Fusion polypeptide and use thereof

Abstract

Provided are a LAG3 variant, a fusion polypeptide, and the use thereof. Also provided is the use of the polypeptide.

Description

A fusion polypeptide and use thereof TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a fusion polypeptide and use thereof. BACKGROUND

[0002] Immune checkpoints are a class of immune-related molecules that exist inhibitory signal molecules, which mainly realize their functions by regulating immune responses in peripheral tissues, including participating in immune defense, immune tolerance and immune tissue damage, etc., and the targeting immune checkpoint related treatment method is to regulate immune response, including innate immune response and adaptive immune response, to realize the intervention of immune system and achieve the purpose of treating diseases. Tumor cells can use immune checkpoint inhibitory signal pathway to realize their immune escape effect. The combination of programmed death receptor-1 (PD-1) and its ligand programmed death receptor protein ligand-1 (PD-L1) is an important event to inhibit anti-tumor immune response, and PD-L1 is expressed in various tumor cells including colon cancer, lung cancer, ovarian cancer and various myeloma, and the expression of PD-L1 is closely related to the prognosis of various cancers. The combination of PD-L1 and PD-1 can lead to T cell apoptosis, immune signal inhibition, cell exhaustion and secretion of immunosuppressive factors, etc., so the function of T cells involved in tumor infiltration is ineffective, which helps tumor cells to escape the monitoring of the immune system.

[0003] At present, the blocking antibodies targeting PD-L1 on the market include Atezolizumab developed by Roche, Avelumab developed by Merck KGaA / Pfizer and Durvalumab developed by AstraZeneca. The blocking antibodies targeting PD-1 on the market include Nivolumab developed by BMS, Permbrolizumab developed by Merck, Karlizumab developed by Hengrui Medicine, Tislelizumab developed by Beike Zhihui and Sindilimumab developed by Sindabio. Although these antibodies have shown tumor treatment effects, the average treatment efficiency is only about 20%, and a considerable part of tumor patients do not respond to anti-PD-L1 antibody and anti-PD-1 antibody treatment. Therefore, enhancing the effectiveness of tumor treatment is still a major problem that needs to be solved urgently in tumor treatment. SUMMARY

[0004] In view of the limitation of the effectiveness of the current PD-1 / PD-L1 pathway blocking in regulating T cell activation for treating tumors, the present application proposes to develop new agents with synergistic PD-1 / PD-L1 pathway blocking to enhance T cell activation for tumor clinical treatment. The development of new agents with synergistic PD-1 / PD-L1 pathway blocking, enhanced T cell activation and / or improved antigen presenting cell activation for tumor clinical treatment can potentially bring more benefits to more tumor patients.

[0005] The present application provides a fusion polypeptide, the fusion polypeptide of the present application can comprise (i) a fusion polypeptide derived from an anti-programmed death ligand-1 (PD-L1) antibody / anti-programmed death-1 (PD-1) antibody related binding antigen fragment, or / and (ii) an immunoglobulin Fc domain, or / and (iii) a CD80 extracellular domain (ECD), or / and (iv) 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 diseases (e.g., cancer).

[0006] The present application provides a class of dual and / or multi-functional fusion polypeptides that can simultaneously interfere with, inhibit or block the PD-1 / PD-L1 signal transduction pathway and / or synergistically stimulate antigen presenting cell activation and / or synergistically stimulate T cell activation, which can effectively stimulate T cells to enhance immune response and / or stimulate antigen presenting cells, and can have a potential effect of killing tumors, thereby improving the efficacy in treating diseases caused by T cell function inhibition, such as tumors, especially in tumor patients who are non-responsive or weakly responsive to PD-1 antibodies and / or PD-L1 antibodies.

[0007] In one aspect, the present application provides a fusion polypeptide, the polypeptide comprising a first domain and a second domain, the first domain being capable of blocking PD-1 / PD-L1 signal, and the second domain comprising CD86 or a functionally active fragment thereof.

[0008] In one embodiment of the fusion polypeptide, the first domain is capable of binding to PD-L1 and / or PD-1.

[0009] In a fusion polypeptide of one embodiment, the first domain comprises HCDR1, HCDR2, and / or HCDR3 of a heavy chain of an antibody, the 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.

[0010] In a fusion polypeptide of one embodiment, the first domain comprises a heavy chain variable region VH of an antibody heavy chain, the 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.

[0011] In a fusion polypeptide of one embodiment, the first domain comprises a heavy chain of an antibody, the 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.

[0012] In a fusion polypeptide of one embodiment, the first domain comprises LCDR1, LCDR2, and / or LCDR3 of a light chain of an antibody light chain, the 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.

[0013] In a fusion polypeptide of one embodiment, the first domain comprises an antibody or antigen binding fragment thereof. In a fusion polypeptide of one embodiment, the first domain comprises a light chain variable region VL of an antibody light chain, the 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.

[0014] In an embodiment of the fusion polypeptide, 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.

[0015] In an 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.

[0016] In an embodiment of the fusion polypeptide, the antigen binding fragment is selected from one or more of the group consisting of a Fab, a Fab’, a Fv fragment, a F(ab’)2, a F(ab)2, a scFv, a di-scFv, a VHH, and a dAb.

[0017] In an embodiment of the fusion polypeptide, the first domain comprises an antibody or an antigen binding fragment thereof selected from the group consisting of Sugemalimab, Durvalumab, Atezolizumab, Avelumab, Envafolimab, Permbrolizumab, and Nivolumab.

[0018] In an embodiment of the fusion polypeptide, the first domain comprises Sugemalimab or an antigen binding fragment thereof.

[0019] In an embodiment of the fusion polypeptide, the second domain is selected from the group consisting of a human-derived CD86 or a functionally active fragment thereof and a mouse-derived CD86 or a functionally active fragment thereof.

[0020] In an embodiment of the fusion polypeptide, the second domain is capable of binding CD28 and / or CTLA4.

[0021] In an embodiment of the fusion polypeptide, the second domain comprises an IgV domain of CD86 or a functionally active fragment thereof.

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

[0023] In an embodiment of the fusion polypeptide, the second domain comprises an amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 3.

[0024] In an embodiment of the fusion polypeptide, the first domain is directly or indirectly linked to the second domain.

[0025] In an embodiment of the fusion polypeptide, the first domain heavy chain is directly or indirectly connected to the second domain.

[0026] In an embodiment of the fusion polypeptide, the C-terminus of the first domain heavy chain is directly or indirectly connected to the N-terminus of the second domain.

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

[0028] In an embodiment of the fusion polypeptide, the first domain light chain is directly or indirectly connected to the second domain.

[0029] In an embodiment of the fusion polypeptide, the C-terminus of the first domain light chain is directly or indirectly connected to the N-terminus of the second domain.

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

[0031] In an embodiment of the fusion polypeptide, the indirect connection comprises a connection via a linker.

[0032] In an 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.

[0033] 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, 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, 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.

[0034] 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.

[0035] In another aspect, the present application provides a fusion polypeptide comprising a first domain capable of blocking PD-1 / PD-L1 signal, a second domain comprising CD86 or a functionally active fragment thereof, and a third domain capable of activating innate immune response.

[0036] In an embodiment of the fusion polypeptide, the third domain is capable of binding to MHCII molecule on antigen presenting cells.

[0037] In an embodiment of the fusion polypeptide, the third domain comprises LAG3 or a functionally active fragment thereof.

[0038] In an embodiment of the fusion polypeptide, the first domain is capable of binding to PD-L1 and / or PD-1.

[0039] In an embodiment of the fusion polypeptide, the first domain comprises HCDR1, HCDR2 and / or HCDR3 of a heavy chain of an antibody, the 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.

[0040] In an embodiment of the fusion polypeptide, the first domain comprises a heavy chain variable region VH of an antibody heavy chain, the 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.

[0041] In an embodiment of the fusion polypeptide, 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.

[0042] In an embodiment of the fusion polypeptide, the first domain comprises a LCDR1, a LCDR2 and / or a LCDR3 of a light chain of 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.

[0043] In an embodiment of the fusion polypeptide, the first domain comprises a VL of a light chain variable region of 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.

[0044] In an embodiment of the fusion polypeptide, 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.

[0045] In an embodiment of the fusion polypeptide, the first domain comprises an antibody or an antigen binding fragment thereof.

[0046] In an 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.

[0047] In an embodiment of the fusion polypeptide, the antigen binding fragment is selected from one or more of the following group: a Fab, a Fab’, a Fv fragment, a F(ab’)2, a F(ab)2, a scFv, a di-scFv, a VHH and a dAb.

[0048] In an embodiment of the fusion polypeptide, the first domain comprises an antibody or an antigen binding fragment thereof selected from the group consisting of Sugemalimab, Durvalumab, Atezolizumab, Avelumab, Envafolimab, Permbrolizumab and Nivolumab.

[0049] In an embodiment of the fusion polypeptide, the first domain comprises Sugemalimab or an antigen binding fragment thereof.

[0050] In an 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.

[0051] In an embodiment of the fusion polypeptide, the second domain is capable of binding CD28 and / or CTLA4.

[0052] In an embodiment of the fusion polypeptide, the second domain comprises an IgV domain of CD86 or a functionally active fragment thereof.

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

[0054] In an embodiment of the fusion polypeptide, the second domain comprises an amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 3.

[0055] In an 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.

[0056] In an embodiment of the fusion polypeptide, the third domain comprises an extracellular domain of LAG3 or a functionally active fragment thereof.

[0057] In an embodiment of the fusion polypeptide, the third domain comprises IgD1, IgD2, IgD3 and / or IgD4 of LAG3 or a functionally active fragment thereof.

[0058] In an 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.

[0059] In an 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.

[0060] In an embodiment of the fusion polypeptide, the first domain and the second domain are directly or indirectly connected.

[0061] In an embodiment of the fusion polypeptide, the first domain heavy chain is directly or indirectly connected to the second domain.

[0062] In one embodiment of the fusion polypeptide, the C-terminus of the first domain heavy chain is directly or indirectly connected to the N-terminus of the second domain.

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

[0064] In one embodiment of the fusion polypeptide, the first domain light chain is directly or indirectly connected to the second domain.

[0065] In one embodiment of the fusion polypeptide, the C-terminus of the first domain light chain is directly or indirectly connected to the N-terminus of the second domain.

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

[0067] In one embodiment of the fusion polypeptide, the C-terminus of the first domain heavy chain is connected to the N-terminus of the second domain and the C-terminus of the first domain light chain is connected to the N-terminus of the second domain.

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

[0069] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly connected to a third domain.

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

[0071] In one embodiment of the fusion polypeptide, the C-terminus of the first domain heavy chain is directly or indirectly connected to the N-terminus of the third domain.

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

[0073] In one embodiment of the fusion polypeptide, the first domain light chain is directly or indirectly connected to the third domain.

[0074] In one embodiment of the fusion polypeptide, the C-terminus of the first domain light chain is directly or indirectly connected to the N-terminus of the third domain.

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

[0076] In one embodiment of the fusion polypeptide, the second domain and the third domain are directly or indirectly connected.

[0077] 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.

[0078] 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.

[0079] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly connected to the second domain, and the second domain is directly or indirectly connected to the third domain.

[0080] 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.

[0081] In one embodiment of the fusion polypeptide, the C-terminus of the first domain heavy chain 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 C-terminus of the first domain light chain 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 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.

[0084] In one embodiment of the fusion polypeptide, the N-terminus of the first domain heavy 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.

[0085] In one embodiment of the fusion polypeptide, the N-terminus of the first domain 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.

[0086] In one embodiment of the fusion polypeptide, the first domain is directly or indirectly connected to the third domain, and the third domain is directly or indirectly connected to the second domain.

[0087] In a fusion polypeptide of one embodiment, 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.

[0088] In a fusion polypeptide of one embodiment, 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 a fusion polypeptide of one embodiment, 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.

[0090] In a fusion polypeptide of one embodiment, 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.

[0091] In a fusion polypeptide of one embodiment, 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.

[0092] In a fusion polypeptide of one embodiment, 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.

[0093] In a fusion polypeptide of one embodiment, the first domain is directly or indirectly connected to the second domain, and the first domain is directly or indirectly connected to the third domain.

[0094] In a fusion polypeptide of one embodiment, 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.

[0095] In a fusion polypeptide of one embodiment, 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.

[0096] In a fusion polypeptide of one embodiment, 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.

[0097] In one embodiment of the fusion polypeptide, the C-terminus of the first domain 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.

[0098] In one embodiment of the fusion polypeptide, the C-terminus of the first domain 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.

[0099] In one embodiment of the fusion polypeptide, the C-terminus of the first domain 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.

[0100] In one embodiment of the fusion polypeptide, the C-terminus of the first domain 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.

[0101] In one embodiment of the fusion polypeptide, the C-terminus of the first domain 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.

[0102] In one embodiment of the fusion polypeptide, the C-terminus of the first domain 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.

[0103] In one embodiment of the fusion polypeptide, the C-terminus of the first domain 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.

[0104] In one embodiment of the fusion polypeptide, the indirect connection comprises a connection by a linker.

[0105] 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.

[0106] 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, 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.

[0107] 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, SEQ ID NO: 173.

[0108] In another aspect, the present application provides a CD86 variant polypeptide comprising an amino acid substitution mutation of humanized CD86.

[0109] In one embodiment, the CD86 variant polypeptide comprises an IgV domain amino acid substitution mutant of humanized CD86 extracellular domain.

[0110] In one embodiment, the CD86 variant polypeptide comprises an IgV domain amino acid substitution mutant of CD86, the mutation site of which comprises one or more of the following amino acid site mutations: A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, H90F. In the present disclosure, the representation of mutation site is based on wild sequence, for example, "A13I of the mutation site of the IgV domain amino acid substitution mutant of CD86" means that the A at the 13th amino acid site of the IgV domain of wild CD86 is mutated to I;

[0111] In one embodiment, the CD86 variant polypeptide comprises an IgV domain amino acid substitution mutant of CD86 comprising 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, 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 / 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.

[0112] In one embodiment, the CD86 variant polypeptide comprises an IgV domain amino acid substitution mutant of CD86 Q25I / F33L / H90I.

[0113] In one embodiment, the CD86 variant polypeptide comprises an extracellular domain amino acid substitution mutant of humanized CD86.

[0114] In an embodiment, the CD86 variant polypeptide comprises an extracellular domain amino acid substitution mutant of CD86 comprising one or more of the following amino acid site mutations: A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, H90F.

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

[0116] In an embodiment, the CD86 variant polypeptide comprises an extracellular domain amino acid substitution mutant of CD86 comprising one or more of the following amino acid site mutations: A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, H90F.

[0117] In an 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.

[0118] In another aspect, the present application provides a fusion polypeptide comprising a first domain comprising a CD86 variant polypeptide described herein and a second domain comprising an antibody or antigen binding fragment thereof or an immunoglobulin domain.

[0119] In an embodiment, the fusion polypeptide, the second domain comprises an antibody or antigen binding fragment thereof.

[0120] In an embodiment, the fusion polypeptide, the second domain is capable of binding one or more of the target selected from the group consisting of 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.

[0121] In an embodiment, the fusion polypeptide, the second domain is capable of binding PD-L1, PD-1 and / or PD-L2.

[0122] In an embodiment, the fusion polypeptide, the second domain comprises HCDR1, HCDR2 and / or HCDR3 of a heavy chain of an antibody, the 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.

[0123] In an embodiment, the fusion polypeptide, the second domain comprises a heavy chain variable region VH of a heavy chain of an antibody, the 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.

[0124] 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 17.

[0125] In an embodiment of the fusion polypeptide, the second 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.

[0126] In an embodiment of the fusion polypeptide, the second domain comprises LCDR1, LCDR2, and / or LCDR3 of 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.

[0127] In an embodiment of the fusion polypeptide, the second domain comprises a light chain variable region VL of 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.

[0128] In an embodiment of the fusion polypeptide, the second 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.

[0129] In an 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 an embodiment of the fusion polypeptide, the second domain comprises Sugemalimab or an antigen binding fragment thereof.

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

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

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

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

[0135] In an embodiment of the fusion polypeptide, the second domain comprises a heavy chain of an antibody, the antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 616.

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

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

[0138] In an embodiment of the fusion polypeptide, the second domain comprises a light chain of an antibody, the antibody light chain comprising the amino acid sequence set forth in SEQ ID NO: 617.

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

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

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

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

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

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

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

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

[0147] In an embodiment of the fusion polypeptide, the second domain is selected from the group consisting of the amino acid sequences of 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 an embodiment of the fusion polypeptide, the first domain is directly or indirectly linked to the second domain.

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

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

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

[0152] In an 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 an 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] In another aspect, the present application provides a fusion polypeptide comprising a first domain, a second domain, and a third domain, wherein the first domain comprises a CD86 variant described herein, the second domain comprises an antibody or an antigen binding fragment thereof, and the third domain is an antibody or an antigen binding fragment thereof which is the same or different from the second domain, or a functional protein or an active fragment thereof.

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

[0156] In an embodiment, the fusion polypeptide comprises an antibody or an antigen binding fragment thereof in the second domain.

[0157] In an embodiment, the fusion polypeptide comprises an antibody or an antigen binding fragment thereof in the second domain.

[0158] In a fusion polypeptide of one embodiment, the second domain comprises HCDR1, HCDR2, and / or HCDR3 of a heavy chain of an antibody, the 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.

[0159] In a fusion polypeptide of one embodiment, the second domain comprises a heavy chain variable region VH of an antibody heavy chain, the 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.

[0160] In a fusion polypeptide of one embodiment, the second domain comprises a heavy chain of an antibody heavy chain, the 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.

[0161] In a fusion polypeptide of one embodiment, the second domain comprises LCDR1, LCDR2, and / or LCDR3 of a light chain of an antibody light chain, the 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.

[0162] In a fusion polypeptide of one embodiment, the second domain comprises a light chain variable region VL of an antibody light chain, the 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.

[0163] In an embodiment of the fusion polypeptide, the second 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.

[0164] In an 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 an embodiment of the fusion polypeptide, the second domain comprises Sugemalimab or an antigen binding fragment thereof.

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

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

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

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

[0170] In an embodiment of the fusion polypeptide, the second domain comprises a heavy chain of an antibody, the antibody heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 616.

