeptide according to any one of claims 19 to 25, wherein the CD86 variant polypeptide comprises an amino acid substitution mutant of the extracellular domain of humanized CD86.

27. The fusion polypeptide according to any one of claims 19 to 26, wherein the CD86 variant polypeptide comprises an amino acid substitution mutant of the extracellular domain of CD86, wherein the amino acid substitution mutant of the extracellular domain of CD86 comprises one or more mutations in the amino acid positions selected from the group consisting of A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, and H90F.

28. The fusion polypeptide according to any one of claims 19 to 27, wherein the CD86 variant polypeptide comprises an amino acid substitution mutant of the extracellular domain of CD86, wherein the amino acid substitution mutant comprises one or more groups selected from the combination shown in the following group: Q25I / F33L / H90I, 25V / F33L / H90I, Q25I / F33V / H90I, Q25V / F33V / H90I, Q25I / F33L / H90V, Q25V / F33L / H90V, Q25I / F33V / H90V, Q25V / F33V / H90V, A13L / Q25I / F33L / H90 I. A13I / Q25I / F33L / H90I, A13L / Q25V / F33L / H90I, A13I / Q25V / F33L / H90I, Q25I / F33L / I89 L / H90I, Q25I / F33L / M60R / H90I, Q25V / F33L / I89L / H90I, Q25V / F33L / M60R / H90I, Q25F / F3 3L / H90I, Q25I / F33L / H90F, Q25I / F33A / H90I, Q25I / H90I, Q25I, H90I, Q25V / H90V, Q25V / H 90I, Q25F / H90I, Q25F / H90V, A13F / Q25I / F33L / H90I, A13M / Q25I / F33L / H90I, Q25A / F33L / H90I, Q25M / F33L / H90I, Q25I / F33M / H90I, Q25I / F33L / I89V / H90I, Q25I / F33L / I89F / H90I, Q25I / F33L / I89M / H90I, and Q25I / F33L / H90M.

29. The fusion polypeptide according to any one of claims 19 to 28, wherein the CD86 variant polypeptide comprises the extracellular domain amino acid substitution mutant Q25I / F33L / H90I of CD86.

30. The fusion polypeptide according to any one of claims 19 to 29, wherein the CD86 variant polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 178 to SEQ ID NO:

243.

31. The fusion polypeptide of any one of claims 1 to 30, wherein the first domain is directly or indirectly linked to the second domain.

32. The fusion polypeptide of any one of claims 1 to 31, wherein the first domain comprises an antibody heavy chain, and the first domain antibody heavy chain is directly or indirectly connected to the second domain.

33. The fusion polypeptide according to any one of claims 1 to 32, wherein the first domain comprises an antibody heavy chain, and the C-terminus of the antibody heavy chain of the first domain is directly or indirectly connected to the N-terminus of the second domain.

34. The fusion polypeptide according to any one of claims 1 to 33, wherein the first domain comprises an antibody heavy chain, and the N-terminus of the antibody heavy chain of the first domain is directly or indirectly connected to the C-terminus of the second domain.

35. The fusion polypeptide of any one of claims 1 to 34, wherein the first domain comprises an antibody light chain, and the first domain antibody light chain is directly or indirectly connected to the second domain.

36. The fusion polypeptide according to any one of claims 1 to 35, wherein the first domain comprises an antibody light chain, and the C-terminus of the antibody light chain of the first domain is directly or indirectly connected to the N-terminus of the second domain.

37. The fusion polypeptide of any one of claims 1 to 36, wherein the first domain comprises an antibody light chain, and the N-terminus of the antibody light chain of the first domain is directly or indirectly connected to the C-terminus of the second domain.

38. The fusion polypeptide of any one of claims 1 to 37, wherein the first domain comprises an antibody heavy chain and an antibody light chain, the C-terminus of the antibody heavy chain of the first domain is directly or indirectly connected to the N-terminus of the second domain, and the C-terminus of the antibody light chain of the first domain is directly or indirectly connected to the N-terminus of the second domain.

39. The fusion polypeptide of any one of claims 1 to 38, wherein the first domain comprises an antibody heavy chain and an antibody light chain, wherein the N-terminus of the antibody heavy chain of the first domain is directly or indirectly connected to the C-terminus of the second domain, and the N-terminus of the antibody light chain of the first domain is directly or indirectly connected to the C-terminus of the second domain.

40. The fusion polypeptide of any one of claims 1-39, wherein the indirect linkage comprises linkage via a linker.

41. The fusion polypeptide of claim 40, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO:

35.

42. The fusion polypeptide of any one of claims 1 to 41, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86 and SEQ ID NO:

87.

43. The fusion polypeptide of any one of claims 1 to 42, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 418 to SEQ ID NO: 450, SEQ ID NO: 451 to SEQ ID NO: 483, and SEQ ID NO: 484 to SEQ ID NO:

615.

44. The fusion polypeptide of any one of claims 1 to 43, further comprising a third domain capable of activating an innate immune response.

45. The fusion polypeptide of claim 44, wherein the third domain is capable of binding to MHC II molecules on antigen-presenting cells.

46. ​​The fusion polypeptide of any one of claims 44-45, wherein the third domain comprises LAG3 or a functionally active fragment thereof.

47. The fusion polypeptide according to any one of claims 44 to 46, wherein the third domain is selected from the group consisting of human-derived LAG3 or a functionally active fragment thereof and mouse-derived LAG3 or a functionally active fragment thereof.

48. The fusion polypeptide of any one of claims 44 to 47, wherein the third domain comprises the extracellular domain of LAG3 or a functionally active fragment thereof.

49. The fusion polypeptide according to any one of claims 44 to 48, wherein the third domain comprises IgD1, IgD2, IgD3 and / or IgD4 of LAG3 or a functionally active fragment thereof.

50. The fusion polypeptide according to any one of claims 44 to 49, wherein the third domain comprises IgD1, IgD1-IgD2, IgD1-IgD2-IgD3, and / or IgD1-IgD2-IgD3-IgD4 of LAG3 or a functionally active fragment thereof.

51. The fusion polypeptide of any one of claims 44-50, wherein the third domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO:

28.

52. The fusion polypeptide of any one of claims 44-51, wherein the LAG3 or a functionally active fragment thereof comprises a LAG3 variant polypeptide comprising truncations and / or mutations based on human LAG3.

53. The fusion polypeptide of claim 52, wherein the LAG3 variant polypeptide comprises a truncation based on a wild-type human LAG3 extracellular region polypeptide.

54. The fusion polypeptide of any one of claims 52-53, wherein the LAG3 variant polypeptide comprises 0-124 amino acids truncated at the N-terminus based on the wild-type human LAG3 extracellular region polypeptide and terminates at amino acid positions 121-167 at the C-terminus based on the wild-type human LAG3 extracellular region polypeptide.

55. The fusion polypeptide of any one of claims 52-54, wherein the LAG3 variant polypeptide comprises 0, 5, 21, 37, 45, 60, 71, 74, 79, 84, 89, 94, 99, 104, 109, or 114 amino acids truncated at the N-terminus based on a wild-type human LAG3 extracellular region polypeptide.

56. The fusion polypeptide of any one of claims 52-55, wherein the LAG3 variant polypeptide comprises 0, 5, 21, 37, 45, 60, 71, 74, 81 or 99 amino acids truncated at the N-terminus based on a wild-type human LAG3 extracellular region polypeptide.

57. The fusion polypeptide of any one of claims 52-56, wherein the LAG3 variant polypeptide comprises 0, 5, 21, 37, 45, 60, 71 or 74 amino acids truncated at the N-terminus based on a wild-type human LAG3 extracellular region polypeptide.

58. The fusion polypeptide of any one of claims 52-57, wherein the LAG3 variant polypeptide comprises a wild-type human LAG3 extracellular region polypeptide ending at amino acid position 121, 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, or 167 at the C-terminus.

59. The fusion polypeptide of any one of claims 52-58, wherein the LAG3 variant polypeptide comprises a wild-type human LAG3 extracellular region polypeptide ending at amino acid position 121, 122, 129, 136, 146, 156, 161, or 167 at the C-terminus.

60. The fusion polypeptide of any one of claims 52-59, wherein the LAG3 variant polypeptide comprises a wild-type human LAG3 extracellular region polypeptide ending at amino acid position 121, 146, 156, 161, or 167 at the C-terminus.

61. The fusion polypeptide of any one of claims 52-60, wherein the LAG3 variant polypeptide comprises a wild-type human LAG3 extracellular domain polypeptide terminating at amino acid position 156, 161, or 167 at the C-terminus.

62. The fusion polypeptide of any one of claims 52-61, wherein the LAG3 variant polypeptide comprises 0, 5, 21, 37, 45, 60, 71, 74, 81, or 99 amino acids truncated at the N-terminus based on a wild-type human LAG3 extracellular region polypeptide and terminates at amino acid position 121, 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, or 167 at the C-terminus based on a wild-type human LAG3 extracellular region polypeptide.

63. The fusion polypeptide of any one of claims 52-62, wherein the LAG3 variant polypeptide comprises 0, 5, 21, 37, 45, 60, 71, or 74 amino acids truncated at the N-terminus based on a wild-type human LAG3 extracellular region polypeptide and terminates at amino acid position 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, or 167 at the C-terminus based on a wild-type human LAG3 extracellular region polypeptide.

64. The fusion polypeptide of any one of claims 52-63, wherein the LAG3 variant polypeptide comprises an amino acid position mutation.

65. The fusion polypeptide of any one of claims 52 to 64, wherein the LAG3 variant polypeptide comprises an amino acid mutation at position 97, wherein the amino acid mutation is at amino acid Arg.

66. The fusion polypeptide of any one of claims 52-65, wherein the LAG3 variant polypeptide comprises an amino acid mutation, wherein the amino acid mutation site is a mutation from the Arg amino acid at position 97 to a Glu amino acid.

67. The fusion polypeptide of any one of claims 52-66, wherein the LAG3 variant polypeptide is selected from the group consisting of amino acid sequences shown in SEQ ID NO: 244 to SEQ ID NO:

349.

68. The fusion polypeptide of any one of claims 52-67, wherein the LAG3 variant polypeptide comprises an amino acid mutation at one of R110, R113, R119, R129, G130, and / or R141.

69. The fusion polypeptide of any one of claims 52-68, wherein the LAG3 variant polypeptide is truncated at the N-terminus by 81 amino acids and terminates at amino acid position 121 at the C-terminus, and mutations are introduced at R110, and / or R113, and / or R119.

70. The fusion polypeptide of any one of claims 52-69, wherein the LAG3 variant polypeptide is truncated at 99 amino acids at the N-terminus and terminates at amino acid position 146 at the C-terminus, and mutations are introduced at R129, and / or G130, and / or R141.

71. The fusion polypeptide of any one of claims 52-70, wherein the LAG3 variant polypeptide is truncated at the N-terminus by 81 amino acids and terminates at amino acid position 146 at the C-terminus, and mutations are introduced at R110, and / or R113, and / or R119, and / or R129, and / or G130, and / or R141.

72. The fusion polypeptide of any one of claims 52-71, wherein the LAG3 variant polypeptide comprises an amino acid site mutation, wherein the R110 site is mutated to K110.

73. The fusion polypeptide of any one of claims 52-72, wherein the LAG3 variant polypeptide comprises an amino acid site mutation, wherein the R113 site is mutated to K113.

74. The fusion polypeptide of any one of claims 52-73, wherein the LAG3 variant polypeptide comprises an amino acid site mutation, wherein the R119 site is mutated to K119.

75. The fusion polypeptide of any one of claims 52-74, wherein the LAG3 variant polypeptide comprises an amino acid site mutation, wherein the R129 site is mutated to K129.

76. The fusion polypeptide of any one of claims 52-75, wherein the LAG3 variant polypeptide comprises an amino acid site mutation, wherein the G130 site is mutated to P130, or A130, or T130, or Y130, or S130.