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

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

[0173] In an embodiment of the fusion polypeptide, the second domain comprises a light chain of an antibody, the antibody light chain comprising an amino acid sequence set forth in SEQ ID NO: 617. In an 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 an embodiment of the fusion polypeptide, the antigen binding fragment is selected from one or more of the group consisting of a Fab, a Fab', a Fv fragment, a F(ab')2, a F(ab)2, a scFv, a di-scFv, a VHH, and a dAb.

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

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

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

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

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

[0180] In an embodiment of the fusion polypeptide, the second domain comprises an Fc domain of a human-derived immunoglobulin IgG4.

[0181] In an embodiment of the fusion polypeptide, the third domain is a functional protein or an active fragment thereof, the functional protein or the active fragment thereof being capable of activating an innate immune response.

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

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

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

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

[0186] In an 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 an 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 an embodiment of the fusion polypeptide, the third domain comprises a LAG3 variant polypeptide.

[0189] In an embodiment of the fusion polypeptide, the third domain comprises a LAG3 variant polypeptide comprising a truncation and / or mutation based on a wild type human-derived LAG3 extracellular region polypeptide.

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

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

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

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

[0194] In an embodiment of the fusion polypeptide, the third domain comprises a LAG3 variant polypeptide comprising a C-terminal truncation between amino acid positions 122-167 based on the wild-type human LAG3 extracellular region polypeptide.

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

[0196] In an embodiment of the fusion polypeptide, the third domain comprises a LAG3 variant polypeptide comprising a C-terminal truncation at amino acid position 122, 129, 136, 146, 156, 161, or 167 based on the wild-type human LAG3 extracellular region polypeptide.

[0197] In an embodiment of the fusion polypeptide, the third domain comprises a LAG3 variant polypeptide comprising a C-terminal truncation at amino acid position 156, 161, or 167 based on the wild-type human LAG3 extracellular region polypeptide.

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

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

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

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

[0202] In an 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 an embodiment of the fusion polypeptide, the first domain is directly or indirectly linked to the second domain.

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

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

[0206] In an 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 an 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 an 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 an embodiment of the fusion polypeptide, the indirect linkage comprises linkage via a linker.

[0210] In an 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 an embodiment of the fusion polypeptide, 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 truncation and / or mutation based on human LAG3. The present application provides an innovative engineering of LAG3 polypeptide to obtain a functional LAG3 variant polypeptide, and methods thereof for modulating antigen presenting cell immune response, thereby achieving treatment or prevention of diseases such as infection, tumor, etc. caused by suppression of immune cell function.

[0213] In one embodiment, the LAG3 variant polypeptide comprises a truncation based on a wild-type human LAG3 ectodomain polypeptide.

[0214] In one embodiment, the LAG3 variant polypeptide comprises a truncation of 0-124 amino acids at the N-terminus based on the wild-type human LAG3 extracellular region polypeptide, for example 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, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or 124 amino acids.and the C-terminus terminates at a position corresponding to any of amino acid positions 122-167, e.g., 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, of the wild-type LAG3 ectodomain polypeptide, e.g., the C-terminus terminates at a position corresponding to amino acid position 122, 123, 124, 125, 126, 127, 128, 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 of the wild-type LAG3 ectodomain polypeptide.

[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 fewer amino acids at the N-terminus than the wild-type human LAG3 ectodomain polypeptide, and the C-terminus terminates at position 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, or 167 of the wild-type LAG3 ectodomain polypeptide.

[0216] In one embodiment, the LAG3 variant polypeptide comprises a truncation of 125-145 amino acids, e.g., 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, or 145 amino acids, at the N-terminus based on the wild-type human LAG3 extracellular region polypeptide; and terminates at the C-terminus at a position corresponding to any one of amino acid positions 148-167 of the wild-type LAG3 extracellular region polypeptide, e.g., a position corresponding to any one of 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, e.g., terminates at the C-terminus at a position corresponding to amino acid position 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, or 167 of the wild-type LAG3 extracellular region polypeptide.

[0217] In one embodiment, the LAG3 variant polypeptide comprises a truncation of 0-124 amino acids at the N-terminus, e.g., 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, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or 124 amino acids, based on the wild-type human LAG3 extracellular region polypeptide; and terminates at the C-terminus at a position that is between amino acid positions 1-121 of the wild-type LAG3 extracellular region polypeptide, e.g., 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, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 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,for example, the position corresponding to any of the positions of 1-121, 2-121, 3-121, 4-121, 5-121, 6-121, 7-121, 8-121, 9-121, 10-121, 11-121, 12-121, 13-121, 14-121, 15-121, 16-121, 17-121, 18-121, 19-121, 20-121, 21-121, 22-121, 23-121, 24-121, 25-121, 26-121, 27-121, 28-121, 29-121, 30-121, 31-121, 32-121, 33-121, 34-121, 35-121, 36-121, 37-121, 38-121, 39-121, 40-121, 41-121, 42-121, 43-121, 44-121, 45-121, 46-121, 47-121, 48-121, 49-121, 50-121, 51-121, 52-121, 53-121, 54-121, 55-121, 56-121, 57-121, 58-121, 59-121, 60-121, 61-121, 62-121, 63-121, 64-121, 65-121, 66-121, 67-121, 68-121, 69-121, 70-121, 71-121, 72-121, 73-121, 74-121, 75-121, 76-121, 77-121, 78-121, 79-121, 80-121, 81-121, 82-121, 83-121, 84-121, 85-121, 86-121, 87-121, 88-121, 89-121, 90-121, 91-121, 92-121, 93-121, 94-121, 95-121, 96-121, 97-121, 98-121, 99-121, 100-121, 101-121, 102-121, 103-121, 104-121, 105-121, 106-121, 107-121, 108-121, 109-121, 110-121, 111-121, 112-121, 113-121, 114-121, 115-121, 116-121, 117-121, 118-121, 119-121, or 120-121,For example, the C-terminus ends at a position corresponding to amino acid position 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, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or 121 of the wild-type LAG3 ectodomain polypeptide.

[0218] In one embodiment of the LAG3 variant, the LAG3 variant polypeptide comprises an amino acid mutation at position 97, which is an Arg amino acid.

[0219] In one embodiment of the LAG3 variant, the LAG3 variant polypeptide comprises an amino acid mutation at position 97, which is an Arg amino acid to a Glu amino acid.

[0220] In certain preferred embodiments, the truncated LAG3 variant polypeptide variant has at least one feature selected from the group consisting of:

[0221] (1) N-terminally truncated by 74 amino acids and C-terminally ends at a position corresponding to amino acid position 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, or 167 of the wild-type LAG3 ectodomain polypeptide; wherein, preferably, the C-terminus ends at a position corresponding to amino acid position 122, 129, 136, 146, 156, 161 of the wild-type LAG3 ectodomain polypeptide.

[0222] (2) N-terminal truncated by 74 amino acids, Arg amino acid at position 97 is mutated to Glu amino acid, and C-terminal terminates at a position corresponding to amino acid position 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; preferably, the C-terminal terminates at a position corresponding to amino acid position 122, 129, 136, 146, 156, 161 of the wild-type LAG3 extracellular region polypeptide.

[0223] (3) N-terminal truncated by 5 amino acids, and C-terminal terminates at a position corresponding to amino acid position 156, 161 or 167 of the wild-type LAG3 extracellular region polypeptide.

[0224] (4) N-terminal truncated by 5 amino acids, Arg amino acid at position 97 is mutated to Glu amino acid, and C-terminal terminates at a position corresponding to amino acid position 156, 161 or 167 of the wild-type LAG3 extracellular region polypeptide.

[0225] (5) N-terminal truncated by 21 amino acids, and C-terminal terminates at a position corresponding to amino acid position 156, 161 or 167 of the wild-type LAG3 extracellular region polypeptide.

[0226] (6) N-terminal truncated by 21 amino acids, Arg amino acid at position 97 is mutated to Glu amino acid, and C-terminal terminates at a position corresponding to amino acid position 156, 161 or 167 of the wild-type LAG3 extracellular region polypeptide.

[0227] (7) N-terminal truncated by 37 amino acids, and C-terminal terminates at a position corresponding to amino acid position 156, 161 or 167 of the wild-type LAG3 extracellular region polypeptide.

[0228] (8) N-terminal truncated by 37 amino acids, Arg amino acid at position 97 is mutated to Glu amino acid, and C-terminal terminates at a position corresponding to amino acid position 156, 161 or 167 of the wild-type LAG3 extracellular region polypeptide.

[0229] (9) N-terminal truncated by 45 amino acids, and C-terminal terminates at a position corresponding to amino acid position 156, 161 or 167 of the wild-type LAG3 extracellular region polypeptide.

[0230] (10) N-terminal truncated by 45 amino acids, Arg amino acid at position 97 is mutated to Glu amino acid, and C-terminal terminates at a position corresponding to amino acid position 156, 161 or 167 of the wild-type LAG3 extracellular region polypeptide.

[0231] (11) N-terminal truncation of 60 amino acids and C-terminal truncation at a position corresponding to amino acid position 156, 161 or 167 of the wild-type LAG3 ectodomain polypeptide.

[0232] (12) N-terminal truncation of 60 amino acids, mutation of the Arg amino acid at position 97 to a Glu amino acid, and C-terminal truncation at a position corresponding to amino acid position 156, 161 or 167 of the wild-type LAG3 ectodomain polypeptide.

[0233] (13) N-terminal truncation of 71 amino acids and C-terminal truncation at a position corresponding to amino acid position 156, 161 or 167 of the wild-type LAG3 ectodomain polypeptide.

[0234] (14) N-terminal truncation of 71 amino acids, mutation of the Arg amino acid at position 97 to a Glu amino acid, and C-terminal truncation at a position corresponding to amino acid position 156, 161 or 167 of the wild-type LAG3 ectodomain polypeptide.

[0235] (15) N-terminal truncation of 79 amino acids and C-terminal truncation at a position corresponding to amino acid position 156, 161 or 167 of the wild-type LAG3 ectodomain polypeptide.

[0236] (16) N-terminal truncation of 84 amino acids and C-terminal truncation at a position corresponding to amino acid position 156, 161 or 167 of the wild-type LAG3 ectodomain polypeptide.

[0237] (17) N-terminal truncation of 89 amino acids and C-terminal truncation at a position corresponding to amino acid position 156, 161 or 167 of the wild-type LAG3 ectodomain polypeptide.

[0238] (18) N-terminal truncation of 94 amino acids and C-terminal truncation at a position corresponding to amino acid position 156, 161 or 167 of the wild-type LAG3 ectodomain polypeptide.

[0239] (19) N-terminal truncation of 99 amino acids and C-terminal truncation at a position corresponding to amino acid position 156, 161 or 167 of the wild-type LAG3 ectodomain polypeptide.

[0240] (20) N-terminal truncation of 104 amino acids and C-terminal truncation at a position corresponding to amino acid position 156, 161 or 167 of the wild-type LAG3 ectodomain polypeptide.

[0241] (21) N-terminal truncation of 109 amino acids and C-terminal truncation at a position corresponding to amino acid position 156, 161 or 167 of the wild-type LAG3 ectodomain polypeptide.

[0242] (22) N-terminal truncation of 114 amino acids and C-terminal truncation at a position corresponding to amino acid position 156, 161, or 167 of the wild-type LAG3 ectodomain polypeptide.

[0243] (23) N-terminal truncation of 0 amino acids and C-terminal truncation at a position corresponding to amino acid position 122, 129, 136, 146, 156, 161, or 167 of the wild-type LAG3 ectodomain polypeptide.

[0244] (24) N-terminal truncation of 0 amino acids, mutation of the Arg amino acid at position 97 to a Glu amino acid, and C-terminal truncation at a position corresponding to amino acid position 122, 129, 136, 146, 156, 161, or 167 of the wild-type LAG3 ectodomain polypeptide.

[0245] In one embodiment, the LAG3 variant polypeptide is selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 244 to SEQ ID NO: 349.

[0246] In another aspect, the present application provides a LAG3 variant polypeptide comprising a truncation and / or mutation based on a human LAG3.

[0247] In certain embodiments, the LAG3 variant polypeptide comprises a truncation based on a wild-type human LAG3 ectodomain polypeptide.

[0248] In certain embodiments, the LAG3 variant polypeptide comprises a truncation based on a wild-type human LAG3 ectodomain polypeptide that is truncated at the N-terminus by 0-124 amino acids and truncated at the C-terminus between amino acid positions 121-167 based on the wild-type human LAG3 ectodomain polypeptide.

[0249] In certain embodiments, the LAG3 variant polypeptide comprises a truncation based on a wild-type human LAG3 ectodomain polypeptide that is truncated at the N-terminus by 0, 5, 21, 37, 45, 60, 71, 74, 79, 81, 84, 89, 94, 99, 104, 109, or 121 amino acids.

[0250] In certain embodiments, the LAG3 variant polypeptide comprises a truncation based on a wild-type human LAG3 ectodomain polypeptide that is truncated at the N-terminus by 0, 5, 21, 37, 45, 60, 71, 74, 81, or 99 amino acids.

[0251] In certain embodiments, the LAG3 variant polypeptide comprises truncation of 0, 5, 21, 37, 45, 60, 71, 74, 81, or 99 amino acids from the N-terminus and termination at the C-terminus at amino acid position 121, 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, or 167, based on the wild-type human LAG3 extracellular region polypeptide.

[0252] In certain embodiments, the LAG3 variant polypeptide comprises truncation of 0, 5, 21, 37, 45, 60, 71, 74, 81, or 99 amino acids from the N-terminus and termination at the C-terminus at amino acid position 121, 122, 129, 136, 146, 156, 161, or 167, based on the wild-type human LAG3 extracellular region polypeptide.

[0253] In certain embodiments, the LAG3 variant polypeptide comprises truncation of 0, 5, 21, 37, 45, 60, 71, 74, 81, or 99 amino acids from the N-terminus and termination at the C-terminus at amino acid position 121, 146, 156, 161, or 167, based on the wild-type human LAG3 extracellular region polypeptide.

[0254] In certain embodiments, the LAG3 variant polypeptide comprises truncation of 0, 5, 21, 37, 45, 60, 71, 74, 81, or 99 amino acids from the N-terminus and termination at the C-terminus at amino acid position 121, 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, or 167, based on the wild-type human LAG3 extracellular region polypeptide.

[0255] In certain embodiments, the LAG3 variant polypeptide comprises an amino acid site mutation.

[0256] In certain embodiments, the LAG3 variant polypeptide comprises an amino acid site mutation at R110, R113, R119, R129, G130, and / or R141.

[0257] In certain embodiments, the LAG3 variant polypeptide is truncated by 81 amino acids from the N-terminus and terminates at the C-terminus at amino acid position 121, and comprises a mutation at the R110, and / or R113, and / or R119 site.

[0258] In certain embodiments, the LAG3 variant polypeptide is truncated by 99 amino acids from the N-terminus and terminates at the C-terminus at amino acid position 146, and comprises a mutation at the R129, and / or G130, and / or R141 site.

[0259] In certain embodiments, the LAG3 variant polypeptide is truncated at the N-terminus by 81 amino acids and terminates at the C-terminus at amino acid position 146, and comprises a mutation at the R110, and / or R113, and / or R119, and / or R129, and / or G130, and / or R141 site.

[0260] In certain embodiments, the LAG3 variant polypeptide comprises a mutation at the R110 site to K110.

[0261] In certain embodiments, the LAG3 variant polypeptide comprises a mutation at the R113 site to K113.

[0262] In certain embodiments, the LAG3 variant polypeptide comprises a mutation at the R119 site to K119.

[0263] In certain embodiments, the LAG3 variant polypeptide comprises a mutation at the R129 site to K129.

[0264] In certain embodiments, the LAG3 variant polypeptide comprises a mutation at the G130 site to P130, or A130, or T130, or Y130, or S130.

[0265] In certain embodiments, the LAG3 variant polypeptide comprises a mutation at the R141 site to K141.

[0266] In certain embodiments, the LAG3 variant polypeptide comprises a mutation at the R141 site to K141.

[0267] In certain embodiments, the LAG3 variant polypeptide comprises a mutation at the R141 site to K141.

[0268] In certain embodiments, the LAG3 variant polypeptide comprises a mutation at the R141 site to K141.

[0269] In an embodiment of the fusion polypeptide, the second domain is capable of binding 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.

[0270] In an embodiment of the fusion polypeptide, the second domain is capable of binding PD-L1, PD-1, and / or PD-L2.

[0271] In an embodiment of the fusion polypeptide, the second domain comprises HCDR1, HCDR2, and / or HCDR3 of a heavy chain of an antibody, the 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.

[0272] In an embodiment of the fusion polypeptide, the second domain comprises a heavy chain variable region VH of an antibody heavy chain, the 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.

[0273] In an embodiment of the fusion polypeptide, the second domain comprises an antibody heavy chain, the 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.

[0274] In an embodiment of the fusion polypeptide, the second domain comprises a LCDR1, a LCDR2 and / or a LCDR3 of a light chain of 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.

[0275] In an embodiment of the fusion polypeptide, the second domain comprises a VL of a light chain variable region of 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.

[0276] In an embodiment of the fusion polypeptide, the second 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.

[0277] In an embodiment of the fusion polypeptide, the second domain comprises an antibody or an antigen binding fragment thereof selected from the group consisting of Sugemalimab, Durvalumab, Atezolizumab, Avelumab, Envafolimab, Permbrolizumab and Nivolumab.

[0278] In an embodiment of the fusion polypeptide, the second domain comprises Sugemalimab or an antigen binding fragment thereof. In an embodiment of the fusion polypeptide, the antigen binding fragment is selected from one or more of the following group: Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, VHH and dAb.

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

[0280] In an embodiment of the fusion polypeptide, the second domain comprises an immunoglobulin IgG antibody comprising one or more selected from the group consisting of human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b and mouse IgG3.

[0281] In an embodiment, the fusion polypeptide comprises a second domain comprising an immunoglobulin Fc domain.

[0282] In an embodiment, the fusion polypeptide comprises a second domain comprising an Fc domain of an immunoglobulin IgG antibody.

[0283] In an embodiment, the fusion polypeptide comprises a second domain comprising an Fc domain of an immunoglobulin IgG antibody, the Fc domain of the immunoglobulin IgG antibody comprising a human IgGl Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgGl Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain, or a mouse IgG3 Fc domain.