77. The fusion polypeptide of any one of claims 52-76, wherein the LAG3 variant polypeptide comprises an amino acid site mutation, wherein the R141 site is mutated to K141.

78. The fusion polypeptide of any one of claims 52-77, wherein the amino acid sequence of the LAG3 variant polypeptide is selected from the group consisting of SEQ ID NO: 1285 to SEQ ID NO: 1318.

79. The fusion polypeptide of any one of claims 44 to 78, wherein the first domain is directly or indirectly linked to the third domain.

80. The fusion polypeptide of any one of claims 44 to 79, wherein the first domain comprises an antibody heavy chain, and the first domain antibody heavy chain is directly or indirectly connected to the third domain.

81. The fusion polypeptide according to any one of claims 44 to 80, wherein the first domain comprises an antibody heavy chain, and the C-terminus of the antibody heavy chain of the first domain is directly or indirectly connected to the N-terminus of the third domain.

82. The fusion polypeptide according to any one of claims 44 to 81, wherein the first domain comprises an antibody heavy chain, and the N-terminus of the antibody heavy chain of the first domain is directly or indirectly connected to the C-terminus of the third domain.

83. The fusion polypeptide of any one of claims 44 to 82, wherein the first domain comprises an antibody light chain, and the first domain light chain is directly or indirectly connected to the third domain.

84. The fusion polypeptide of any one of claims 44 to 83, wherein the first domain comprises an antibody light chain, and the C-terminus of the light chain of the first domain is directly or indirectly connected to the N-terminus of the third domain.

85. The fusion polypeptide of any one of claims 44-84, wherein the first domain comprises an antibody light chain, and the N-terminus of the light chain of the first domain is directly or indirectly connected to the C-terminus of the third domain.

86. The fusion polypeptide of any one of claims 44 to 85, wherein the second domain and the third domain are directly or indirectly linked.

87. The fusion polypeptide according to any one of claims 44 to 86, wherein the C-terminus of the second domain is directly or indirectly linked to the N-terminus of the third domain.

88. The fusion polypeptide according to any one of claims 44 to 87, wherein the N-terminus of the second domain is directly or indirectly linked to the C-terminus of the third domain.

89. The fusion polypeptide of any one of claims 44 to 88, wherein the first domain is directly or indirectly linked to the second domain, and the second domain is directly or indirectly linked to the third domain.

90. The fusion polypeptide of any one of claims 44 to 89, wherein the C-terminus of the first domain is directly or indirectly linked to the N-terminus of the second domain, and the C-terminus of the second domain is directly or indirectly linked to the N-terminus of the third domain.

91. The fusion polypeptide of any one of claims 44-90, wherein the first domain comprises an antibody heavy chain, the C-terminus of the antibody heavy chain of the first domain is directly or indirectly connected to the N-terminus of the second domain, and the C-terminus of the second domain is directly or indirectly connected to the N-terminus of the third domain.

92. The fusion polypeptide of any one of claims 44-91, wherein the first domain comprises an antibody light chain, the C-terminus of the antibody light chain of the first domain is directly or indirectly connected to the N-terminus of the second domain, and the C-terminus of the second domain is directly or indirectly connected to the N-terminus of the third domain.

93. The fusion polypeptide of any one of claims 44 to 92, wherein the N-terminus of the first domain is directly or indirectly connected to the C-terminus of the second domain, and the N-terminus of the second domain is directly or indirectly connected to the C-terminus of the third domain.

94. The fusion polypeptide of any one of claims 44-93, wherein the first domain comprises an antibody heavy chain, the N-terminus of the antibody heavy chain of the first domain is directly or indirectly connected to the C-terminus of the second domain, and the N-terminus of the second domain is directly or indirectly connected to the C-terminus of the third domain.

95. The fusion polypeptide of any one of claims 44-94, wherein the first domain comprises an antibody light chain, the N-terminus of the first domain antibody light chain is directly or indirectly connected to the C-terminus of the second domain, and the N-terminus of the second domain is directly or indirectly connected to the C-terminus of the third domain.

96. The fusion polypeptide of any one of claims 44-95, wherein the first domain is directly or indirectly linked to the third domain, and the third domain is directly or indirectly linked to the second domain.

97. The fusion polypeptide of any one of claims 44 to 96, wherein the C-terminus of the first domain is directly or indirectly linked to the N-terminus of the third domain, and the C-terminus of the third domain is directly or indirectly linked to the N-terminus of the second domain.

98. The fusion polypeptide of any one of claims 44-97, wherein the first domain comprises an antibody heavy chain, the C-terminus of the first domain antibody heavy chain is directly or indirectly connected to the N-terminus of the third domain, and the C-terminus of the third domain is directly or indirectly connected to the N-terminus of the second domain.

99. A fusion polypeptide as described in any one of claims 44 to 98, wherein the first domain comprises an antibody light chain, the C-terminus of the first domain antibody light chain is directly or indirectly connected to the N-terminus of the third domain, and the C-terminus of the third domain is directly or indirectly connected to the N-terminus of the second domain.

100. The fusion polypeptide of any one of claims 44 to 99, wherein the N-terminus of the first domain is directly or indirectly linked to the C-terminus of the third domain, and the N-terminus of the third domain is directly or indirectly linked to the C-terminus of the second domain.

101. The fusion polypeptide of any one of claims 44-100, wherein the first domain comprises an antibody heavy chain, the N-terminus of the antibody heavy chain of the first domain is directly or indirectly connected to the C-terminus of the third domain, and the N-terminus of the third domain is directly or indirectly connected to the C-terminus of the second domain.

102. The fusion polypeptide of any one of claims 44-101, wherein the first domain comprises an antibody light chain, the N-terminus of the antibody light chain of the first domain is directly or indirectly connected to the C-terminus of the third domain, and the N-terminus of the third domain is directly or indirectly connected to the C-terminus of the second domain.

103. The fusion polypeptide of any one of claims 44 to 102, wherein the first domain is directly or indirectly linked to the second domain, and the first domain is directly or indirectly linked to the third domain.