[0284] In an embodiment, the fusion polypeptide comprises a second domain comprising an Fc domain of a human immunoglobulin IgG4.

[0285] In an embodiment, the fusion polypeptide comprises a second domain selected from the group consisting of the amino acid sequences of 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.

[0286] In an embodiment, the fusion polypeptide comprises a first domain directly or indirectly linked to a second domain.

[0287] In an embodiment, the fusion polypeptide comprises a first domain directly or indirectly linked to a second domain at the N-terminus.

[0288] In an embodiment, the fusion polypeptide comprises a first domain directly or indirectly linked to a second domain at the C-terminus.

[0289] In an embodiment, the fusion polypeptide comprises an indirect linkage comprising linkage by a linker.

[0290] In an embodiment, the fusion polypeptide comprises a linker comprising 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.

[0291] In an 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.

[0292] In certain embodiments, the fusion polypeptide comprises an antibody heavy chain, preferably the antibody heavy chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 4; the antibody heavy chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1353 to SEQ ID NO: 1386.

[0293] In certain embodiments, the fusion polypeptide comprises a light chain, the light chain comprises an antibody light chain polypeptide, preferably the antibody light chain polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0294] In certain embodiments, the fusion polypeptide heavy chain and the fusion polypeptide light chain combine to form the fusion polypeptide.

[0295] In certain embodiments, the first domain is directly or indirectly linked to the second domain.

[0296] In certain embodiments, the first domain is directly or indirectly linked to the N-terminus of the second domain.

[0297] In certain embodiments, the first domain is directly or indirectly linked to the C-terminus of the second domain.

[0298] In certain embodiments, the second domain can comprise an antibody heavy chain, the LAG3 variant polypeptide can be directly or indirectly linked to the antibody heavy chain of the second domain.

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

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

[0301] In certain embodiments, the second domain can comprise an antibody heavy chain, the LAG3 variant polypeptide can be directly or indirectly linked to the N-terminus of the antibody heavy chain of the second domain.

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

[0303] In certain embodiments, the second domain can comprise an antibody light chain, the LAG3 variant polypeptide can be directly or indirectly linked to the antibody light chain of the second domain.

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

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

[0306] In certain embodiments, the second domain can comprise an antibody light chain, and the LAG3 variant polypeptide can be directly or indirectly connected to the C-terminus of the antibody light chain of the second domain.

[0307] In certain embodiments, the second domain can comprise an antibody light chain, and the LAG3 variant polypeptide can be directly or indirectly connected to the C-terminus of the antibody light chain of the second domain.

[0308] In certain embodiments, the indirect connection comprises connection via a linker.

[0309] In certain embodiments, the second domain comprises an immunoglobulin Fc domain.

[0310] In certain embodiments, the second domain comprises an Fc domain of an immunoglobulin IgG antibody.

[0311] In certain embodiments, the second domain comprises an Fc domain of an immunoglobulin IgG antibody, which comprises a human IgGl Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgGl Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain, or a mouse IgG3 Fc domain.

[0312] In certain embodiments, the second domain comprises an Fc domain of a human-derived immunoglobulin IgG4.

[0313] In certain embodiments, the LAG3 variant polypeptide is covalently connected to the second domain with or without a linker peptide.

[0314] In certain embodiments, the second domain is selected from the group consisting of the amino acid sequences of SEQ ID NO: 1313 to SEQ ID NO: 1352.

[0315] In certain embodiments, the fusion polypeptide further comprises a third domain, which is the same or different antibody or antigen-binding fragment or functional protein or active fragment thereof as the second domain.

[0316] In another aspect, the present application provides a fusion polypeptide comprising a first domain, a second domain, and a third domain, the first domain comprising a LAG3 variant polypeptide described herein, the second domain comprising an antibody or antigen binding fragment thereof or an immunoglobulin domain, and the third domain being the same or different antibody or antigen binding fragment or functional protein or active fragment thereof as the second domain.

[0317] In an embodiment of the fusion polypeptide, the second domain comprises an antibody or antigen binding fragment thereof.

[0318] In an embodiment of the fusion polypeptide, the second domain is capable of binding one or more of the target set consisting of: 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.

[0319] In an embodiment of the fusion polypeptide, the second domain is capable of binding PD-L1, PD-1, and / or PD-L2. In an embodiment of the fusion polypeptide, the second domain comprises HCDR1, HCDR2, and / or HCDR3 of a heavy chain of an antibody, the 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.

[0320] In an embodiment of the fusion polypeptide, the second domain comprises a heavy chain variable region VH of a heavy chain of an antibody, the 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.

[0321] In an embodiment of the fusion polypeptide, the second 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.

[0322] In an embodiment of the fusion polypeptide, the second domain comprises a LCDR1, a LCDR2, and / or a LCDR3 of a light chain of 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.

[0323] In an embodiment of the fusion polypeptide, the second domain comprises a light chain variable region VL of 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.

[0324] In an embodiment of the fusion polypeptide, the second 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.

[0325] In an embodiment of the fusion polypeptide, the second domain comprises an antibody or an antigen binding fragment thereof selected from the group consisting of Sugemalimab, Durvalumab, Atezolizumab, Avelumab, Envafolimab, Permbrolizumab, and Nivolumab.

[0326] In an embodiment of the fusion polypeptide, the second domain comprises Sugemalimab or an antigen binding fragment thereof.

[0327] In an 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.

[0328] In an embodiment of the fusion polypeptide, the antigen binding fragment is selected from one or more of the following group: Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, VHH, and dAb.

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

[0330] In an embodiment of the fusion polypeptide, the second domain comprises an immunoglobulin IgG antibody, the immunoglobulin IgG comprising one or more selected from the group consisting of human IgGl, human IgG2, human IgG3, human IgG4, mouse IgGl, mouse IgG2a, mouse IgG2b, and mouse IgG3.

[0331] In an embodiment of the fusion polypeptide, the second domain comprises an immunoglobulin Fc domain.

[0332] In an embodiment of the fusion polypeptide, the second domain comprises an immunoglobulin IgG Fc domain, the immunoglobulin Fc domain comprising a human IgGl Fc domain, a human IgG2 Fc domain, a human IgG3 Fc domain, a human IgG4 Fc domain, a mouse IgGl Fc domain, a mouse IgG2a Fc domain, a mouse IgG2b Fc domain, or a mouse IgG3 Fc domain.

[0333] In an embodiment of the fusion polypeptide, the immunoglobulin Fc domain comprises an Fc domain of human immunoglobulin IgG4.

[0334] In an embodiment of the fusion polypeptide, the second domain is selected from the group of amino acid sequences consisting of 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.

[0335] In an embodiment of the fusion polypeptide, the third domain is a functional protein or an active fragment thereof, the functional protein or the active fragment thereof being capable of activating an innate immune response.

[0336] In an embodiment of the fusion polypeptide, the third domain is capable of binding CD28 and / or CTLA4.

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

[0338] In a fusion polypeptide of one embodiment, the third domain is selected from the group consisting of human CD86 or a functionally active fragment thereof, mouse CD86 or a functionally active fragment thereof, human CD80 or a functionally active fragment thereof, and mouse CD80 or a functionally active fragment thereof.

[0339] In a fusion polypeptide of one embodiment, the third domain comprises an IgV domain of CD80, an IgV domain of CD86, or an active fragment thereof.

[0340] In a fusion polypeptide of one embodiment, the third domain comprises an extracellular domain of CD80, an extracellular domain of CD86, or an active fragment thereof.

[0341] In a fusion polypeptide of one embodiment, the third domain comprises a CD86 variant polypeptide.

[0342] In a fusion polypeptide of one embodiment, the third domain comprises a CD86 variant polypeptide, the mutant comprising an amino acid substitution mutation of humanized CD86.

[0343] In a fusion polypeptide of one embodiment, the third domain comprises a CD86 variant polypeptide, the CD86 variant polypeptide comprising an IgV domain amino acid substitution mutant of CD86 and / or an extracellular domain amino acid substitution mutant of humanized CD86.

[0344] In a fusion polypeptide of one embodiment, the third domain comprises a CD86 variant polypeptide, the CD86 variant polypeptide comprising an IgV domain amino acid substitution mutant of CD86, the mutation site of the IgV domain amino acid substitution mutant of CD86 comprising one or more of the 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, H90F.

[0345] In a fusion polypeptide of one embodiment, the third domain comprises a CD86 variant polypeptide, the CD86 variant polypeptide comprising an extracellular domain amino acid substitution mutant of CD86, the extracellular domain amino acid substitution mutant of CD86 comprising one or more of the 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, H90F.

[0346] In one embodiment of the fusion polypeptide, the third domain is selected from the group consisting of the amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 3, SEQ ID NO: 178 to SEQ ID NO: 243.

[0347] In certain embodiments, the IgV or extracellular domain amino acid substitution mutant of CD86 can be selected from the group consisting of the following combinations of amino acid mutation sites: 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, 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 / 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.

[0348] In certain embodiments, the IgV or extracellular domain amino acid substitution mutant of CD86 comprises the following amino acid mutation site: Q25I / F33L / H90I.

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

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

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

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

[0353] In an embodiment of the fusion polypeptide, the first domain is directly or indirectly connected to the second domain, and the second domain is directly or indirectly connected to the third domain.

[0354] In an embodiment of the fusion polypeptide, the first domain is directly or indirectly connected to the third domain, and the second domain is directly or indirectly connected to the third domain.

[0355] In an embodiment of the fusion polypeptide, the indirect connection comprises a connection by a linker.

[0356] In an 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.

[0357] In an embodiment of the fusion polypeptide, the fusion polypeptide comprises 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.

[0358] In certain embodiments, the fusion polypeptide comprises a heavy chain of a multifunctional LAG3 variant fusion Sugemalimab-CD86 IgV Q25I / F33L / H90I antibody polypeptide complex, the heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 1387-SEQ ID NO: 1420.

[0359] In certain embodiments, the fusion polypeptide comprises a heavy chain of a multifunctional LAG3 variant fusion Sugemalimab-CD86 IgV Q25I / F33L / H90I antibody polypeptide complex, the heavy chain amino acid sequence selected from the group consisting of SEQ ID NO: 1421 and SEQ ID NO: 1426.

[0360] In certain embodiments, the nucleic acid encoding the fusion polypeptide comprises a nucleotide sequence set forth in any one of SEQ ID NO: 1422-SEQ ID NO: 1425, SEQ ID NO: 1427-SEQ ID NO: 1433.

[0361] In another aspect, the application provides an immunoconjugate comprising a fusion polypeptide described herein, a CD86 variant polypeptide described herein, and / or a LAG3 variant described herein.

[0362] In another aspect, the application provides a nucleic acid molecule encoding a fusion polypeptide described herein, a CD86 variant polypeptide described herein, and / or a LAG3 variant described herein.

[0363] In another aspect, the application provides a vector comprising a nucleic acid molecule described herein.

[0364] In another aspect, the application provides a cell comprising and / or expressing a fusion polypeptide described herein, a CD86 variant polypeptide described herein, a LAG3 variant described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, and / or a vector described herein.

[0365] In another aspect, the application provides a composition comprising a fusion polypeptide described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, a vector described herein, and / or a cell described herein, and optionally a pharmaceutically acceptable carrier.

[0366] In another aspect, the application provides a method of making a fusion polypeptide described herein, a CD86 variant polypeptide described herein, and a LAG3 variant described herein, comprising culturing a cell described herein under conditions such that the fusion polypeptide is expressed.

[0367] In another aspect, the application provides a method of blocking the interaction of a PD-L1 protein with a PD-1, comprising administering an effective amount of a fusion polypeptide described herein, a CD86 variant polypeptide described herein, a LAG3 variant described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein, and / or a composition described herein.

[0368] In another aspect, the application provides a method of stimulating antigen presenting cells and / or activating T cells, comprising administering an effective amount of a fusion polypeptide described herein, a CD86 variant polypeptide described herein, a LAG3 variant described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein, and / or a composition described herein.

[0369] According to the method described herein, the stimulating antigen presenting cells comprises a member selected from the group consisting of increasing expression of costimulatory molecules by antigen presenting cells, maturing antigen presenting cells by changing morphology, increasing secretion of chemotactic factors by antigen presenting cells, and increasing phagocytic ability of antigen presenting cells.

[0370] In another aspect, the present application provides a method of inhibiting the growth and / or proliferation of a tumor or tumor cell, comprising administering an effective amount of a fusion polypeptide described herein, a CD86 variant polypeptide described herein, a LAG3 variant described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein, and / or a composition described herein.

[0371] In another aspect, the present application provides the use of a fusion polypeptide described herein, a CD86 variant polypeptide described herein, a LAG3 variant described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein, and / or a composition described herein for the preparation of a medicament for preventing, ameliorating and / or treating a tumor.

[0372] According to the use described herein, wherein the tumor comprises a solid tumor and / or a hematological tumor.

[0373] According to the use described herein, wherein the tumor is selected from the group consisting of a colon tumor, a breast tumor, a lung tumor, a gastric tumor, a melanoma, a head and neck tumor, a lymphoma, a nasopharyngeal tumor, a cervical tumor, an esophageal tumor, a renal tumor, a skin squamous carcinoma, an endometrial tumor, a liver tumor, a bladder tumor, a urothelial tumor, and a skin tumor.

[0374] In another aspect, the present application provides a fusion polypeptide described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein, and / or a composition described herein for use in preventing, ameliorating and / or treating a tumor.

[0375] In another aspect, the present application provides a fusion polypeptide described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein, and / or a composition described herein for use in preventing, ameliorating and / or treating a tumor, wherein the tumor comprises a solid tumor and a hematological tumor.

[0376] In another aspect, the present application provides a fusion polypeptide described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein, and / or a composition described herein for use in preventing, ameliorating and / or treating a tumor, wherein the tumor is selected from the group consisting of a colon tumor, a breast tumor, a lung tumor, a gastric tumor, a melanoma, a head and neck tumor, a lymphoma, a nasopharyngeal tumor, a cervical tumor, an esophageal tumor, a renal tumor, a skin squamous carcinoma, an endometrial tumor, a liver tumor, a bladder tumor, a urothelial tumor, and a skin tumor.

[0377] In another aspect, the present application provides a method of preventing, ameliorating and / or treating a tumor, which can comprise administering to a subject in need thereof a fusion polypeptide described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein, and / or a composition described herein.

[0378] According to the method described herein, wherein the tumor comprises a solid tumor and a blood tumor.

[0379] According to the method described herein, wherein the tumor is selected from the group consisting of a colon tumor, a breast tumor, a lung tumor, a stomach tumor, a melanoma, a head and neck tumor, a lymphoma, a nasopharyngeal tumor, a cervical tumor, an esophageal tumor, a renal tumor, a skin squamous carcinoma, an endometrial tumor, a liver tumor, a bladder tumor, a urothelial tumor, and a skin tumor.

[0380] Other aspects and advantages of the present application can be readily ascertained by one skilled in the art from the following detailed description. Only the preferred embodiments of the application are shown and described in the following detailed description. As will be realized by those skilled in the art, the application is capable of modifications in various obvious aspects, all without departing from the application as recited in the claims. Accordingly, the drawings and descriptions should be regarded as illustrative in nature and not restrictive. BRIEF DESCRIPTION OF DRAWINGS

[0381] The specific features of the application involved herein are shown in the appended claims. The features and advantages of the application involved herein can be better understood by referring to the detailed description of the exemplary embodiments and the accompanying drawings. A brief description of the drawings is as follows:

[0382] Figure 1 shows an exemplary structural diagram of the fusion polypeptide described herein. The present application provides an example that can include any one or more subunits in the first domain (i) derived from any anti-PD-L1 and / or anti-PD-1 antibody antigen recognition region, the immunoglobulin Fc region (ii), the second domain (iii) and / or the third domain (iv) (herein shown with one subunit of the second domain (iii) and / or the third domain (iv), although two or more in series of the same subunit can be considered, and although any mixed combination of subunits in (iv) in series can be extended); the first domain is connected to the second domain via a linker or covalently through the immunoglobulin Fc region, and similarly, the third domain, the subunit of the second domain can be connected via a linker or covalently.

[0383] Figure 2 shows a result diagram of detecting the expression of the fusion polypeptide complex of the present application using SDS-PAGE.

[0384] Figure 3 shows the results of the fusion polypeptide complex of the present application binding to human PDL1 protein detected by ELISA.

[0385] Figure 4 shows the results of the fusion polypeptide complex of the present application binding to human CTLA4 protein detected by ELISA.

[0386] Figure 5 shows the results of the fusion polypeptide complex of the present application binding to human CD28 protein detected by ELISA.

[0387] Figure 6 shows the results of the fusion polypeptide complex of the present application regulating Jurkat T cell activation.

[0388] Figure 7 shows the results of the fusion polypeptide complex of the present application regulating antigen presenting cells up-regulating expression of costimulatory molecules.

[0389] Figure 8 shows the results of the fusion polypeptide complex of the present application regulating antigen presenting cells morphological differentiation.

[0390] Figure 9 shows the results of the fusion polypeptide complex of the present application regulating antigen presenting cells secreting CCL4.

[0391] Figure 10 shows the results of the CD86 mutant fusion polypeptide complex expression detected by SDS-PAGE.

[0392] Figure 11 shows the results of the CD86 mutant fusion polypeptide complex expression detected by AKTA.

[0393] Figure 12 shows the results of the fusion polypeptide complex of the present application regulating antigen presenting cells secreting CCL4.

[0394] Figure 13 shows the results of the fusion polypeptide complex of the present application inhibiting tumor growth.

[0395] Figure 14 shows the results of the LAG3 mutant fusion polypeptide complex expression detected by SDS-PAGE.

[0396] Figure 15 shows the results of the fusion polypeptide complex of the present application regulating antigen presenting cells secreting CCL4.

[0397] Figure 16 shows the results of the fusion polypeptide complex of the present application regulating primary PBMC cells secreting CCL4.

[0398] Figure 17 shows the results of the fusion polypeptide complex of the present application regulating primary DCs cells morphological differentiation.

[0399] Figure 18 shows the results of the fusion polypeptide complex of the present application regulating primary DCs cells up-regulating expression of costimulatory molecules.

[0400] Figure 19 shows a graph of the results of the modulation of TNFa secretion by primary DCs cells by the fusion polypeptide complexes of the application.