104. The fusion polypeptide of any one of claims 44 to 103, wherein the C-terminus of the first domain is directly or indirectly linked to the N-terminus of the second domain, and the N-terminus of the first domain is directly or indirectly linked to the C-terminus of the third domain.

105. The fusion polypeptide of any one of claims 44-104, wherein the first domain comprises an antibody heavy chain, the C-terminus of the first domain antibody heavy chain is directly or indirectly connected to the N-terminus of the second domain, and the N-terminus of the first domain heavy chain is directly or indirectly connected to the C-terminus of the third domain.

106. The fusion polypeptide of any one of claims 44-105, wherein the first domain comprises an antibody light chain, the C-terminus of the first domain antibody light chain is directly or indirectly connected to the N-terminus of the second domain, and the N-terminus of the first domain light chain is directly or indirectly connected to the C-terminus of the third domain.

107. The fusion polypeptide of any one of claims 44-106, wherein the first domain comprises an antibody heavy chain, the C-terminus of the first domain antibody heavy chain is directly or indirectly connected to the N-terminus of the second domain, and the N-terminus of the first domain light chain is directly or indirectly connected to the C-terminus of the third domain.

108. The fusion polypeptide of any one of claims 44-107, wherein the first domain comprises an antibody light chain, the C-terminus of the first domain antibody light chain is directly or indirectly connected to the N-terminus of the second domain, and the N-terminus of the first domain heavy chain is directly or indirectly connected to the C-terminus of the third domain.

109. The fusion polypeptide of any one of claims 44 to 108, wherein the N-terminus of the first domain is directly or indirectly connected to the C-terminus of the second domain, and the C-terminus of the first domain is directly or indirectly connected to the N-terminus of the third domain.

110. The fusion polypeptide of any one of claims 44-109, wherein the first domain comprises an antibody heavy chain, the N-terminus of the first domain antibody heavy chain is directly or indirectly connected to the C-terminus of the second domain, and the C-terminus of the first domain heavy chain is directly or indirectly connected to the N-terminus of the third domain.

111. The fusion polypeptide of any one of claims 44-110, wherein the first domain comprises an antibody light chain, the N-terminus of the first domain antibody light chain is directly or indirectly connected to the C-terminus of the second domain, and the C-terminus of the first domain light chain is directly or indirectly connected to the N-terminus of the third domain.

112. The fusion polypeptide of any one of claims 44 to 111, wherein the first domain comprises an antibody heavy chain and an antibody light chain, the N-terminus of the first domain antibody heavy chain is directly or indirectly connected to the C-terminus of the second domain, and the C-terminus of the first domain antibody light chain is directly or indirectly connected to the N-terminus of the third domain.

113. The fusion polypeptide of any one of claims 44 to 112, wherein the first domain comprises an antibody heavy chain and an antibody light chain, the N-terminus of the first domain antibody light chain is directly or indirectly connected to the C-terminus of the second domain, and the C-terminus of the first domain antibody heavy chain is directly or indirectly connected to the N-terminus of the third domain.

114. The fusion polypeptide of any one of claims 44-113, wherein the indirect linkage comprises linkage via a linker.

115. The fusion polypeptide of claim 114, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO:

35.

116. The fusion polypeptide of any one of claims 44-115, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116 NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 171, SEQ ID NO:

173.

117. The fusion polypeptide of any one of claims 44-116, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 751 to SEQ ID NO: 1024, SEQ ID NO: 1249, SEQ ID NO: 1251, SEQ ID NO: 1253, and SEQ ID NO: 1255.

118. The fusion polypeptide of any one of claims 44-117, comprising a multifunctional LAG3 variant fused to the heavy chain of the Sugemalimab-CD86 IgV Q25I / F33L / H90I antibody polypeptide complex, wherein the heavy chain amino acid sequence is selected from the group consisting of SEQ ID NO: 1387-SEQ ID NO: 1420.

119. The fusion polypeptide of any one of claims 44-118, comprising the following sequences: SEQ ID NO: 1421, SEQ ID NO: 1426.

120. A CD86 variant polypeptide comprising an amino acid substitution mutation of humanized CD86.

121. The CD86 variant polypeptide of claim 120, comprising an amino acid substitution mutant of the humanized CD86 extracellular domain IgV domain.

122. The CD86 variant polypeptide according to any one of claims 120-121, comprising an amino acid substitution mutant of the IgV domain of CD86, wherein the mutation site of the amino acid substitution mutant of the IgV domain of CD86 comprises one or more amino acid site mutations selected from the group consisting of A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, and H90F.

123. The CD86 variant polypeptide of any one of claims 120-122, comprising a combination of 1, 2, 3, 4, 5 or more amino acid position mutations selected from the group consisting of A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, and H90F.

124. The CD86 variant polypeptide according to any one of claims 120 to 123, comprising an amino acid substitution mutant of the IgV domain of CD86, wherein the amino acid substitution mutant comprises one or more groups selected from the combination shown in the following group: Q25I / F33L / H90I, Q25V / F33L / H90I, Q25I / F33V / H90I, Q25V / F33V / H90I, Q25I / F33L / H90V, Q25V / F33L / H90V, Q25I / F33V / H90V, Q25V / F33V / H90V, A13L / Q25I / F33L / H90I, A13I / Q25 I / F33L / H90I, A13L / Q25V / F33L / H90I, A13I / Q25V / F33L / H90I, Q25I / F33L / I89L / H90I, Q25I / F33 L / M60R / H90I, Q25V / F33L / I89L / H90I, Q25V / F33L / M60R / H90I, Q25F / F33L / H90I, Q25I / F33L / H9 0F, Q25I / F33A / H90I, Q25I / H90I, Q25I, H90I, Q25V / H90V, Q25V / H90I, Q25F / H90I, Q25F / H90V, A 13F / Q25I / F33L / H90I, A13M / Q25I / F33L / H90I, Q25A / F33L / H90I, Q25M / F33L / H90I, Q25I / F33M / H90I, Q25I / F33L / I89V / H90I, Q25I / F33L / I89F / H90I, Q25I / F33L / I89M / H90I, and Q25I / F33L / H90M.