[0401] Figure 20 shows a graph of the results of the modulation of T cell activation by the fusion polypeptide complexes of the application.

[0402] Figure 21 shows a graph of the results of the modulation of the morphology of primary DCs cells by the LAG3 variant polypeptide complexes of the application.

[0403] Figure 22 shows a graph of the results of the modulation of the upregulation of the expression of costimulatory molecules by primary DCs cells by the LAG3 variant polypeptide complexes of the application.

[0404] Figure 23 shows a graph of the results of the modulation of TNFa secretion by primary DCs cells by the LAG3 variant polypeptide complexes of the application.

[0405] Figure 24 shows the LC-MS mass analysis of the deglycosylation reduction of the LAG3 variant polypeptide complexes of the application.

[0406] Figure 25 shows a graph of the results of the tumor inhibition curve of the fusion polypeptide complexes of the application in the MC38 / hPD-L1 tumor model. DETAILED DESCRIPTION

[0407] The present application will be readily understood by the following detailed description in conjunction with the accompanying drawings, and the foregoing information. Those skilled in the art will appreciate that the following description is provided to enable them to make and use the application.

[0408] Terminology

[0409] In the present application, the term "modulation of T cell immune response" generally refers to the modulation of the action of lymphoid T cells by the bifunctional and / or multifunctional fusion polypeptides disclosed in the present application. For example, it can extend to any other related T cell modulator that affects the expression of the T cell NFAT transcription factor, the secretion of cytokines IL-2, IFN-γ, TNFα and Granzyme B, cell proliferation and cell killing of target cells including tumor cells, after treating T cells, which can extend 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, etc.; also including CD8 T cells involved in covering but not limited to tumor tissue infiltrating CD8 T cells, effector CD8 T cells, immune memory CD8 T cells, etc.

[0410] In the present application, the term "stimulating antigen presenting cells" generally refers to the activation of antigen presenting cells regulated by the bifunctional and / or multifunctional fusion polypeptides disclosed in the present application. For example, the effects on antigen presenting cells after treating antigen presenting cells can include but are not limited to the expression of co-stimulatory molecules ICOSL, CD40L, CD137L, OX40L, CD80, CD83 and / or CD86, the activation and differentiation of antigen presenting cells, the antigen phagocytosis and presentation, the production of chemokines CCL4, CCL22, CCL17, etc., the expression of cytokines IL-1 beta, TNFa, IFN gamma, IL-6, IL-12, etc., the recruitment of T cell migration to secondary lymphoid organs, the enhancement of T cell immune response, etc. which can be expanded to any other antigen presenting cell modulators, including but not limited to B lymphocyte lines and primary B lymphocytes, monocyte lines and primary monocytes, macrophage lines and primary macrophages, dendritic cell (DC) lines and primary DC cells, etc. The antigen phagocytosis includes but is not limited to the phagocytosis of bacteria, viruses, proteins, polysaccharides, etc.

[0411] In the present application, the term "releasing the inhibition of T cells by PD-L1 / PD-1" generally refers to the bifunctional and / or multifunctional fusion polypeptides disclosed in the present application binding to PDL1 protein and blocking the binding of PD-L1 to PD-1, and thus improving the inhibition of T cells by PD-L1 / PD-1, including but not limited to the expression of NFAT transcription factor in T cells, the secretion of cytokines IL-2, IFN-gamma, TNF alpha and Granzyme B, etc., cell proliferation and cell killing of target cells, etc.

[0412] In the present application, the term "anti-tumor activity" generally refers to any biological activity that reduces or prevents the proliferation or activity of tumor cells in vivo and / or in vitro. In one embodiment, the anti-tumor activity is the anti-tumor effect of the bifunctional and / or multifunctional fusion polypeptides described in the present application.

[0413] In the present application, the term "CD86" generally refers to a class of molecules that activate cells. For example, CD86 in the present application includes full-length, variants, and / or functionally active fragments thereof. CD86 in the present application can mean a polypeptide or fragment thereof having at least about 85% amino acid identity to the protein encoded by the NCBI Accession No. Gene ID: 942 gene, and having CD28 (the protein encoded by the NCBI Accession No. Gene ID: 940 gene) and / or CTLA4 (the protein encoded by the NCBI Accession No. Gene ID: 13 gene) binding activity, 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" mean a polypeptide or fragment thereof having an amino acid sequence of at least about 85% amino acid identity to the domain in the extracellular region of the CD86 protein, and having 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 IgV domain" mean a polypeptide or fragment thereof having an amino acid sequence of at least about 85% amino acid identity to the IgV-like domain in the domain in the extracellular region of the CD86 protein, and having CD28 and / or CTLA4 binding activity, an exemplary CD86 extracellular domain IgV domain amino acid sequence is provided below (SEQ ID NO: 3), the tandem number of the CD86 extracellular domain or the CD86 extracellular domain IgV domain disclosed in the present application is not limited to the exemplary number shown, and any tandem combination comprising this functional domain can be considered as within the scope of the present application.

[0414] In the present application, the term "anti-PD-L1 antibody" generally refers to an antibody that selectively binds 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 antibodies against PD-L1 such as Durvalumab (MEDI4736) developed by AstraZeneca, Sugemalimab (CS1001) developed by Guoke Pharmaceutical, JS003 developed by Junshi Biopharmaceutical, Envafolimab (KN035) developed by Kangtai Biopharmaceutical, Atezolizumab (MPDL3280A) developed by Roche, and Avelumab (MSB0010718C) co-developed by Merck KGaA / Pfizer, exemplary anti-PD-L1 antibodies Sugemalimab (CS1001) heavy chain polypeptide amino acid sequence (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) are provided below.

[0415] In the present application, the term "anti-PD-1 antibody" means an antibody that selectively binds and has the activity of blocking a PD-1 polypeptide. For example, 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; including but not limited to anti-PD-1 antibodies such as Permbrolizumab (MK-3475) developed by Merck and Nivolumab (BMS-936558) developed by Bristol-Myers Squibb (BMS), exemplary anti-PD-1 antibody 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.

[0416] In the present application, the term "LAG3" generally refers to a class of proteins or polypeptides. For example, LAG3 in the present application includes full length, variants, and / or functionally active fragments thereof. For example, LAG3 can indicate a polypeptide having at least about 85% amino acid identity to the protein encoded by the NCBI Gene ID: 3902 gene of LAG3 (Lymphocyte activation gene 3), and having MHCII (or HLA-DR) and / or FGL1 (the protein encoded by the NCBI Gene ID: 2267 gene) binding activity, and / or activating antigen presenting cells, or fragments thereof, an exemplary human LAG3 amino acid sequence is provided below (SEQ ID NO: 19); the term "LAG3 extracellular region" means a polypeptide having an amino acid sequence of the extracellular region in the full length LAG3 protein (the polypeptide of NCBI amino acid 23-450 in the LAG3 protein; the extracellular region starts at the 23rd amino acid, thus for example, a truncation of 74, i.e., the truncation can start at the 97th), and having at least about 85% amino acid identity, and having MHCII and / or FGL1 binding activity, and / or activating antigen presenting cells, or fragments thereof, an exemplary human LAG3 extracellular domain amino acid sequence is provided below (SEQ ID NO: 20) and a tandem human LAG3 extracellular domain amino acid covalently linked by a linker (SEQ ID NO: 21); the tandem number disclosed in the present application is not limited to the exemplary number shown, any tandem combination comprising this functional domain can be considered within the scope of the present application.

[0417] In the present application, the term "LAG3-IgD1" means a polypeptide or fragment thereof having an amino acid sequence of an Ig-like V-type domain of the extracellular region of human LAG3 and at least about 85% amino acid identity, an exemplary human LAG3-IgD1 amino acid sequence is provided below (SEQ ID NO: 22); the term "LAG3-IgD2" means a polypeptide or fragment thereof having an amino acid sequence of an Ig-like C2-type 1 domain of the extracellular region of human LAG3 and at least about 85% amino acid identity, an exemplary human LAG3-IgD2 amino acid sequence is provided below (SEQ ID NO: 23); the term "LAG3-IgD3" means a polypeptide or fragment thereof having an amino acid sequence of an Ig-like C2-type 2 domain of the extracellular region of human LAG3 and at least about 85% amino acid identity, an exemplary human LAG3-IgD3 amino acid sequence is provided below (SEQ ID NO: 24); the term "LAG3-IgD4" means a polypeptide or fragment thereof having an amino acid sequence of an Ig-like C2-type 3 domain of the extracellular region of human LAG3 and 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" means a polypeptide or fragment thereof comprising an amino acid sequence of human LAG3-IgD1 and human LAG3-IgD2 and 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: 26); the number of tandem disclosed in the present application is not limited to the exemplary number shown, any tandem combination comprising the functional domain of LAG3-D1 / D2 is considered to be within the scope of the present application; the term "LAG3-D1 / D2 / D3" means a polypeptide or fragment thereof comprising an amino acid sequence of human LAG3-IgD1, human LAG3-IgD2 and human LAG3-IgD3 and 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 tandem disclosed in the present application is not limited to the exemplary number shown, any tandem combination comprising the functional domain of LAG3-D1 / D2 / D3 is considered to be within the scope of the present application;The term "LAG3-D1 / D2 / D3 / D4" means a polypeptide or fragment thereof comprising the amino acid sequence of human LAG3-IgD1, human LAG3-IgD2, human LAG3-IgD3 and human LAG3-IgD4 with at least about 85% amino acid identity, and having MHCII, and / or FGL1 binding activity, and / or activating antigen presenting cell activity. Exemplary human LAG3-D1 / D2 / D3 / D4 amino acid sequences are provided below (SEQ ID NO: 28). The number of tandem combinations disclosed herein is not limited to the exemplified number. Any combination of tandem LAG3-D1 / D2 / D3 / D4 functional domains is considered to be within the scope of the disclosure.

[0418] In the present application, the term "fusion polypeptide" generally refers to a polypeptide resulting from the fusion of two or more proteins or polypeptides. The term "fusion polypeptide" can be used interchangeably with "fusion protein" in the present application. A fusion polypeptide can comprise a fusion polypeptide complex. A fusion polypeptide can be artificially produced by recombinant DNA technology. For example, genes or nucleic acid molecules encoding the two or more proteins or polypeptides can be linked to each other to form a fusion gene or a fused nucleic acid molecule, which can encode the fusion polypeptide. Translation of the fusion gene can produce a single polypeptide, which can have the properties of at least one, or even each, of the two or more proteins or polypeptides before fusion.

[0419] In the present application, the term "dual and / or multi-functional fusion polypeptide" generally refers to a polypeptide or protein resulting from the fusion of one or more polypeptides or domains thereof from different sources. The anti-PD-L1 antibody and / or the anti-PD-1 antibody are covalently linked to a functional fusion polypeptide comprising at least one of the CD86 extracellular domain, and / or the LAG3 extracellular domain, and / or a functional domain comprising at least one LAG3 extracellular structure through a linker. The dual and / or multi-functional fusion polypeptide disclosed herein includes, but is 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 a functional fusion polypeptide comprising at least one of the CD86 extracellular domain, and / or the LAG3 extracellular domain, and / or a functional domain comprising at least one LAG3 extracellular structure through a linker, or a combination thereof, is considered to be within the scope of the disclosure.

[0420] In the present application, the term "antibody-related antigen-binding fragment" generally refers to a fragment of an antibody comprising amino acids responsible for specific binding to an antigen. It can be a fragment of an antibody molecule that determines the key difference, also known as an antigen-binding domain, or also known as an "epitope" or "antigenic determinant"; the antigen-binding domain is usually composed of an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), however, it does not necessarily include both, the antigen-binding domain of the antibody disclosed in the present application is not limited to the traditional VH and VL composed domain, but also includes the antigen-binding domain contained in any other kind of antibody but not limited to, such as recombinant antibody, single domain antibody, heavy chain antibody, chimeric antibody, bispecific antibody and other unconventional antibodies and their combinations.

[0421] Further, the anti-PD-1 antibody and / or anti-PD-L1 antibody disclosed in the present application is also not limited to a traditional natural antibody, and should include any other kind of antibody but not limited to, for example, recombinant antibody, single domain antibody, heavy chain antibody, chimeric antibody, bispecific antibody and other unconventional antibodies and their combinations having anti-PD-1 antibody and / or anti-PD-L1 characteristics.

[0422] In the present application, the term "Fab" generally refers to an antibody fragment composed of VL, VH, CL and CH1 domains.

[0423] In the present application, the term "Fab'" generally refers to an antibody fragment having several additional residues at the carboxy terminus of the CH1 domain compared to the Fab fragment. For example, Fab' can include one or more cysteines from the hinge region of an antibody.

[0424] In the present application, the term "F(ab)2" generally refers to an antigen-binding fragment obtained by linking pairs of Fab fragments via cysteines.

[0425] In the present application, the term "dAb fragment" generally refers to an antibody fragment composed of a VH domain (Ward et al., Nature 341 :544 546 (1989)).

[0426] In the present application, the term "complementarity determining region CDR" generally refers to the 3 hypervariable regions (HVR) of the light chain variable region (VL) and the heavy chain variable region (VH), which are called hypervariable regions because they can form precise complementarity with antigenic determinants in spatial structure.

[0427] In the present application, the term "Fv fragment" generally refers to an antibody fragment composed of VL and VH domains of a single arm of an antibody.

[0428] In the present application, the term "scFv" generally refers to a molecule that is formed by linking the variable region of the heavy chain and the variable region of the light chain of an antibody through a short peptide linker, also known as a single-chain antibody.

[0429] In the present application, the term "immunoglobulin" means an antibody comprising an antigen-specific binding fragment and a constant region fragment, the antigen-specific binding fragment being the fragment that determines the key difference in the immunoglobulin, which can also be referred to as an antigen binding domain, or also referred to as an "epitope" or "antigenic determinant"; the antigen binding domain is generally composed of the variable region of the heavy chain (VH) and the variable region of the light chain (VL), however, it does not necessarily include both, the antigen binding domain of the antibody disclosed in the present application is not limited to the traditional VH and VL composed domain, but also includes the antigen binding domain contained in any other kind of antibody but not limited to, such as recombinant antibody, single domain antibody, heavy chain antibody, chimeric antibody, bispecific antibody and other unconventional antibodies and combinations thereof. The constant region refers to the common structural region of the immunoglobulin, which comprises the constant region of the antibody light chain and the constant region of the heavy chain.

[0430] In the present application, the term "immunoglobulin Fc domain" generally refers to the Fc fragment formed by papain hydrolysis of a traditional antibody IgG into one Fc fragment and two identical Fab fragments. The Fc domain can comprise the CH2, CH3 and hinge region fragments of the antibody heavy chain, the traditional Fc fragment has the function of binding to the Fc fragment receptor to mediate related biological effects, and site-specific mutations can change its binding ability to the corresponding target receptor, thus affecting its biological function, and the immunoglobulin Fc domain disclosed in the present application should include but not limited to the traditional Fc fragment and other any form of Fc mutant. The biological functions that the immunoglobulin Fc domain can provide include but are not limited to prolonging the half-life, improving the stability of the molecule, facilitating the expression and detection of the fusion protein, mediating the transplacental and mucosal barrier, mediating antibody-dependent cell-mediated cytotoxicity (ADCC), mediating inflammatory response, mediating antibody-dependent cell-mediated phagocytosis (ADCP), mediating complement-dependent cytotoxicity (CDC), mediating promotion of dendritic cell (DC) maturation, regulating cytokine secretion, regulating B cell proliferation and differentiation. The Fc domain of an exemplary human immunoglobulin IgG1 (SEQ ID NO: 20) and the Fc domain of an exemplary human immunoglobulin IgG4 (SEQ ID NO: 21) are provided below.

[0431] In the present application, the terms "polypeptide" and "protein" have the same meaning and can be used interchangeably. In the present application, amino acids are generally represented by the single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0432] In the present application, the protein, polypeptide and / or amino acid sequence referred to should also be understood to encompass at least the range of variants or homologues of the said protein or polypeptide having the same or similar function.

[0433] In the present application, the "variant" generally refers to a peptide that is substantially similar to the said peptide, which can be a protein or polypeptide having one or more amino acids substituted, deleted or added in the amino acid sequence of the said protein and / or the said polypeptide (e.g., an antibody or fragment thereof of a specific binding protein). For example, the functional variant can comprise a protein or polypeptide that has been altered by at least 1, for example, 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 can substantially maintain the biological properties of the said protein or the said polypeptide before the alteration (e.g., substitution, deletion or addition). For example, the functional variant can maintain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the said protein or the said polypeptide before the alteration. For example, the substitution can be a conservative substitution.

[0434] In the present application, the homologue can be a protein or polypeptide having at least about 85% (e.g., having 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 to the amino acid sequence of the said protein and / or the said polypeptide (e.g., an antibody or fragment thereof of a specific binding protein).

[0435] In the present application, the term "homology" generally refers to the similarity, analogy, or correlation between two or more sequences. The "percent sequence homology" can be calculated by comparing two sequences to be aligned in a comparison window, determining the number of positions at which the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys, and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percent sequence homology. The alignment for determining percent sequence homology can be achieved in various ways that are known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve the maximum alignment over the full length or a specified region of a sequence being compared. The homology can also be determined, but not limited to, by FASTA and BLAST.

[0436] Generally, in a polypeptide chain, an amino group is connected to another carboxyl group in the polypeptide chain to form a chain, but at both ends of the protein, there are remaining amino acid residues that do not form a peptide bond, which are the end of the polypeptide chain carrying a free amino group and the end of the polypeptide chain carrying a carboxyl group, respectively. In the present 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 the present application, the term "C-terminus" generally refers to the end of the polypeptide chain where the amino acid residue carries a free carboxyl group.

[0437] In the present application, the term "nucleic acid molecule" generally refers to an isolated form of nucleotide, deoxyribonucleotide, or ribonucleotide, or an analog thereof, of any length, isolated from its natural environment or artificially synthesized.

[0438] In the present application, the term "immunoconjugate" generally refers to a polypeptide molecule conjugated with one or more heterologous molecules, including but not limited to a cytotoxin.

[0439] In the present application, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and expressed.

[0440] In the present application, the term "linker" generally refers to a linking molecule that links one or more polypeptides or domains thereof. For example, a linker can have conformational flexibility, 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 linker disclosed in the present application can be extended to any short peptide having 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).