125. The CD86 variant polypeptide according to any one of claims 120 to 124, comprising the IgV domain amino acid substitution mutant Q25I / F33L / H90I of CD86.

126. The CD86 variant polypeptide of any one of claims 120-125, comprising a humanized Amino acid substitution mutants of the extracellular domain of CD86.

127. The CD86 variant polypeptide according to any one of claims 120 to 126, comprising an amino acid substitution mutant of the extracellular domain of CD86, wherein the amino acid substitution mutant of the extracellular domain of CD86 comprises one or more mutations in amino acid positions selected from the group consisting of A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, and H90F.

128. The CD86 variant polypeptide according to any one of claims 120 to 127, comprising an amino acid substitution mutant of the extracellular domain of CD86, wherein the amino acid substitution mutant comprises one or more groups selected from the combination shown in the following group: Q25I / F33L / H90I, 25V / F33L / H90I, Q25I / F33V / H90I, Q25V / F33V / H90I, Q25I / F33L / H90V, Q25V / F33L / H90V, Q25I / F33V / H90V, Q25V / F33V / H90V, A13L / Q25I / F33L / H90I, A13I / Q25 I / F33L / H90I, A13L / Q25V / F33L / H90I, A13I / Q25V / F33L / H90I, Q25I / F33L / I89L / H90I, Q25I / F33 L / M60R / H90I, Q25V / F33L / I89L / H90I, Q25V / F33L / M60R / H90I, Q25F / F33L / H90I, Q25I / F33L / H9 0F, Q25I / F33A / H90I, Q25I / H90I, Q25I, H90I, Q25V / H90V, Q25V / H90I, Q25F / H90I, Q25F / H90V, A 13F / Q25I / F33L / H90I, A13M / Q25I / F33L / H90I, Q25A / F33L / H90I, Q25M / F33L / H90I, Q25I / F33M / H90I, Q25I / F33L / I89V / H90I, Q25I / F33L / I89F / H90I, Q25I / F33L / I89M / H90I, and Q25I / F33L / H90M.

129. The CD86 variant polypeptide according to any one of claims 120 to 128, comprising the extracellular domain amino acid substitution mutant Q25I / F33L / H90I of CD86.

130. The CD86 variant polypeptide of any one of claims 120-129, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 178 to SEQ ID NO:

243.

131. A fusion polypeptide comprising a first domain and a second domain, wherein the first domain comprises the CD86 variant polypeptide according to any one of claims 120-130, and the second domain comprises an antibody or an antigen-binding fragment thereof or an immunoglobulin Fc domain.

132. The fusion polypeptide of claim 131, wherein the second domain comprises an antibody or an antigen-binding fragment thereof.

133. The fusion polypeptide of any one of claims 131-132, wherein the antibody is selected from the group consisting of an immunoglobulin IgG antibody, a recombinant antibody, a chimeric antibody, a heavy chain antibody, a single domain antibody, and a bispecific antibody.

134. The fusion polypeptide of any one of claims 131-133, wherein the second domain is capable of binding to one or more targets shown in the following group: PD-L1, PD-L2, PD-1, OX40, 4-1BB, ICOS, TIGIT, CTLA4, LAG3, CD3, VEGF, VEGFR, CD47, HGF, Trop2, EpCAM, CCR8, CCR4, CCR5, GPRC5D, BCMA, CD19, CD20, HER-2neu, DLL1, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CD123, CD33, CD30, CD38, NKG2A, Nkp36 and Tim3.

135. The fusion polypeptide of any one of claims 131 to 134, wherein the second domain is capable of binding to CD3.

136. The fusion polypeptide of any one of claims 131-135, wherein the second domain comprises a CD3B219 antibody or an antigen-binding fragment thereof.

137. The fusion polypeptide of any one of claims 131 to 136, wherein the second domain comprises HCDR1, HCDR2 and / or HCDR3 of an antibody heavy chain comprising the amino acid sequence shown in SEQ ID NO:

616.

138. The fusion polypeptide according to any one of claims 131 to 137, wherein the second domain comprises a heavy chain variable region VH of an antibody, and the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO:

616.

139. The fusion polypeptide of any one of claims 131 to 138, wherein the second domain comprises an antibody heavy chain comprising the amino acid sequence of SEQ ID NO:

616.

140. The fusion polypeptide of any one of claims 131-139, wherein the second domain comprises LCDR1, LCDR2 and / or LCDR3 of an antibody light chain comprising the amino acid sequence of SEQ ID NO:

617.

141. The fusion polypeptide according to any one of claims 131 to 140, wherein the second domain comprises a light chain variable region VL of an antibody, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO:

617.

142. The fusion polypeptide of any one of claims 131-141, wherein the second domain comprises an antibody light chain, and the antibody light chain comprises the amino acid sequence shown in SEQ ID NO:

617.

143. The fusion polypeptide of claims 131-142, wherein the immunoglobulin Fc domain comprises an IgG antibody Fc domain, and the immunoglobulin IgG antibody comprises one or more selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b, and mouse IgG3.

144. The fusion polypeptide of any one of claims 131-143, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 350 to SEQ ID NO: 382, ​​SEQ ID NO: 384 to SEQ ID NO: 416, SEQ ID NO: 618 to SEQ ID NO: 624, and SEQ ID NO: 626 to SEQ ID NO:

632.

145. The fusion polypeptide according to any one of claims 131 to 144, further comprising a third domain, wherein the third domain is an antibody or antigen-binding fragment thereof, or a functional protein or active fragment thereof, which is the same as or different from the second domain.

146. The fusion polypeptide according to any one of claims 131 to 145, wherein the third domain is a functional protein or an active fragment thereof, and the functional protein or the active fragment thereof can activate an innate immune response.

147. The fusion polypeptide of any one of claims 131-146, wherein the third domain is capable of binding to an MHC II molecule on an antigen presenting cell.

148. The fusion polypeptide of any one of claims 131-147, wherein the third domain comprises LAG3 or a functionally active fragment thereof.