[0441] In the present application, the term "CD3" generally refers to a protein complex and is composed of four distinct chains. In mammals, the complex contains one CD3y chain, one CD35 chain, and two CD3e chains. These chains associate with the T cell receptor (TCR) and the zeta-chain to generate activation signals in T lymphocytes. The TCR, zeta-chain, and CD3 molecules some form a TCR complex. As used herein, the term "CD3" refers to any native CD3 from any human source. The term also encompasses "full-length" and unprocessed protein as well as any form of the protein or one or more CD3 chains (polypeptides) resulting from processing in the cell (e.g., mature polypeptide). The term also encompasses naturally occurring variants and isoforms of CD3, e.g., 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 CD3y chain, CD35 chain, and CD3e chain and sequences.

[0442] In the present application, the term "anti-CD3 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize a single CD3 subunit (e.g., B, d, y, or z), as well as antibodies and antigen-binding fragments thereof that specifically recognize a dimeric complex of two CD3 subunits (e.g., y / e, d / e, and z / z CD3 dimers). For example, monoclonal antibodies, including human, humanized, chimeric, or murine antibodies, that are directed against the CD3 receptor in the T cell antigen receptor of mature T cells. For example, the anti-CD3 antibody can be a CD3B219 antibody or an antigen-binding fragment thereof.

[0443] In the present application, the term "amino acid mutation" generally refers to amino acid substitutions, deletions, insertions, and modifications. Any combination of substitutions, deletions, insertions and modifications can be made to arrive at a final construct, so long as the final construct possesses the desired attributes. In one embodiment, the amino acid mutation is a substitution. The term "amino acid mutation at a position" refers to a substitution or deletion of the specified residue or insertion of at least one amino acid residue adjacent to the specified residue. The substitution of an amino acid can be conservative or non-conservative. For example, "A13I" can indicate a substitution of alanine (A, Ala) at position 13 to isoleucine (I, lie).

[0444] In the present application, the term "active fragment" generally refers to a nucleic acid or amino acid fragment or variant that possesses some biological activity or function. For example, a functionally active fragment can retain or partially retain the ability of a full-length protein to bind to another molecule.

[0445] "CD86 active fragment" in the present application means a polypeptide or fragment thereof having CD28 and / or CTLA4 binding activity of the CD86 IgV or CD86 ECD protein and its derivative variant polypeptides described in the present application, including but not limited to features such as regulating T cell activity, immune system activation, and anti-tumor activity.

[0446] "LAG3 active fragment" in the present application means a polypeptide or fragment thereof having the ability to activate antigen-presenting cells of the LAG3 protein and its derivative variant polypeptides described in the present application, including but not limited to features such as regulating antigen-presenting cell activity, immune system activation, and anti-tumor activity.

[0447] The term "CD86 variant polypeptide" refers to a protein or polypeptide that has one or more amino acid substitutions, deletions or additions in the amino acid sequence of a wild-type CD86. For example, the functional variant can comprise a protein or polypeptide that has been altered by at least 1, for example 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 can substantially maintain the biological properties of CD86 before the alteration (e.g., substitution, deletion or addition). For example, the functional variant can maintain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide before the alteration. For example, the substitution can be a conservative substitution. "CD86 variant polypeptide" means an active fragment derived from a CD86 protein and having the functional properties possessed by a CD86 protein, including but not limited to having CD28 and / or CTLA4 binding activity.

[0448] The term "fused polypeptide complex of variant polypeptides" is also referred to as "variant polypeptide complex", "polypeptide complex", "fused polypeptide complex" or "fusion complex" in the present disclosure. "CD86 mutant fused polypeptide complex" means a functional fused polypeptide complex of a CD86 ECD mutant or a CD86 IgV mutant linked to an antibody or antigen binding fragment thereof, or an immunoglobulin Fc domain or a functional polypeptide fragment, with or without a linker peptide, wherein the antibody in the functional fused polypeptide complex of a disclosed CD86 mutant linked to an antibody or antigen binding fragment thereof includes but is not limited to an anti-PD-Ll antibody, an anti-PD-1 antibody, an anti-TIGIT antibody, an anti-CTLA4 antibody, an anti-CEA antibody, an anti-BCMA antibody, an anti-LAG3 antibody, an anti-CD3 antibody, an anti-Her2 antibody, an anti-Her3 antibody, an anti-VEGF antibody, an anti-VEGFR antibody, an anti-EGFR antibody, an anti-c-Met antibody, an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD38 antibody, an anti-TROP-2 antibody, an anti-CD40 antibody, an anti-4-1BB antibody, an anti-CD30 antibody, and the like; any form of antibody covalently linked to a disclosed CD86 ECD mutant or CD86 IgV mutant with or without a linker peptide, or a functional fusion protein or a combination thereof, should be considered within the scope of the present disclosure.

[0449] The term "LAG3 variant polypeptide" refers to a protein or polypeptide that has one or more amino acid substitutions, deletions or additions in the amino acid sequence of a wild-type LAG3. For example, the functional variant can include a protein or polypeptide that has been altered by at least 1, e.g., 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 can substantially retain the biological properties of LAG3 prior to alteration. For example, the functional variant can 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 alteration. For example, the substitutions can be conservative substitutions. "LAG3 variant polypeptide" means an active fragment derived from a LAG3 protein and having functional properties possessed by a LAG3 protein, including but not limited to the function of promoting activation and maturation of antigen presenting cells. The term "LAG3 variant polypeptide" means an active fragment derived from a LAG3 protein and having functional properties possessed by a LAG3 protein. The functional properties possessed by a LAG3 protein include but are not limited to the function of having MHCII (or HLA-DR) and / or FGL1 binding, promoting activation and maturation of antigen presenting cells.

[0450] In the present application, the term "LAG3 variant fusion polypeptide complex" generally refers to a polypeptide or protein after fusion of one or more polypeptides or domains thereof from different sources. The term "fusion polypeptide complex of variant polypeptides" is also referred to as "variant polypeptide complex", "polypeptide complex", "fusion polypeptide complex" or "fusion complex" in the present application. The LAG3 variant polypeptide is covalently linked to at least one subunit functional domain of the Fc domain, and / or to at least one subunit functional domain of the antibody or antigen binding fragment thereof, through or without the linker peptide. The LAG3 variant fusion polypeptide complex disclosed in the present application includes, but is not limited to, a functional fusion polypeptide or a combination thereof in which the LAG3 variant polypeptide is covalently linked to at least one subunit functional domain of any form of antibody, functional protein fragment and / or Fc domain.

[0451] The activation of the antigen presenting cells includes, but is not limited to, up-regulation of the expression of the costimulatory receptors on the antigen presenting cells, enhancement of the antigen phagocytosis, enhancement of the secretion of cytokines and chemokines, etc.

[0452] The costimulatory receptors include, but are not limited to, ICOSL, CD40L, CD137L, OX40L, CD80, CD83 and CD86, etc.

[0453] The antigen phagocytosis includes, but is not limited to, phagocytosis of bacteria, viruses, proteins, polysaccharides, etc.

[0454] The cytokines include, but are not limited to, IL-1beta, TNFa, IFNγ, IL-6, IL-12, etc.

[0455] The chemokines include, but are not limited to, CCL1, CCL2, CCL3, CCL4, CCL5, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, etc.

[0456] In the present application, the expression "N-terminal truncation of X amino acids" refers to the deletion of the amino acid residues at positions 1-X at the N-terminus of the extracellular segment of a protein. For example, the expression "N-terminal truncation of 74 amino acids of the LAG3 extracellular region polypeptide" can refer to a variant polypeptide obtained by deleting the amino acid residues at positions 23-96 at the N-terminus of the wild-type LAG3 extracellular region polypeptide. For example, the expression "N-terminal truncation of 74 amino acids of the LAG3 extracellular region polypeptide" can refer to a polypeptide obtained based on the amino acid at position 97 of the wild-type human LAG3 polypeptide. For example, the expression "N-terminal truncation of 74 amino acids of the LAG3 extracellular region polypeptide" can be denoted as "LAG3 97-450".

[0457] In the present application, the expression "C-terminus terminates at amino acid position X" means that all of the amino acid residues after (i.e., starting from amino acid position X+1) amino acid position X are deleted. For example, the expression "C-terminus terminates at LAG3 ectodomain polypeptide amino acid position 156" can mean a variant polypeptide obtained by deleting all of the amino acid residues after (i.e., starting from amino acid position 157) amino acid position NCBI number 156 of the wild-type LAG3 ectodomain polypeptide. For example, the expression "C-terminus terminates at LAG3 ectodomain polypeptide amino acid position 156" can mean that the C-terminus of the wild-type LAG3 ectodomain polypeptide is truncated by 294 amino acids. For example, the expression "C-terminus terminates at LAG3 ectodomain polypeptide amino acid position 156" can be expressed as "LAG3 23-156". For example, the expression "N-terminus is truncated by 74 amino acids and C-terminus terminates at amino acid position 156" can be expressed as "LAG3 97-156".

[0458] In the present application, the term "comprising" generally means including, but not excluding other elements.

[0459] In the present application, the term "about" generally means a variation of 0.5-10% above or below the specified numerical value, for example, a variation 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 numerical value.

[0460] DETAILED DESCRIPTION

[0461] In one aspect, the present application provides a fusion polypeptide, which can comprise a first domain capable of blocking PD-1 / PD-L1 signal and a second domain comprising CD86 or a functionally active fragment thereof. For example, the fusion polypeptide can comprise one or more of the first domain and one or more of the second domain.

[0462] For example, the first domain can be capable of binding to PD-L1 and / or PD-1.

[0463] For example, the first domain can comprise HCDR3 of an antibody heavy chain, which can 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 can comprise HCDR2 of an antibody heavy chain, which can 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 can comprise HCDR1 of an antibody heavy chain, which can 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.

[0466] For example, the first domain can comprise a heavy chain variable region VH of an antibody heavy chain, which can 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.

[0467] For example, the first domain can comprise an antibody heavy chain, which can 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.

[0468] For example, the first domain can comprise LCDR3 of an antibody light chain, which can 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 can comprise LCDR2 of an antibody light chain, which can 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 can comprise LCDR1 of an antibody light chain, which can 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.

[0471] For example, the first domain can comprise a light chain variable region VL of an antibody light chain, which can 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.

[0472] For example, the first domain can comprise an antibody light chain, which can 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.

[0473] For example, the first domain can comprise HCDR3, HCDR2, and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 4. For example, the antibody can be Sugemalimab.

[0474] For example, the first domain can comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 5. For example, the antibody can be Sugemalimab.

[0475] For example, the first domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 4, and the first domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 5. For example, the antibody can be Sugemalimab.

[0476] For example, the first domain can comprise a heavy chain variable region VH of an antibody heavy chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 4, and the first domain can comprise a light chain variable region VL of an antibody light chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 5. For example, the antibody can be Sugemalimab.

[0477] For example, the first domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 6. For example, the antibody can be JS003.

[0478] For example, the first domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 7. For example, the antibody can be JS003.

[0479] For example, the first domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 6, and the first domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 7. For example, the antibody can be JS003.

[0480] For example, the first domain can comprise a heavy chain variable region VH of an antibody heavy chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 6, and the first domain can comprise a light chain variable region VL of an antibody light chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 7. For example, the antibody can be JS003.

[0481] For example, the first domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 8. For example, the antibody can be Durvalumab.

[0482] For example, the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise an amino acid sequence as shown in SEQ ID NO: 9. For example, the antibody may be Durvalumab.

[0483] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of the antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 8, and the first domain may comprise LCDR3, LCDR2, and LCDR1 of the 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.

[0484] For example, the first domain may include a heavy chain variable region VH of the antibody heavy chain, which may contain the amino acid sequence shown in SEQ ID NO: 8, and the first domain may include a light chain variable region VL of the antibody light chain, which may contain the amino acid sequence shown in SEQ ID NO: 9. For example, the antibody may be Durvalumab.

[0485] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of the 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.

[0486] For example, the first domain may comprise LCDR3, LCDR2, and LCDR1 of an antibody light chain, wherein the antibody light chain may comprise an amino acid sequence as shown in SEQ ID NO: 11. For example, the antibody may be Atezolizumab.

[0487] For example, the first domain may comprise HCDR3, HCDR2, and HCDR1 of the antibody heavy chain, wherein the antibody heavy chain may comprise the amino acid sequence shown in SEQ ID NO: 10, and the first domain may comprise LCDR3, LCDR2, and LCDR1 of the 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.

[0488] For example, the first domain can comprise a heavy chain variable region VH of an antibody heavy chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 10, and the first domain can comprise a light chain variable region VL of an antibody light chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 11. For example, the antibody can be Atezolizumab.

[0489] For example, the first domain can comprise a HCDR3, a HCDR2, and a HCDR1 of an antibody heavy chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 12. For example, the antibody can be Avelumab.

[0490] For example, the first domain can comprise a LCDR3, a LCDR2, and a LCDR1 of an antibody light chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 13. For example, the antibody can be Avelumab.

[0491] For example, the first domain can comprise a HCDR3, a HCDR2, and a HCDR1 of an antibody heavy chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 12, and the first domain can comprise a LCDR3, a LCDR2, and a LCDR1 of an antibody light chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 13. For example, the antibody can be Avelumab.

[0492] For example, the first domain can comprise a heavy chain variable region VH of an antibody heavy chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 12, and the first domain can comprise a light chain variable region VL of an antibody light chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 13. For example, the antibody can be Avelumab.

[0493] For example, the first domain can comprise a HCDR3, a HCDR2, and a HCDR1 of an antibody, which can comprise an amino acid sequence as set forth in SEQ ID NO: 14. For example, the antibody can be Envafolimab.

[0494] For example, the first domain can comprise a HCDR3, a HCDR2, and a HCDR1 of an antibody heavy chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 15. For example, the antibody can be Permbrolizumab.

[0495] For example, the first domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 16. For example, the antibody can be Permbrolizumab.

[0496] For example, the first domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 15, and the first domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 16. For example, the antibody can be Permbrolizumab.

[0497] For example, the first domain can comprise a heavy chain variable region VH of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 15, and the first domain can comprise a light chain variable region VL of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 16. For example, the antibody can be Permbrolizumab.

[0498] For example, the first domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 17. For example, the antibody can be Nivolumab.

[0499] For example, the first domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 18. For example, the antibody can be Nivolumab.

[0500] For example, the first domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 17, and the first domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 18. For example, the antibody can be Nivolumab.

[0501] For example, the first domain can comprise a heavy chain variable region VH of an antibody heavy chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 17, and the first domain can comprise a light chain variable region VL of an antibody light chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 18. For example, the antibody can be Nivolumab.

[0502] For example, the first domain can comprise an antibody or an antigen binding fragment thereof.

[0503] 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.

[0504] For example, the antigen binding fragment can be selected from one or more of the group consisting of a Fab, a Fab', a Fv fragment, a F(ab')2, a F(ab)2, a scFv, a di-scFv, a VHH, and a dAb.

[0505] For example, the first domain can comprise an antibody or an antigen binding fragment thereof selected from the group consisting of

[0506] For example, the first domain can comprise Sugemalimab or an antigen binding fragment thereof.

[0507] For example, the first domain can comprise Sugemalimab or an antigen binding fragment thereof.

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

[0509] For example, the second domain can be selected from the group consisting of a CD86 or a functionally active fragment thereof derived from a human and a CD86 or a functionally active fragment thereof derived from a mouse.

[0510] For example, the second domain can bind to CD28, and / or CTLA4.

[0511] For example, the second domain can comprise an IgV domain of CD86 or a functionally active fragment thereof.

[0512] For example, the second domain can comprise an extracellular domain of CD86 or a functionally active fragment thereof.

[0513] For example, the second domain can comprise an amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 3.

[0514] For example, the first domain and the second domain can be directly or indirectly connected.

[0515] For example, the N-terminus of the first domain and the second domain can be directly or indirectly connected.

[0516] For example, the C-terminus of the first domain and the second domain can be directly or indirectly connected.

[0517] For example, the first domain can comprise an antibody heavy chain, and the antibody heavy chain of the first domain can be directly or indirectly connected to the second domain.

[0518] For example, the first domain can comprise 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 second domain.

[0519] For example, the first domain can comprise 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 second domain.

[0520] For example, the first domain can comprise an antibody light chain, and the antibody light chain of the first domain can be directly or indirectly connected to the second domain.

[0521] For example, the first domain can comprise an antibody light chain, and the C-terminus of the antibody light chain of the first domain can be directly or indirectly connected to the N-terminus of the second domain.

[0522] For example, the first domain can comprise an antibody light chain, 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.

[0523] For example, the indirect connection can comprise a connection through a linker.

[0524] For example, the linker can 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.

[0525] 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, 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, 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.

[0526] 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.

[0527] In an aspect, the present application also provides a fusion polypeptide, which can comprise a first domain capable of blocking PD-1 / PD-L1 signal, a second domain comprising CD86 or a functionally active fragment thereof, and a third domain capable of activating innate immune response. For example, the fusion polypeptide can comprise one or more of the first domain, one or more of the second domain, and one or more of the third domain.

[0528] For example, the first domain is capable of binding to PD-L1 and / or PD-1.

[0529] For example, the first domain can comprise HCDR3 of an antibody heavy chain, which can 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 can comprise HCDR2 of an antibody heavy chain, which can 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.

[0531] For example, the first domain can comprise HCDR1 of an antibody heavy chain, which can 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.

[0532] For example, the first domain can comprise heavy chain variable region VH of an antibody heavy chain, which can 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.

[0533] For example, the first domain can comprise an antibody heavy chain, which can 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.

[0534] For example, the first domain can comprise an antibody light chain LCDR3, which can 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 can comprise an antibody light chain LCDR2, which can 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 can comprise an antibody light chain LCDR1, which can 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.

[0537] For example, the first domain can comprise an antibody light chain VL, which can 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.

[0538] For example, the first domain can comprise an antibody light chain, which can 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.

[0539] For example, the first domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 4. For example, the antibody can be Sugemalimab.

[0540] For example, the first domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 5. For example, the antibody can be Sugemalimab.

[0541] For example, the first domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 4, and the first domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 5. For example, the antibody can be Sugemalimab.

[0542] For example, the first domain can comprise a heavy chain variable region VH of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 4, and the first domain can comprise a light chain variable region VL of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 5. For example, the antibody can be Sugemalimab.

[0543] For example, the first domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 6. For example, the antibody can be JS003.

[0544] For example, the first domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 7. For example, the antibody can be JS003.

[0545] For example, the first domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 6, and the first domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 7. For example, the antibody can be JS003.

[0546] For example, the first domain can comprise a heavy chain variable region VH of an antibody heavy chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 6, and the first domain can comprise a light chain variable region VL of an antibody light chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 7. For example, the antibody can be JS003.

[0547] For example, the first domain can comprise a HCDR3, a HCDR2, and a HCDR1 of an antibody heavy chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 8. For example, the antibody can be Durvalumab.

[0548] For example, the first domain can comprise a LCDR3, a LCDR2, and a LCDR1 of an antibody light chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 9. For example, the antibody can be Durvalumab.

[0549] For example, the first domain can comprise a HCDR3, a HCDR2, and a HCDR1 of an antibody heavy chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 8, and the first domain can comprise a LCDR3, a LCDR2, and a LCDR1 of an antibody light chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 9. For example, the antibody can be Durvalumab.

[0550] For example, the first domain can comprise a heavy chain variable region VH of an antibody heavy chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 8, and the first domain can comprise a light chain variable region VL of an antibody light chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 9. For example, the antibody can be Durvalumab.

[0551] For example, the first domain can comprise a HCDR3, a HCDR2, and a HCDR1 of an antibody heavy chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 10. For example, the antibody can be Atezolizumab.

[0552] For example, the first domain can comprise a LCDR3, a LCDR2, and a LCDR1 of an antibody light chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 11. For example, the antibody can be Atezolizumab.

[0553] For example, the first domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 10, and the first domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 11. For example, the antibody can be Atezolizumab.

[0554] For example, the first domain can comprise a heavy chain variable region VH of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 10, and the first domain can comprise a light chain variable region VL of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 11. For example, the antibody can be Atezolizumab.

[0555] For example, the first domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 12. For example, the antibody can be Avelumab.

[0556] For example, the first domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 13. For example, the antibody can be Avelumab.

[0557] For example, the first domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 12, and the first domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 13. For example, the antibody can be Avelumab.

[0558] For example, the first domain can comprise a heavy chain variable region VH of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 12, and the first domain can comprise a light chain variable region VL of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 13. For example, the antibody can be Avelumab.

[0559] For example, the first domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody, wherein the antibody can comprise an amino acid sequence as set forth in SEQ ID NO: 14. For example, the antibody can be Envafolimab.

[0560] For example, the first domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 15. For example, the antibody can be Permbrolizumab.

[0561] For example, the first domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 16. For example, the antibody can be Permbrolizumab.

[0562] For example, the first domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 15, and the first domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 16. For example, the antibody can be Permbrolizumab.

[0563] For example, the first domain can comprise a heavy chain variable region VH of an antibody heavy chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 15, and the first domain can comprise a light chain variable region VL of an antibody light chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 16. For example, the antibody can be Permbrolizumab.

[0564] For example, the first domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 17. For example, the antibody can be Nivolumab.

[0565] For example, the first domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 18. For example, the antibody can be Nivolumab.

[0566] For example, the first domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, wherein the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 17, and the first domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, wherein the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 18. For example, the antibody can be Nivolumab.

[0567] For example, the first domain can comprise a heavy chain variable region VH of an antibody heavy chain, the antibody heavy chain can comprise an amino acid sequence as set forth in SEQ ID NO: 17, and the first domain can comprise a light chain variable region VL of an antibody light chain, the antibody light chain can comprise an amino acid sequence as set forth in SEQ ID NO: 18. For example, the antibody can be Nivolumab.

[0568] For example, the first domain can comprise an antibody or an antigen binding fragment thereof.

[0569] 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.

[0570] For example, the antigen binding fragment can be selected from one or more of the following group: Fab, Fab', Fv fragment, F(ab')2, F(ab)2, scFv, di-scFv, VHH and dAb.

[0571] For example, the first domain can comprise an antibody or an antigen binding fragment thereof selected from the group consisting of Sugemalimab, Durvalumab, Atezolizumab, Avelumab, Envafolimab, Permbrolizumab and Nivolumab.

[0572] For example, the first domain can comprise Sugemalimab or an antigen binding fragment thereof.

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

[0574] For example, the second domain can be selected from the group consisting of a human-derived CD86 or a functionally active fragment thereof and a mouse-derived CD86 or a functionally active fragment thereof.

[0575] For example, the second domain can bind to CD28, and / or CTLA4.

[0576] For example, the second domain can comprise an IgV domain of CD86 or a functionally active fragment thereof.

[0577] For example, the second domain can comprise an extracellular domain of CD86 or a functionally active fragment thereof.

[0578] For example, the second domain can comprise an amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 3.

[0579] For example, the third domain can be capable of binding to an MHCII molecule on an antigen presenting cell, and / or activating antigen presentation.

[0580] For example, the third domain can comprise LAG3 or a functionally active fragment thereof.

[0581] For example, the third domain can be selected from the group consisting of a human-derived LAG3 or a functionally active fragment thereof and a mouse-derived LAG3 or a functionally active fragment thereof.

[0582] For example, the third domain can comprise an extracellular domain of LAG3 or a functionally active fragment thereof.

[0583] For example, the third domain can comprise IgD1, IgD2, IgD3 and / or IgD4 of LAG3 or a functionally active fragment thereof.

[0584] For example, the third domain can comprise IgD1, IgD1-IgD2, IgD1-IgD2-IgD3, and / or IgD1-IgD2-IgD3-IgD4 of LAG3 or a functionally active fragment thereof.

[0585] For example, the third domain can 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.

[0586] For example, the first domain and the second domain can be directly or indirectly connected.

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

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

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

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

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

[0592] For example, the first domain comprises an antibody light chain, and the C-terminus of the first domain antibody light chain can be directly or indirectly connected to the N-terminus of the second domain.

[0593] For example, the first domain comprises an antibody light chain, and the C-terminus of the first domain antibody light chain can be directly or indirectly connected to the N-terminus of the second domain.

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

[0595] For example, the first domain and the third domain can be directly or indirectly connected.

[0596] For example, the first domain comprises an antibody light chain, and the C-terminus of the first domain antibody light chain can be directly or indirectly connected to the N-terminus of the second domain.

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

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

[0599] For example, the first domain comprises an antibody light chain, and the C-terminus of the first domain antibody light chain can be directly or indirectly connected to the N-terminus of the second domain.

[0600] For example, the first domain comprises an antibody light chain, and the C-terminus of the first domain antibody light chain can be directly or indirectly connected to the N-terminus of the second domain.

[0601] For example, the first domain comprises an antibody light chain, and the C-terminus of the first domain antibody light chain can be directly or indirectly connected to the N-terminus of the second domain.

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

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

[0604] For example, the N-terminus of the second domain can be directly or indirectly connected to the C-terminus of the third domain.

[0605] For example, the first domain can be directly or indirectly connected to the second domain, and the second domain can be directly or indirectly connected to the third domain.

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

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

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

[0609] For example, the N-terminus of the first domain can be directly or indirectly connected to the C-terminus of the second domain, and the N-terminus of the second domain can be directly or indirectly connected to the C-terminus of the third domain.

[0610] For example, the first domain comprises an antibody heavy chain, the N-terminus of the first domain antibody heavy chain can be directly or indirectly connected to the C-terminus of the second domain, and the N-terminus of the second domain can be directly or indirectly connected to the C-terminus of the third domain.

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

[0612] For example, the first domain can be directly or indirectly connected to the third domain, and the third domain can be directly or indirectly connected to the second domain.

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

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

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

[0616] For example, the N-terminus of the first structural domain can be directly or indirectly connected to the C-terminus of the third structural domain, and the N-terminus of the third structural domain can be directly or indirectly connected to the C-terminus of the second structural domain.

[0617] For example, the first domain contains an antibody heavy chain, the N-terminus of the antibody heavy chain in the first domain can be directly or indirectly connected to the C-terminus of the third domain, and the N-terminus of the third domain can be directly or indirectly connected to the C-terminus of the second domain.

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

[0619] For example, the first structural domain and the second structural domain can be directly or indirectly connected, and the first structural domain and the third structural domain can be directly or indirectly connected.

[0620] For example, the C-terminus of the first structural domain can be directly or indirectly connected to the N-terminus of the second structural domain, and the N-terminus of the first structural domain can be directly or indirectly connected to the C-terminus of the third structural domain.

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

[0622] For example, the first domain contains an antibody light chain, the C-terminus of the antibody light chain in the first domain can be directly or indirectly connected to the N-terminus of the second domain, and the N-terminus of the light chain in the first domain can be directly or indirectly connected to the C-terminus of the third domain.

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

[0625] For example, the N-terminus of the first domain can be directly or indirectly connected to the C-terminus of the second domain, and the C-terminus of the first domain can be directly or indirectly connected to the N-terminus of the third domain.

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

[0627] For example, the first domain comprises an antibody light chain, the N-terminus of the first domain antibody light chain can be directly or indirectly connected to the C-terminus of the second domain, and the C-terminus of the first domain light chain can be directly or indirectly connected to the N-terminus of the third domain.

[0628] For example, the first domain comprises an antibody heavy chain and an antibody light chain, the N-terminus of the first domain antibody heavy chain can be directly or indirectly connected to the C-terminus of the second domain, and the C-terminus of the first domain antibody light chain can be directly or indirectly connected to the N-terminus of the third domain.

[0629] For example, the first domain comprises an antibody heavy chain and an antibody light chain, the N-terminus of the first domain antibody light chain can be directly or indirectly connected to the C-terminus of the second domain, and the C-terminus of the first domain antibody heavy chain can be directly or indirectly connected to the N-terminus of the third domain.

[0630] For example, the indirect connection can comprise a connection through a linker.

[0631] For example, the linker can 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.

[0632] For example, the fusion polypeptide can comprise 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, 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.

[0633] For example, the fusion polypeptide can 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, SEQ ID NO: 173.

[0634] In one aspect, the present application discloses a dual and / or multi-functional fusion polypeptide comprising a dual fusion polypeptide, which comprises a first domain consisting of (i) an antigen binding fragment having specific binding to a first cell target, and / or (ii) a Fc domain, and / or a second domain having specific binding to a second cell target, and / or a third domain having specific binding to a third cell target, wherein the second cell target binds to the second domain via or not via a linker covalently linked to the C-terminus of the first domain polypeptide, or to the N-terminus of the first domain polypeptide, and wherein the third cell target binds to the third domain via or not via a linker covalently linked to the C-terminus of the first domain polypeptide, or to the N-terminus of the first domain polypeptide; wherein it is explicitly stated that the first domain of the present application can be extended to certain Fc domain-free anti-PD-L1 and / or anti-PD-1 antibodies, including but not limited to single domain antibodies, recombinant antibodies, and single chain antibodies, etc. For example, the C-terminus of the heavy chain polypeptide or the light chain polypeptide of the first domain is covalently linked to the second domain and / or the third domain via a linker, which is considered to be within the scope of the claims of the present application; similarly, the N-terminus of the heavy chain polypeptide or the light chain polypeptide of the first domain is covalently linked to the second domain and / or the third domain via a linker, which is also considered to be within the scope of the claims of the present application. Therefore, the dual and / or multi-functional fusion polypeptide can simultaneously bind to the first cell target, the second cell target, and / or the third cell target.

[0635] In one aspect, the present application provides a specific polypeptide complex constituting a dual and / or multi-functional fusion polypeptide, which comprises a first polypeptide and a second polypeptide constituting the polypeptide complex, and it is explicitly stated that the second polypeptide can be absent in certain specific antibodies.

[0636] In another aspect, the second polypeptide of the polypeptide complex of the present application is a light chain of the first domain; the first polypeptide of the polypeptide complex of the present application comprises, in order from the N-terminus to the C-terminus of the polypeptide, a third domain and / or a second domain, a linker, a heavy chain of the first domain, a linker, and a third domain and / or a second domain covalently linked together, and the first polypeptide and the second polypeptide constitute a specific polypeptide complex of a dual and / or multi-functional fusion polypeptide; wherein the linkers can each independently be present or absent; in some cases, only one of the second domain or the third domain is present.

[0637] In another aspect, the second polypeptide of the polypeptide complex of the present application is a first domain light chain; the first polypeptide of the polypeptide complex of the present application comprises, in order from the N-terminus to the C-terminus of the polypeptide, a third domain and / or a second domain, a linker, a third domain and / or a second domain, and a linker and a first domain heavy chain covalently linked together, the first polypeptide and the second polypeptide forming a specific polypeptide complex of a bi- and / or multi-functional fusion polypeptide; wherein the linkers can each independently be present or absent; in some cases, only one of the second domain or the third domain is present.

[0638] In another aspect, the second polypeptide of the polypeptide complex of the present application is a first domain light chain; the first polypeptide of the polypeptide complex of the present application comprises, in order from the N-terminus to the C-terminus of the polypeptide, a third domain and / or a second domain, a linker, a third domain and / or a second domain, and a linker and a first domain heavy chain covalently linked together, the first polypeptide and the second polypeptide forming a specific polypeptide complex of a bi- and / or multi-functional fusion polypeptide; wherein the linkers can each independently be present or absent; in some cases, only one of the second domain or the third domain is present.

[0639] In another aspect, the second polypeptide of the polypeptide complex of the present application is a first domain light chain; the first polypeptide of the polypeptide complex of the present application comprises, in order from the N-terminus to the C-terminus of the polypeptide, a third domain and / or a second domain, a linker, a third domain and / or a second domain, and a linker and a first domain heavy chain covalently linked together, the first polypeptide and the second polypeptide forming a specific polypeptide complex of a bi- and / or multi-functional fusion polypeptide; wherein the linkers can each independently be present or absent; in some cases, only one of the second domain or the third domain is present.

[0640] In another aspect, the second polypeptide of the polypeptide complex of the present application is a first domain light chain; the first polypeptide of the polypeptide complex of the present application comprises, in order from the N-terminus to the C-terminus of the polypeptide, a third domain and / or a second domain, a linker, a third domain and / or a second domain, and a linker and a first domain heavy chain covalently linked together, the first polypeptide and the second polypeptide forming a specific polypeptide complex of a bi- and / or multi-functional fusion polypeptide; wherein the linkers can each independently be present or absent; in some cases, only one of the second domain or the third domain is present.

[0641] In another aspect, the first polypeptide of the polypeptide complex of the present application is a first domain heavy chain; the second polypeptide of the polypeptide complex of the present application comprises, in order from the N-terminus to the C-terminus of the polypeptide, a third domain and / or a second domain, a linker, a third domain and / or a second domain, a linker, and a first domain light chain covalently linked together, the first polypeptide and the second polypeptide forming a specific polypeptide complex of a bi- and / or multi-functional fusion polypeptide; wherein the linkers can each independently be present or absent; in some cases, only one of the second domain or the third domain is present.

[0642] In another aspect, the first polypeptide of the polypeptide complex of the present application is a first domain heavy chain; the second polypeptide of the polypeptide complex of the present application comprises, in order from the N-terminus to the C-terminus of the polypeptide, a third domain and / or a second domain, a linker, a third domain and / or a second domain, a linker, and a first domain light chain covalently linked together, the first polypeptide and the second polypeptide forming a specific polypeptide complex of a bi- and / or multi-functional fusion polypeptide; wherein the linkers can each independently be present or absent; in some cases, only one of the second domain or the third domain is present.

[0643] In another aspect, the first polypeptide of the polypeptide complex of the present application is a first domain heavy chain; the second polypeptide of the polypeptide complex of the present application comprises, in order from the N-terminus to the C-terminus of the polypeptide, a third domain and / or a second domain, a linker, a third domain and / or a second domain, a linker, and a first domain light chain covalently linked together, the first polypeptide and the second polypeptide forming a specific polypeptide complex of a bi- and / or multi-functional fusion polypeptide; wherein the linkers can each independently be present or absent; in some cases, only one of the second domain or the third domain is present.

[0644] In another aspect, the first polypeptide of the polypeptide complex of the present application is a first domain heavy chain; the second polypeptide of the polypeptide complex of the present application comprises, in order from the N-terminus to the C-terminus of the polypeptide, a third domain and / or a second domain, a linker, a third domain and / or a second domain, a linker, and a first domain light chain covalently linked together, the first polypeptide and the second polypeptide forming a specific polypeptide complex of a bi- and / or multi-functional fusion polypeptide; wherein the linkers can each independently be present or absent; in some cases, only one of the second domain or the third domain is present.

[0645] In another aspect, the first polypeptide of the polypeptide complex of the present application is a first domain heavy chain; the second polypeptide of the polypeptide complex of the present application comprises, in order from the N-terminus to the C-terminus of the polypeptide, a third domain and / or a second domain, a linker, a third domain and / or a second domain, a linker, and a first domain light chain covalently linked together, the first polypeptide and the second polypeptide forming a specific polypeptide complex of a bi- and / or multi-functional fusion polypeptide; wherein the linkers can each independently be present or absent; in some cases, only one of the second domain or the third domain is present.

[0646] In another aspect, the polypeptide complex of the present application comprises only the structure of the first polypeptide, which comprises, in order from the N-terminus to the C-terminus of the polypeptide, a first domain heavy chain, a linker, a third domain and / or a second domain, the linker and the third domain and / or the second domain being covalently linked to form a specific polypeptide complex of a dual and / or multifunctional fusion polypeptide; wherein the linkers can each independently be present or absent; in some cases, only one of the second domain or the third domain is present.

[0647] In another aspect, the polypeptide complex of the present application comprises only the structure of the first polypeptide, which comprises, in order from the N-terminus to the C-terminus of the polypeptide, a first domain heavy chain, a linker, a third domain and / or a second domain, the linker and the third domain and / or the second domain being covalently linked to form a specific polypeptide complex of a dual and / or multifunctional fusion polypeptide; wherein the linkers can each independently be present or absent; in some cases, only one of the second domain or the third domain is present.