149. A LAG3 variant polypeptide comprising truncations and / or mutations based on human LAG3.

150. The fusion polypeptide or LAG3 variant polypeptide of claim 149, comprising a truncation of a wild-type human LAG3 extracellular domain polypeptide.

151. The LAG3 variant polypeptide of any one of claims 149-150, comprising 0-124 amino acids truncated at the N-terminus based on a wild-type human LAG3 extracellular region polypeptide and terminating at amino acid positions 121-167 based on a wild-type human LAG3 extracellular region polypeptide at the C-terminus.

152. The LAG3 variant polypeptide of any one of claims 149-151, comprising 0, 5, 21, 37, 45, 60, 71, 74, 79, 84, 89, 94, 99, 104, 109, or 114 amino acids truncated at the N-terminus based on a wild-type human LAG3 extracellular region polypeptide.

153. The LAG3 variant polypeptide of any one of claims 149-152, comprising 0, 5, 21, 37, 45, 60, 71, 74, 81, or 99 amino acids truncated at the N-terminus based on a wild-type human LAG3 extracellular region polypeptide.

154. The LAG3 variant polypeptide of any one of claims 149-153, comprising 0, 5, 21, 37, 45, 60, 71 or 74 amino acids truncated at the N-terminus based on a wild-type human LAG3 extracellular region polypeptide.

155. The LAG3 variant polypeptide of any one of claims 149-154, comprising a wild-type human LAG3 extracellular region polypeptide ending at amino acid position 121, 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, or 167 at the C-terminus.

156. The LAG3 variant polypeptide of any one of claims 149-155, comprising a wild-type human LAG3 extracellular region polypeptide ending at amino acid position 121, 122, 129, 136, 146, 156, 161, or 167 at the C-terminus.

157. The LAG3 variant polypeptide of any one of claims 149-156, comprising a wild-type human LAG3 extracellular region polypeptide ending at amino acid position 121, 146, 156, 161, or 167 at the C-terminus.

158. The LAG3 variant polypeptide of any one of claims 149-157, comprising a wild-type human LAG3 extracellular region polypeptide terminating at amino acid position 156, 161, or 167 at the C-terminus.

159. The LAG3 variant polypeptide of any one of claims 149-158, comprising 0, 5, 21, 37, 45, 60, 71, 74, 81 or 99 amino acids truncated at the N-terminus based on a wild-type human LAG3 extracellular region polypeptide and terminating at amino acid position 121, 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 or 167 at the C-terminus based on a wild-type human LAG3 extracellular region polypeptide.

160. The LAG3 variant polypeptide of any one of claims 149-159, comprising 0, 5, 21, 37, 45, 60, 71 or 74 amino acids truncated at the N-terminus based on a wild-type human LAG3 extracellular region polypeptide and terminating at amino acid position 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 or 167 at the C-terminus based on a wild-type human LAG3 extracellular region polypeptide.

161. The LAG3 variant polypeptide of any one of claims 149-160, comprising an amino acid position mutation.

162. The LAG3 variant polypeptide of any one of claims 149-161, comprising an amino acid mutation at position 97, wherein the amino acid mutation is at amino acid position 97 (Arg).

163. The LAG3 variant polypeptide of any one of claims 149-162, comprising an amino acid mutation at position 97, wherein the Arg amino acid is mutated to a Glu amino acid.

164. The LAG3 variant polypeptide of any one of claims 149-163, wherein the LAG3 variant polypeptide is selected from the group consisting of amino acid sequences shown in SEQ ID NO: 244 to SEQ ID NO:

349.

165. The LAG3 variant polypeptide of any one of claims 149-164, comprising an amino acid mutation at position R110, R113, R119, R129, G130, and / or R141.

166. The LAG3 variant polypeptide of any one of claims 149-165, which is truncated at the N-terminus by 81 amino acids, terminates at amino acid position 121 at the C-terminus, and has mutations introduced at R110, and / or R113, and / or R119.

167. The LAG3 variant polypeptide of any one of claims 149-165, which is truncated at the N-terminus by 99 amino acids and terminates at amino acid position 146 at the C-terminus, and has mutations introduced at R129, and / or G130, and / or R141.

168. The LAG3 variant polypeptide of any one of claims 149-165, which is truncated at the N-terminus by 81 amino acids and terminates at amino acid position 146 at the C-terminus, and has mutations introduced at R110, and / or R113, and / or R119, and / or R129, and / or G130, and / or R141.

169. The LAG3 variant polypeptide of any one of claims 149-168, comprising an amino acid position mutation, wherein the R110 position is mutated to K110.

170. The LAG3 variant polypeptide of any one of claims 149-169, comprising an amino acid position mutation, wherein the R113 position is mutated to K113.

171. The LAG3 variant polypeptide of any one of claims 149-170, comprising an amino acid position mutation, wherein the R119 position is mutated to K119.

172. The LAG3 variant polypeptide of any one of claims 149-171, comprising an amino acid position mutation, wherein the R129 position is mutated to K129.

173. The LAG3 variant polypeptide of any one of claims 149-172, comprising an amino acid position mutation, wherein the G130 position is mutated to P130, or A130, or T130, or Y130, or S130.

174. The LAG3 variant polypeptide of any one of claims 149-173, comprising an amino acid position mutation, wherein the R141 position is mutated to K141.

175. The LAG3 variant polypeptide of any one of claims 149-174, wherein the amino acid sequence of the LAG3 variant polypeptide is selected from the group consisting of SEQ ID NO: 1285-SEQ ID NO: 1318.

176. A fusion polypeptide comprising a first domain and a second domain, wherein the first domain comprises the LAG3 variant polypeptide of any one of claims 149-175, and the second domain comprises an antibody or antigen-binding fragment thereof, or an immunoglobulin Fc domain.

177. The fusion polypeptide of claim 176, wherein the second domain comprises an antibody or an antigen-binding fragment thereof.

178. The fusion polypeptide of any one of claims 176-177, wherein the antibody is selected from the group consisting of an immunoglobulin antibody, a recombinant antibody, a chimeric antibody, a heavy chain antibody, a single domain antibody, and a bispecific antibody.

179. A fusion polypeptide as described in any one of claims 176-178, wherein the antigen binding fragment is selected from one or more of the following groups: Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, VHH and dAb.

180. The fusion polypeptide of any one of claims 176-179, wherein the second domain is capable of binding to one or more of the targets shown in the following group: PD-L1, PD-L2, PD-1, OX40, 4-1BB, ICOS, TIGIT, CTLA4, LAG3, CD3, VEGF, VEGFR, CD47, HGF, Trop2, EpCAM, CCR8, CCR4, CCR5, GPRC5D, BCMA, CD19, CD20, HER-2neu, DLL1, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CD123, CD33, CD30, CD38, NKG2A, Nkp36 and Tim3.

181. The fusion polypeptide of any one of claims 176-180, wherein the second domain is capable of binding to PD-L1, PD-1 and / or PD-L2.

182. The fusion polypeptide of any one of claims 176-181, wherein the second domain comprises HCDR1, HCDR2 and / or HCDR3 of an antibody heavy chain, preferably wherein the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO:

17.

183. The fusion polypeptide of any one of claims 176-182, wherein the second domain comprises the heavy chain variable region VH of an antibody heavy chain, preferably the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO:

17.

184. The fusion polypeptide of any one of claims 176-183, wherein the second domain comprises an antibody heavy chain, preferably the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO:

17.

185. The fusion polypeptide of any one of claims 176-184, wherein the second domain comprises LCDR1, LCDR2 and / or LCDR3 of an antibody light chain, preferably wherein the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16 and SEQ ID NO:

18.

186. The fusion polypeptide of any one of claims 176-185, wherein the second domain comprises the light chain variable region VL of an antibody light chain, preferably the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO:

18.

187. The fusion polypeptide of any one of claims 176-186, wherein the second domain comprises an antibody light chain, preferably the antibody light chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, and SEQ ID NO:

18.

188. The fusion polypeptide of any one of claims 176-187, wherein the second domain comprises an antibody or antigen-binding fragment thereof selected from the group consisting of Sugemalimab, Durvalumab, Atezolizumab, Avelumab, Envafolimab, Permbrolizumab, and Nivolumab.

189. The fusion polypeptide of any one of claims 176-188, wherein the second domain comprises Sugemalimab or an antigen-binding fragment thereof.

190. The fusion polypeptide of any one of claims 176-189, comprising a heavy chain, wherein the heavy chain amino acid sequence is selected from the group consisting of SEQ ID NO: 1353 to SEQ ID NO: 1386.

191. The fusion polypeptide of any one of claims 176-190, wherein the fusion polypeptide comprises a light chain, and the light chain comprises an antibody light chain polypeptide, preferably the fusion polypeptide light chain sequence is selected from the group comprising: SEQ ID NO:

5.

192. The fusion polypeptide of any one of claims 176-191, wherein the fusion polypeptide heavy chain and the fusion polypeptide light chain are combined to form the fusion polypeptide.

193. The fusion polypeptide of any one of claims 176-192, wherein the first domain is directly or indirectly linked to the second domain.

194. The fusion polypeptide of any one of claims 176-193, wherein the first domain is directly or indirectly connected to the N-terminus of the second domain.

195. The fusion polypeptide of any one of claims 176-194, wherein the first domain is directly or indirectly connected to the C-terminus of the second domain.

196. The fusion polypeptide of any one of claims 176-195, wherein the second domain may comprise an antibody heavy chain, and the LAG3 variant polypeptide may be directly or indirectly linked to the antibody heavy chain of the second domain.

197. According to the fusion polypeptide of any one of claims 176-196, the second domain may comprise an antibody heavy chain, and the LAG3 variant polypeptide may be directly or indirectly linked to the C-terminus of the antibody heavy chain of the second domain.

198. According to the fusion polypeptide of any one of claims 176-197, the second domain may comprise an antibody heavy chain, and the N-terminus of the LAG3 variant polypeptide may be directly or indirectly linked to the C-terminus of the antibody heavy chain of the second domain.

199. According to any one of claims 176-198, the second domain may comprise an antibody heavy chain, and the LAG3 variant polypeptide may be directly or indirectly linked to the N-terminus of the antibody heavy chain of the second domain.

200. The fusion polypeptide of any one of claims 176-199, wherein the second domain may comprise an antibody heavy chain, and the C-terminus of the LAG3 variant polypeptide may be directly or indirectly linked to the N-terminus of the antibody heavy chain of the second domain.

201. The fusion polypeptide of any one of claims 176-200, wherein the second domain may comprise an antibody light chain, and the LAG3 variant polypeptide may be directly or indirectly linked to the antibody light chain of the second domain.

202. The fusion polypeptide of any one of claims 176-201, wherein the second domain may comprise an antibody light chain, and the LAG3 variant polypeptide may be directly or indirectly linked to the C-terminus of the antibody light chain of the second domain.

203. The fusion polypeptide of any one of claims 176-202, wherein the second domain may comprise an antibody light chain, and the N-terminus of the LAG3 variant polypeptide may be directly or indirectly linked to the C-terminus of the antibody light chain of the second domain.

204. The fusion polypeptide of any one of claims 176-203, wherein the second domain may comprise an antibody light chain, and the LAG3 variant polypeptide may be directly or indirectly linked to the N-terminus of the antibody light chain of the second domain.

205. The fusion polypeptide of any one of claims 176-204, wherein the second domain may comprise an antibody light chain, and the C-terminus of the LAG3 variant polypeptide may be directly or indirectly linked to the N-terminus of the antibody light chain of the second domain.

206. The fusion polypeptide of any one of claims 176-205, wherein the indirect connection comprises connection via a linker.

207. The fusion polypeptide of any one of claims 176-206, wherein the second domain comprises an immunoglobulin Fc domain.

208. The fusion polypeptide of claim 207, wherein the immunoglobulin Fc domain comprises an IgG antibody Fc domain, and the immunoglobulin IgG antibody comprises one or more selected from human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b, and mouse IgG3.