[0648] In another aspect, the polypeptide complex of the present application comprises only the structure of the first polypeptide, which comprises, in order from the N-terminus to the C-terminus of the polypeptide, a first domain heavy chain, a linker, a third domain and / or a second domain, the linker and the third domain and / or the second domain being covalently linked to form a specific polypeptide complex of a dual and / or multifunctional fusion polypeptide; wherein the linkers can each independently be present or absent; in some cases, only one of the second domain or the third domain is present.

[0649] The (a) antigen binding fragment contained in the first domain of the dual and / or multifunctional fusion polypeptide of the present application can be derived from any anti-PD-L1 and / or anti-PD-1 antibody, including any antibody capable of blocking or reducing the binding of PD-L1 to its receptor, thereby relieving the PD-L1-mediated immunosuppressive effect, including any anti-PD-L1 and / or anti-PD-1 antibody that has been published so far and any anti-PD-L1 and / or anti-PD-1 antibody to be developed in the future.

[0650] The second domain of the dual and / or multi-functional fusion polypeptide comprises an extracellular region of one and / or more ligands, at least including a CD86 extracellular domain or a polypeptide composed of IgV region of CD86 extracellular domain. For example, CD86 is a member of the B7 family of proteins that costimulate activation of T cells. A mature CD86 molecule is composed of an extracellular domain (ECD), a transmembrane domain, and an intracellular domain. The extracellular domain (ECD) is the key region of the molecule that binds to the corresponding receptor on T cells. The CD86 extracellular domain comprises an immunoglobulin-like V (IgV) region and an immunoglobulin-like C2 (IgC2) region. The IgV domain is the key domain that directly participates in the binding of its receptor. The CD86 extracellular domain IgV can bind to CD28 to induce T cell activation, proliferation, and effector function. CTLA4 can also bind to the IgV region of CD86 to participate in immune suppression regulation.

[0651] The third domain of the dual and / or multi-functional fusion polypeptide comprises an extracellular region of one and / or more ligands, at least including a LAG3 extracellular domain or one or more subunits of a polypeptide composed of IgD1, IgD2, IgD3, and IgD4 of LAG3 extracellular domain. For example, LAG3 is a type 1 transmembrane protein in the immune checkpoint and is mainly expressed on activated NK cells and T cells. LAG3 is expressed on tumor-exhausted CD8+ T cells and can be an effective target for tumor immunotherapy. For example, LAG3 has a dual function. On the one hand, LAG3 can negatively regulate T cell proliferation and activation. After LAG3 binds to its ligand, major histocompatibility complex II (MHCII) or ligand fibrinogen-like protein 1 (FGL1), it can inhibit the activation and function of T cells. On the other hand, LAG3 can bind to MHCII molecules on antigen-presenting cells to induce the activation of antigen-presenting cells, such as up-regulating the expression of CD80 / CD83 / CD86, enhancing antigen presentation, and secreting chemokines to recruit T cells, thereby assisting T cell activation. Antibodies that antagonize LAG3 can directly relieve LAG3-mediated T cell inhibition, but also block LAG3-mediated activation of antigen-presenting cells, so they cannot achieve overall enhancement of LAG3-related immune activation effects. The LAG3 extracellular domain chimeric protein provided in the present application can simultaneously achieve the effects of relieving T cell immune suppression and activating antigen-presenting cells, and has potential prospects for tumor treatment.

[0652] Provided below are exemplary first polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of the anti-PD-L1 antibody Sugemalimab covalently linked to a CD86 extracellular domain via an Fc domain linkage linker (SEQ ID NO: 36), the first polypeptide comprising the bifunctional fusion polypeptide complex with a second polypeptide of the light chain of Sugemalimab (SEQ ID NO: 5); first polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of the anti-PD-L1 antibody Sugemalimab covalently linked to an IgV region of a CD86 extracellular domain via an Fc domain linkage linker (SEQ ID NO: 37), the first polypeptide comprising the bifunctional antibody polypeptide complex with a second polypeptide of the light chain of Sugemalimab; first polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of the anti-PD-L1 antibody Sugemalimab covalently linked to a CD86 extracellular domain via an antigen binding region heavy chain N-terminal linkage linker (SEQ ID NO: 38), the first polypeptide comprising the bifunctional fusion polypeptide complex with a second polypeptide of the light chain of Sugemalimab; first polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of the anti-PD-L1 antibody Sugemalimab covalently linked to an IgV region of a CD86 extracellular domain via an antigen binding region heavy chain N-terminal linkage linker (SEQ ID NO: 39), the first polypeptide comprising the bifunctional fusion polypeptide complex with a second polypeptide of the light chain of Sugemalimab; second polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of the anti-PD-L1 antibody Sugemalimab covalently linked to a CD86 extracellular domain via an antigen binding region light chain C-terminal linkage linker (SEQ ID NO: 40), the second polypeptide comprising the bifunctional fusion polypeptide complex with a first polypeptide of the heavy chain of Sugemalimab (SEQ ID NO: 4); second polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of the anti-PD-L1 antibody Sugemalimab covalently linked to an IgV region of a CD86 extracellular domain via an antigen binding region light chain C-terminal linkage linker (SEQ ID NO: 41), the second polypeptide comprising the bifunctional fusion polypeptide complex with a first polypeptide of the heavy chain of Sugemalimab; the second polypeptide comprising the bifunctional antibody polypeptide complex with a first polypeptide of the heavy chain of Sugemalimab; second polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of the anti-PD-L1 antibody Sugemalimab covalently linked to a CD86 extracellular domain via an antigen binding region light chain N-terminal linkage linker (SEQ ID NO: 42), the second polypeptide comprising the bifunctional fusion polypeptide complex with a first polypeptide of the heavy chain of Sugemalimab.Sugemalimab is covalently linked to the second polypeptide amino acid sequence of a bifunctional fusion polypeptide complex (SEQ ID NO: 43) via an antigen binding region light chain N- linked linker, the second polypeptide of which comprises the extracellular domain IgV region of CD86, and the first polypeptide of which comprises the heavy chain of Sugemalimab.

[0653] Provided below are the first polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of the exemplary anti-PD-Ll antibody Durvalumab covalently linked to the CD86 extracellular domain via an Fc domain linkage linker (SEQ ID NO:44), the first polypeptide comprising the bifunctional fusion polypeptide complex with a Durvalumab light chain second polypeptide (SEQ ID NO:9); the first polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of Durvalumab covalently linked to the CD86 extracellular domain IgV region via an Fc domain linkage linker (SEQ ID NO:45), the first polypeptide comprising the bifunctional antibody polypeptide complex with a Durvalumab light chain second polypeptide; the first polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of Durvalumab covalently linked to the CD86 extracellular domain via an antigen binding region heavy chain N-terminal linkage linker (SEQ ID NO:46), the first polypeptide comprising the bifunctional fusion polypeptide complex with a Durvalumab light chain second polypeptide; the first polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of Durvalumab covalently linked to the CD86 extracellular domain IgV region via an antigen binding region heavy chain N-terminal linkage linker (SEQ ID NO:47), the first polypeptide comprising the bifunctional fusion polypeptide complex with a Durvalumab light chain second polypeptide; the second polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of Durvalumab covalently linked to the CD86 extracellular domain via an antigen binding region light chain C-terminal linkage linker (SEQ ID NO:48), the second polypeptide comprising the bifunctional fusion polypeptide complex with a Durvalumab heavy chain first polypeptide (SEQ ID NO:8); the second polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of Durvalumab covalently linked to the CD86 extracellular domain IgV region via an antigen binding region light chain C-terminal linkage linker (SEQ ID NO:49), the second polypeptide comprising the bifunctional fusion polypeptide complex with a Durvalumab heavy chain first polypeptide; the second polypeptide comprising the bifunctional antibody polypeptide complex with a Durvalumab heavy chain first polypeptide; the second polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of Durvalumab covalently linked to the CD86 extracellular domain via an antigen binding region light chain N-terminal linkage linker (SEQ ID NO:50), the second polypeptide comprising the bifunctional fusion polypeptide complex with a Durvalumab heavy chain first polypeptide; the second polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of Durvalumab covalently linked to the CD86 extracellular domain IgV region via an antigen binding region light chain N-terminal linkage linker (SEQ ID NO:51), the second polypeptide comprising the bifunctional fusion polypeptide complex with a Durvalumab heavy chain first polypeptide.

[0654] Provided below are exemplary bifunctional fusion polypeptide complex first polypeptide amino acid sequences for an anti-PD-Ll antibody Atezolizumab covalently linked to a CD86 extracellular domain via an Fc domain linkage linker (SEQ ID NO: 52), said first polypeptide comprising said bifunctional fusion polypeptide complex with a Atezolizumab light chain second polypeptide (SEQ ID NO: 11); an anti-PD-Ll antibody Atezolizumab covalently linked to a CD86 extracellular domain IgV region via an Fc domain linkage linker first polypeptide amino acid sequence (SEQ ID NO: 53), said first polypeptide comprising said bifunctional antibody polypeptide complex with a Atezolizumab light chain second polypeptide; an anti-PD-Ll antibody Atezolizumab covalently linked to a CD86 extracellular domain via an antigen binding region heavy chain N-terminal linkage linker first polypeptide amino acid sequence (SEQ ID NO: 54), said first polypeptide comprising said bifunctional fusion polypeptide complex with a Atezolizumab light chain second polypeptide; an anti-PD-Ll antibody Atezolizumab covalently linked to a CD86 extracellular domain IgV region via an antigen binding region heavy chain N-terminal linkage linker first polypeptide amino acid sequence (SEQ ID NO: 55), said first polypeptide comprising said bifunctional fusion polypeptide complex with a Atezolizumab light chain second polypeptide; an anti-PD-Ll antibody Atezolizumab covalently linked to a CD86 extracellular domain via an antigen binding region light chain C-terminal linkage linker second polypeptide amino acid sequence (SEQ ID NO: 56), said second polypeptide comprising said bifunctional fusion polypeptide complex with a Atezolizumab heavy chain first polypeptide (SEQ ID NO: 10); an anti-PD-Ll antibody Atezolizumab covalently linked to a CD86 extracellular domain IgV region via an antigen binding region light chain C-terminal linkage linker second polypeptide amino acid sequence (SEQ ID NO: 57), said second polypeptide comprising said bifunctional fusion polypeptide complex with a Atezolizumab heavy chain first polypeptide; said second polypeptide comprising said bifunctional antibody polypeptide complex with a Atezolizumab heavy chain first polypeptide; an anti-PD-Ll antibody Atezolizumab covalently linked to a CD86 extracellular domain via an antigen binding region light chain N-terminal linkage linker second polypeptide amino acid sequence (SEQ ID NO: 58), said second polypeptide comprising said bifunctional fusion polypeptide complex with a Atezolizumab heavy chain first polypeptide.Atezolizumab is covalently linked through an antigen binding region light chain N- linked linker to the second polypeptide amino acid sequence of a bifunctional fusion polypeptide complex of the extracellular domain IgV region of CD86 (SEQ ID NO: 59), which second polypeptide constitutes the bifunctional fusion polypeptide complex with the first polypeptide of the Atezolizumab heavy chain.

[0655] The following provides a first polypeptide amino acid sequence of a bifunctional fusion polypeptide complex of an exemplary anti-PD-Ll antibody Avelumab covalently linked to a CD86 extracellular domain via a Fc domain linkage linker (SEQ ID NO: 60), which first polypeptide comprises the bifunctional fusion polypeptide complex with a second polypeptide of the Avelumab light chain (SEQ ID NO: 13); a first polypeptide amino acid sequence of a bifunctional fusion polypeptide complex of Avelumab covalently linked to a CD86 extracellular domain IgV region via a Fc domain linkage linker (SEQ ID NO: 61), which first polypeptide comprises the bifunctional antibody polypeptide complex with a second polypeptide of the Avelumab light chain; a first polypeptide amino acid sequence of a bifunctional fusion polypeptide complex of Avelumab covalently linked to a CD86 extracellular domain via an antigen binding region heavy chain N-terminal linkage linker (SEQ ID NO: 62), which first polypeptide comprises the bifunctional fusion polypeptide complex with a second polypeptide of the Avelumab light chain; a first polypeptide amino acid sequence of a bifunctional fusion polypeptide complex of Avelumab covalently linked to a CD86 extracellular domain IgV region via an antigen binding region heavy chain N-terminal linkage linker (SEQ ID NO: 63), which first polypeptide comprises the bifunctional fusion polypeptide complex with a second polypeptide of the Avelumab light chain; a second polypeptide amino acid sequence of a bifunctional fusion polypeptide complex of Avelumab covalently linked to a CD86 extracellular domain via an antigen binding region light chain C-terminal linkage linker (SEQ ID NO: 64), which second polypeptide comprises the bifunctional fusion polypeptide complex with a first polypeptide of the Avelumab heavy chain (SEQ ID NO: 12); a second polypeptide amino acid sequence of a bifunctional fusion polypeptide complex of Avelumab covalently linked to a CD86 extracellular domain IgV region via an antigen binding region light chain C-terminal linkage linker (SEQ ID NO: 65), which second polypeptide comprises the bifunctional fusion polypeptide complex with a first polypeptide of the Avelumab heavy chain; which second polypeptide comprises the bifunctional antibody polypeptide complex with a first polypeptide of the Avelumab heavy chain; a second polypeptide amino acid sequence of a bifunctional fusion polypeptide complex of Avelumab covalently linked to a CD86 extracellular domain via an antigen binding region light chain N-terminal linkage linker (SEQ ID NO: 66), which second polypeptide comprises the bifunctional fusion polypeptide complex with a first polypeptide of the Avelumab heavy chain; a second polypeptide amino acid sequence of a bifunctional fusion polypeptide complex of Avelumab covalently linked to a CD86 extracellular domain IgV region via an antigen binding region light chain N-terminal linkage linker (SEQ ID NO: 67), which second polypeptide comprises the bifunctional fusion polypeptide complex with a first polypeptide of the Avelumab heavy chain.

[0656] The following provides an exemplary bifunctional fusion polypeptide complex first polypeptide amino acid sequence of Envafolimab covalently linked to a CD86 extracellular domain via a Fc domain linkage linker (SEQ ID NO: 68), the first polypeptide comprising the bifunctional fusion polypeptide complex; Envafolimab covalently linked to a CD86 extracellular domain IgV region via a Fc domain linkage linker first polypeptide amino acid sequence (SEQ ID NO: 69), the first polypeptide comprising the bifunctional antibody polypeptide complex; Envafolimab covalently linked to a CD86 extracellular domain via an antigen binding region heavy chain N-terminal linkage linker first polypeptide amino acid sequence (SEQ ID NO: 70), the first polypeptide comprising the bifunctional fusion polypeptide complex; Envafolimab covalently linked to a CD86 extracellular domain IgV region via an antigen binding region heavy chain N-terminal linkage linker first polypeptide amino acid sequence (SEQ ID NO: 71), the first polypeptide comprising the bifunctional fusion polypeptide complex;

[0657] Provided below are the first polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of the exemplary anti-PD-1 antibody Permbrolizumab covalently linked to the CD86 extracellular domain via an Fc domain linkage linker (SEQ ID NO: 72), the first polypeptide comprising the bifunctional fusion polypeptide complex with a Permbrolizumab light chain second polypeptide (SEQ ID NO: 16); the first polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of the exemplary anti-PD-1 antibody Permbrolizumab covalently linked to the CD86 extracellular domain IgV region via an Fc domain linkage linker (SEQ ID NO: 73), the first polypeptide comprising the bifunctional antibody polypeptide complex with a Permbrolizumab light chain second polypeptide; the first polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of the exemplary anti-PD-1 antibody Permbrolizumab covalently linked to the CD86 extracellular domain via an antigen binding region heavy chain N-terminal linkage linker (SEQ ID NO: 74), the first polypeptide comprising the bifunctional fusion polypeptide complex with a Permbrolizumab light chain second polypeptide; the first polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of the exemplary anti-PD-1 antibody Permbrolizumab covalently linked to the CD86 extracellular domain IgV region via an antigen binding region heavy chain N-terminal linkage linker (SEQ ID NO: 75), the first polypeptide comprising the bifunctional fusion polypeptide complex with a Permbrolizumab light chain second polypeptide; the second polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of the exemplary anti-PD-1 antibody Permbrolizumab covalently linked to the CD86 extracellular domain via an antigen binding region light chain C-terminal linkage linker (SEQ ID NO: 76), the second polypeptide comprising the bifunctional fusion polypeptide complex with a Permbrolizumab heavy chain first polypeptide (SEQ ID NO: 15); the second polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of the exemplary anti-PD-1 antibody Permbrolizumab covalently linked to the CD86 extracellular domain IgV region via an antigen binding region light chain C-terminal linkage linker (SEQ ID NO: 77), the second polypeptide comprising the bifunctional fusion polypeptide complex with a Permbrolizumab heavy chain first polypeptide; the second polypeptide comprising the bifunctional antibody polypeptide complex with a Permbrolizumab heavy chain first polypeptide; the second polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of the exemplary anti-PD-1 antibody Permbrolizumab covalently linked to the CD86 extracellular domain via an antigen binding region light chain N-terminal linkage linker (SEQ ID NO: 78), the second polypeptide comprising the bifunctional fusion polypeptide complex with a Permbrolizumab heavy chain first polypeptide.Permbrolizumab is covalently linked via an antigen binding region light chain N- linked linker to the second polypeptide amino acid sequence of a bifunctional fusion polypeptide complex of the extracellular domain IgV region of CD86 (SEQ ID NO: 79), which second polypeptide is comprised with the first polypeptide of the heavy chain of Permbrolizumab to form the bifunctional fusion polypeptide complex.