209. The fusion polypeptide of any one of claims 176-208, wherein the second domain comprises an immunoglobulin Fc domain.

210. The fusion polypeptide of any one of claims 176-209, wherein the second domain comprises an Fc domain of an immunoglobulin IgG antibody.

211. The fusion polypeptide of any one of claims 176-210, wherein the second domain comprises the Fc domain of an immunoglobulin IgG antibody, wherein the Fc domain of the immunoglobulin IgG antibody comprises a human IgG1 Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgG1 Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain, or a mouse IgG3 Fc domain.

212. The fusion polypeptide of any one of claims 176-211, wherein the second domain comprises the Fc domain of human immunoglobulin IgG4.

213. The fusion polypeptide of any one of claims 176-212, wherein the second domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO: 18, SEQ ID NO: 29 to SEQ ID NO: 30, and SEQ ID NO: 634 to SEQ ID NO:

639.

214. The fusion polypeptide of any one of claims 176-213, wherein the second domain is selected from the group consisting of amino acid sequences shown in SEQ ID NO: 1319 to SEQ ID NO: 1352.

215. The fusion polypeptide of any one of claims 176-214, wherein the first domain is directly or indirectly linked to the second domain.

216. The fusion polypeptide of any one of claims 176-215, wherein the first domain is directly or indirectly linked to the N-terminus of the second domain.

217. The fusion polypeptide of any one of claims 176-216, wherein the first domain is directly or indirectly linked to the C-terminus of the second domain.

218. The fusion polypeptide of any one of claims 176-217, wherein the indirect connection comprises connection via a linker.

219. The fusion polypeptide of claim 218, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO:

35.

220. The fusion polypeptide of any one of claims 176-219, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 640 to SEQ ID NO: 745, SEQ ID NO: 747 to SEQ ID NO: 750, and SEQ ID NO: 1353 to SEQ ID NO: 1386.

221. The fusion polypeptide of claims 176-220, further comprising a third domain, wherein the third domain is an antibody or antigen-binding fragment or a functional protein or an active fragment thereof that is the same as or different from the second domain.

222. The fusion polypeptide of claim 221, wherein the third domain is a functional protein or an active fragment thereof, and the functional protein or the active fragment thereof can activate the innate immune response.

223. The fusion polypeptide of any one of claims 221-222, wherein the third domain is capable of binding to CD28 and / or CTLA4.

224. The fusion polypeptide of any one of claims 221-223, wherein the third domain comprises CD80, CD86, or an active fragment thereof.

225. An immunoconjugate comprising the fusion polypeptide of any one of claims 1-119, 131-148, and 176-224, the CD86 variant polypeptide of any one of claims 120-130, and / or the LAG3 variant of any one of claims 149-175.

226. A nucleic acid molecule encoding the fusion polypeptide of any one of claims 1-119, 131-148, and 176-224, the CD86 variant polypeptide of any one of claims 120-130, and / or the LAG3 variant of any one of claims 149-175.

227. A vector comprising the nucleic acid molecule of claim 226.

228. A cell comprising and / or expressing the fusion polypeptide of any one of claims 1-119, 131-148, and 176-224, the CD86 variant polypeptide of any one of claims 120-130, the LAG3 variant of any one of claims 149-175, the immunoconjugate of claim 225, the nucleic acid molecule of claim 226, and / or the vector of claim 227.

229. A composition comprising the fusion polypeptide of any one of claims 1-119, 131-148 and 176-224, the CD86 variant polypeptide of any one of claims 120-130, the LAG3 variant of any one of claims 149-175, the immunoconjugate of claim 225, the nucleic acid molecule of claim 226, the vector of claim 227, and / or the cell of claim 228, and optionally a pharmaceutically acceptable carrier.

230. A method of producing the fusion polypeptide of any one of claims 1-119, 131-148, and 176-224, the CD86 variant polypeptide of any one of claims 120-130, and the LAG3 variant of any one of claims 149-175, comprising culturing the cell of claim 228 under conditions that allow expression of the fusion polypeptide.

231. A method for blocking the interaction between PD-L1 protein and PD-1, comprising administering an effective amount of the fusion polypeptide of any one of claims 1-119, 131-148 and 176-224.

232. A method for stimulating the action of antigen presenting cells and / or activated T cells comprising the LAG3 variant of any one of claims 149-175.

233. The method according to claim 232, wherein the stimulation of antigen-presenting cells comprises a step selected from the group consisting of increasing the expression of co-stimulatory molecules in antigen-presenting cells, causing morphological changes and maturation of antigen-presenting cells, increasing the secretion of chemokines in antigen-presenting cells, and enhancing the phagocytic ability of antigen-presenting cells.

234. A method of inhibiting tumor or tumor cell growth and / or proliferation, comprising administering an effective amount of the fusion polypeptide of any one of claims 1-119, 131-148, and 176-224, the CD86 variant polypeptide of any one of claims 120-130, the LAG3 variant of any one of claims 149-175, the immunoconjugate of claim 225, the nucleic acid molecule of claim 226, the vector of claim 227, the cell of claim 228, and / or the composition of claim 229.

235. Use of the fusion polypeptide of any one of claims 1-119, 131-148 and 176-224, the CD86 variant polypeptide of any one of claims 120-130, the LAG3 variant of any one of claims 149-175, the immunoconjugate of claim 225, the nucleic acid molecule of claim 226, the vector of claim 227, the cell of claim 228, and / or the composition of claim 229 in the preparation of a medicament, wherein the medicament is used to prevent, improve and / or treat tumors.

236. The use according to claim 235, wherein the tumor comprises a solid tumor and / or a hematological tumor.

237. The method of any one of claims 235-236, wherein the tumor is selected from the group consisting of colon tumors, breast tumors, lung tumors, stomach tumors, melanomas, head and neck tumors, lymphomas, nasopharyngeal tumors, cervical tumors, esophageal tumors, kidney tumors, skin squamous cell carcinomas, endometrial tumors, liver tumors, bladder tumors, urothelial tumors, and skin tumors.

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