[0658] Provided below are the first polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of the exemplary anti-PD-1 antibody Nivolumab covalently linked to the CD86 extracellular domain via an Fc domain linkage linker (SEQ ID NO: 80), the first polypeptide comprising the bifunctional fusion polypeptide complex with a Nivolumab light chain second polypeptide (SEQ ID NO: 18); the first polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of Nivolumab covalently linked to the CD86 extracellular domain IgV region via an Fc domain linkage linker (SEQ ID NO: 81), the first polypeptide comprising the bifunctional antibody polypeptide complex with a Nivolumab light chain second polypeptide; the first polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of Nivolumab covalently linked to the CD86 extracellular domain via an antigen binding region heavy chain N-terminal linkage linker (SEQ ID NO: 82), the first polypeptide comprising the bifunctional fusion polypeptide complex with a Nivolumab light chain second polypeptide; the first polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of Nivolumab covalently linked to the CD86 extracellular domain IgV region via an antigen binding region heavy chain N-terminal linkage linker (SEQ ID NO: 83), the first polypeptide comprising the bifunctional fusion polypeptide complex with a Nivolumab light chain second polypeptide; the second polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of Nivolumab covalently linked to the CD86 extracellular domain via an antigen binding region light chain C-terminal linkage linker (SEQ ID NO: 84), the second polypeptide comprising the bifunctional fusion polypeptide complex with a Nivolumab heavy chain first polypeptide (SEQ ID NO: 17); the second polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of Nivolumab covalently linked to the CD86 extracellular domain IgV region via an antigen binding region light chain C-terminal linkage linker (SEQ ID NO: 85), the second polypeptide comprising the bifunctional fusion polypeptide complex with a Nivolumab heavy chain first polypeptide; the second polypeptide comprising the bifunctional antibody polypeptide complex with a Nivolumab heavy chain first polypeptide; the second polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of Nivolumab covalently linked to the CD86 extracellular domain via an antigen binding region light chain N linkage linker (SEQ ID NO: 86), the second polypeptide comprising the bifunctional fusion polypeptide complex with a Nivolumab heavy chain first polypeptide; the second polypeptide amino acid sequences of bifunctional fusion polypeptide complexes of Nivolumab covalently linked to the CD86 extracellular domain IgV region via an antigen binding region light chain N linkage linker (SEQ ID NO: 87), the second polypeptide comprising the bifunctional fusion polypeptide complex with a Nivolumab heavy chain first polypeptide.

[0659] Provided below are exemplary first polypeptide amino acid sequences of a multifunctional fusion polypeptide complex of a CD86 extracellular domain covalently linked to a LAG3 extracellular domain via a C-terminal linker to the Fc domain of a heavy chain of Sugemalimab, the first polypeptide covalently linked to a second polypeptide of a light chain of Sugemalimab to form the multifunctional fusion polypeptide complex; a CD86 extracellular domain covalently linked to a LAG3 extracellular domain D1 / D2 / D3 via a C-terminal linker to the Fc domain of a heavy chain of Sugemalimab, the first polypeptide covalently linked to a second polypeptide of a light chain of Sugemalimab to form the multifunctional fusion polypeptide complex; a CD86 extracellular domain covalently linked to a LAG3 extracellular domain D1 / D2 via a C-terminal linker to the Fc domain of a heavy chain of Sugemalimab, the first polypeptide covalently linked to a second polypeptide of a light chain of Sugemalimab to form the multifunctional fusion polypeptide complex; a CD86 extracellular domain covalently linked to a LAG3 extracellular domain D1 via a C-terminal linker to the Fc domain of a heavy chain of Sugemalimab, the first polypeptide covalently linked to a second polypeptide of a light chain of Sugemalimab to form the multifunctional fusion polypeptide complex; a LAG3 extracellular domain covalently linked to a CD86 extracellular domain via a C-terminal linker to the Fc domain of a heavy chain of Sugemalimab, the first polypeptide covalently linked to a second polypeptide of a light chain of Sugemalimab to form the multifunctional fusion polypeptide complex; the first polypeptide covalently linked to a second polypeptide of a light chain of Sugemalimab to form the multifunctional fusion polypeptide complex; a LAG3 extracellular domain D1 / D2 / D3 covalently linked to a CD86 extracellular domain via a C-terminal linker to the Fc domain of a heavy chain of Sugemalimab, the first polypeptide covalently linked to a second polypeptide of a light chain of Sugemalimab to form the multifunctional fusion polypeptide complex;The first polypeptide and the second polypeptide of the Sugemalimab light chain form the multifunctional fusion polypeptide complex; the Sugemalimab heavy chain is covalently linked to the LAG3 extracellular domains D1 / D2 via a linker at the C-terminus of the Fc domain, and the LAG3 extracellular domains D1 / D2 are covalently linked to the CD86 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 form the multifunctional fusion polypeptide complex; the first polypeptide and the second polypeptide of the Sugemalimab light chain form the multifunctional fusion polypeptide complex; the Sugemalimab heavy chain is covalently linked to the LAG3 extracellular domain D1 via a linker at the C-terminus of the Fc domain, and the LAG3 extracellular domain D1 is covalently linked to the CD86 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 form the multifunctional fusion polypeptide complex.

[0660] The following provides an exemplary anti-PD-L1 antibody, Sugemalimab, in which the heavy chain is covalently linked to the CD86 extracellular domain IgV region via a linker at the C-terminus of the Fc domain, and the CD86 extracellular domain IgV region is covalently linked to the LAG3 extracellular domain via a linker at the C-terminus of the LAG3 extracellular domain. The first polypeptide amino acid sequence (SEQ ID NO: 96) and the second polypeptide of the Sugemalimab light chain constitute the multifunctional fusion polypeptide complex. Alternatively, a multifunctional fusion polypeptide complex can be further described, in which the Sugemalimab heavy chain is covalently linked to the CD86 extracellular domain IgV region via a linker at the C-terminus of the Fc domain, and the CD86 extracellular domain IgV region is covalently linked to the LAG3 extracellular domains D1 / D2 / D3 via a linker at the C-terminus of the LAG3 extracellular domain (SEQ ID NO: 96). NO: 97), the first polypeptide and the second polypeptide of the Sugemalimab light chain form the multifunctional fusion polypeptide complex; the Sugemalimab heavy chain is covalently linked to the CD86 extracellular domain IgV region via a linker at the C-terminus of the Fc domain, and the C-terminus of the CD86 extracellular domain IgV region is covalently linked to the LAG3 extracellular domain D1 / D2. The first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex (SEQ ID NO: 98) is also mentioned. NO: 99), the first polypeptide and the second polypeptide of the Sugemalimab light chain form 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 IgV region via a linker. The amino acid sequence of the first polypeptide of the multifunctional fusion polypeptide complex (SEQ ID NO: 100), the first polypeptide and the second polypeptide of the Sugemalimab light chain form the multifunctional fusion polypeptide complex; the first polypeptide and the second polypeptide of the Sugemalimab light chain form the multifunctional fusion polypeptide complex.Sugemalimab heavy chain covalently linked to LAG3 extracellular domain D1 / D2 / D3 via a linker at the C-terminus of the Fc domain, the LAG3 extracellular domain D1 / D2 / D3 covalently linked to a first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the CD86 extracellular domain IgV region via a linker at the C-terminus of the LAG3 extracellular domain D1 / D2 / D3, the first polypeptide covalently linked to a second polypeptide of a Sugemalimab light chain to form the multifunctional fusion polypeptide complex; the first polypeptide covalently linked to a second polypeptide of a Sugemalimab light chain to form the multifunctional fusion polypeptide complex; Sugemalimab heavy chain covalently linked to LAG3 extracellular domain D1 / D2 via a linker at the C-terminus of the Fc domain, the LAG3 extracellular domain D1 / D2 covalently linked to a first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the CD86 extracellular domain IgV region via a linker at the C-terminus of the LAG3 extracellular domain D1 / D2, the first polypeptide covalently linked to a second polypeptide of a Sugemalimab light chain to form the multifunctional fusion polypeptide complex; the first polypeptide covalently linked to a second polypeptide of a Sugemalimab light chain to form the multifunctional fusion polypeptide complex; Sugemalimab heavy chain covalently linked to LAG3 extracellular domain D1 via a linker at the C-terminus of the Fc domain, the LAG3 extracellular domain D1 covalently linked to a first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the CD86 extracellular domain IgV region via a linker at the C-terminus of the LAG3 extracellular domain D1, the first polypeptide covalently linked to a second polypeptide of a Sugemalimab light chain to form the multifunctional fusion polypeptide complex.

[0661] Provided below are first polypeptide amino acid sequences of a multifunctional fusion polypeptide complex of a CD86 ectodomain C-terminal linking linker covalently linked to a heavy chain of an anti-PD-L1 antibody Sugemalimab, a Sugemalimab C-terminal linking linker covalently linked to a LAG3 ectodomain, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a light chain of Sugemalimab; a CD86 ectodomain C-terminal linking linker covalently linked to a heavy chain of an anti-PD-L1 antibody Sugemalimab, a Sugemalimab heavy chain C-terminal linking linker covalently linked to a LAG3 ectodomain D1 / D2 / D3, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a light chain of Sugemalimab; a CD86 ectodomain C-terminal linking linker covalently linked to a heavy chain of an anti-PD-L1 antibody Sugemalimab, a Sugemalimab heavy chain C-terminal linking linker covalently linked to a LAG3 ectodomain D1 / D2, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a light chain of Sugemalimab; a CD86 ectodomain C-terminal linking linker covalently linked to a heavy chain of an anti-PD-L1 antibody Sugemalimab, a Sugemalimab heavy chain C-terminal linking linker covalently linked to a LAG3 ectodomain D1, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a light chain of Sugemalimab; a LAG3 ectodomain C-terminal linking linker covalently linked to a heavy chain of an anti-PD-L1 antibody Sugemalimab, a Sugemalimab C-terminal linking linker covalently linked to a CD86 ectodomain, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a light chain of Sugemalimab; a LAG3 ectodomain D1 / D2 / D3 C-terminal linking linker covalently linked to a heavy chain of an anti-PD-L1 antibody Sugemalimab, a Sugemalimab C-terminal linking linker covalently linked to a CD86 ectodomain, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a light chain of Sugemalimab;the first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the LAG3 ectodomain D1 / D2 C-terminal junction linker covalently linked to an anti-PD-L1 antibody sugemalimab heavy chain, a sugemalimab C-terminal junction linker covalently linked to a CD86 ectodomain (SEQ ID NO: 110), the first polypeptide comprising the multifunctional fusion polypeptide complex with a sugemalimab light chain second polypeptide; the first polypeptide comprising the multifunctional fusion polypeptide complex with a sugemalimab light chain second polypeptide; the first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the LAG3 ectodomain D1 C-terminal junction linker covalently linked to an anti-PD-L1 antibody sugemalimab heavy chain, a sugemalimab C-terminal junction linker covalently linked to a CD86 ectodomain (SEQ ID NO: 111), the first polypeptide comprising the multifunctional fusion polypeptide complex with a sugemalimab light chain second polypeptide.

[0662] Provided below are exemplary first polypeptide amino acid sequences of a multifunctional fusion polypeptide complex of a CD86 ectodomain IgV region C-terminal linking linker covalently linked to an anti-PD-L1 antibody Sugemalimab heavy chain, a Sugemalimab C-terminal linking linker covalently linked to a LAG3 ectodomain, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a Sugemalimab light chain; a CD86 ectodomain IgV region C-terminal linking linker covalently linked to an anti-PD-L1 antibody Sugemalimab heavy chain, a Sugemalimab heavy chain C-terminal linking linker covalently linked to a LAG3 ectodomain D1 / D2 / D3, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a Sugemalimab light chain; a CD86 ectodomain IgV region C-terminal linking linker covalently linked to an anti-PD-L1 antibody Sugemalimab heavy chain, a Sugemalimab heavy chain C-terminal linking linker covalently linked to a LAG3 ectodomain D1 / D2, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a Sugemalimab light chain; a CD86 ectodomain IgV region C-terminal linking linker covalently linked to an anti-PD-L1 antibody Sugemalimab heavy chain, a Sugemalimab heavy chain C-terminal linking linker covalently linked to a LAG3 ectodomain D1, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a Sugemalimab light chain; a LAG3 ectodomain C-terminal linking linker covalently linked to an anti-PD-L1 antibody Sugemalimab heavy chain, a Sugemalimab C-terminal linking linker covalently linked to a CD86 ectodomain IgV region, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a Sugemalimab light chain; a LAG3 ectodomain D1 / D2 / D3 C-terminal linking linker covalently linked to an anti-PD-L1 antibody Sugemalimab heavy chain, a Sugemalimab C-terminal linking linker covalently linked to a CD86 ectodomain IgV region, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a Sugemalimab light chain;the first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of a LAG3 ectodomain D1 / D2 C-terminal junction linker covalently linked to an anti-PD-L1 antibody sugemalimab heavy chain, a sugemalimab C-terminal junction linker covalently linked to a CD86 ectodomain IgV domain, the first polypeptide comprising the multifunctional fusion polypeptide complex with a sugemalimab light chain second polypeptide; the first polypeptide comprising the multifunctional fusion polypeptide complex with a sugemalimab light chain second polypeptide; the first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of a LAG3 ectodomain D1 C-terminal junction linker covalently linked to an anti-PD-L1 antibody sugemalimab heavy chain, a sugemalimab C-terminal junction linker covalently linked to a CD86 ectodomain IgV domain, the first polypeptide comprising the multifunctional fusion polypeptide complex with a sugemalimab light chain second polypeptide.

[0663] Provided below are first polypeptide amino acid sequences of multifunctional fusion polypeptide complexes in which a CD86 ectodomain is covalently linked to a LAG3 ectodomain, the first polypeptide covalently linked to a second polypeptide of a light chain of an anti-PD-L1 antibody, e.g., a light chain of Sugemalimab; a CD86 ectodomain is covalently linked to a LAG3 ectodomain D1 / D2 / D3, the first polypeptide covalently linked to a second polypeptide of a light chain of an anti-PD-L1 antibody, e.g., a light chain of Sugemalimab; a CD86 ectodomain is covalently linked to a LAG3 ectodomain D1 / D2, the first polypeptide covalently linked to a second polypeptide of a light chain of an anti-PD-L1 antibody, e.g., a light chain of Sugemalimab; a CD86 ectodomain is covalently linked to a LAG3 ectodomain D1, the first polypeptide covalently linked to a second polypeptide of a light chain of an anti-PD-L1 antibody, e.g., a light chain of Sugemalimab; a LAG3 ectodomain is covalently linked to a CD86 ectodomain, the first polypeptide covalently linked to a second polypeptide of a light chain of an anti-PD-L1 antibody, e.g., a light chain of Sugemalimab; a LAG3 ectodomain D1 / D2 / D3 is covalently linked to a CD86 ectodomain, the first polypeptide covalently linked to a second polypeptide of a light chain of an anti-PD-L1 antibody, e.g., a light chain of Sugemalimab.a first polypeptide amino acid sequence of a multifunctional fusion polypeptide complex of LAG3 ectodomain D1 / D2 C-terminal linker linker covalently linked to CD86 ectodomain, CD86 ectodomain C-terminal linker linker covalently linked to an anti-PD-L1 antibody Sugemalimab heavy chain, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a Sugemalimab light chain; a first polypeptide amino acid sequence of a multifunctional fusion polypeptide complex of LAG3 ectodomain D1 C-terminal linker linker covalently linked to CD86 ectodomain, CD86 ectodomain C-terminal linker linker covalently linked to an anti-PD-L1 antibody Sugemalimab heavy chain, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a Sugemalimab light chain;

[0664] Provided below are first polypeptide amino acid sequences of multifunctional fusion polypeptide complexes in which a CD86 ectodomain IgV region is covalently linked to a LAG3 ectodomain C-terminal junction linker is covalently linked to an anti-PD-L1 antibody Sugemalimab heavy chain, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a Sugemalimab light chain; a CD86 ectodomain IgV region is covalently linked to a LAG3 ectodomain D1 / D2 / D3 C-terminal junction linker is covalently linked to an anti-PD-L1 antibody Sugemalimab heavy chain, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a Sugemalimab light chain; a CD86 ectodomain IgV region is covalently linked to a LAG3 ectodomain D1 / D2 C-terminal junction linker is covalently linked to an anti-PD-L1 antibody Sugemalimab heavy chain, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a Sugemalimab light chain; a CD86 ectodomain IgV region is covalently linked to a LAG3 ectodomain D1 C-terminal junction linker is covalently linked to an anti-PD-L1 antibody Sugemalimab heavy chain, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a Sugemalimab light chain; a LAG3 ectodomain is covalently linked to a CD86 ectodomain IgV region C-terminal junction linker is covalently linked to an anti-PD-L1 antibody Sugemalimab heavy chain, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a Sugemalimab light chain; a LAG3 ectodomain D1 / D2 / D3 is covalently linked to a CD86 ectodomain IgV region C-terminal junction linker is covalently linked to an anti-PD-L1 antibody Sugemalimab heavy chain, the first polypeptide comprising the multifunctional fusion polypeptide complex with a second polypeptide of a Sugemalimab light chain;The LAG3 extracellular domain D1 / D2 C-terminal linker covalently connects to the CD86 extracellular domain IgV region, and the CD86 extracellular domain IgV region C-terminal linker covalently connects to the first polypeptide amino acid sequence (SEQ ID NO: 134) of a multifunctional fusion polypeptide complex containing the anti-PD-L1 antibody Sugemalimab heavy chain. The first polypeptide and the Sugemalimab light chain second polypeptide constitute the multifunctional fusion polypeptide complex. Alternatively, the LAG3 extracellular domain D1 C-terminal linker covalently connects to the CD86 extracellular domain IgV region, and the CD86 extracellular domain IgV region C-terminal linker covalently connects to the first polypeptide amino acid sequence (SEQ ID NO: 135) of a multifunctional fusion polypeptide complex containing the anti-PD-L1 antibody Sugemalimab heavy chain. The first polypeptide and the Sugemalimab light chain second polypeptide constitute the multifunctional fusion polypeptide complex.

[0665] The following provides an exemplary first polypeptide amino acid sequence (SEQ ID NO: 38) of a multifunctional fusion polypeptide complex in which a CD86 extracellular domain C-terminal linker is covalently linked to a second polypeptide of the Sugemalimab light chain (SEQ ID NO: 136) to form the multifunctional fusion polypeptide complex; a first polypeptide amino acid sequence (SEQ ID NO: 39) of a multifunctional fusion polypeptide complex in which a CD86 extracellular domain IgV region C-terminal linker is covalently linked to a second polypeptide of the Sugemalimab light chain (SEQ ID NO: 136) to form the multifunctional fusion polypeptide complex; and a first polypeptide amino acid sequence (SEQ ID NO: 39) of a multifunctional fusion polypeptide complex in which a CD86 extracellular domain IgV region C-terminal linker is covalently linked to a second polypeptide of the Sugemalimab light chain (SEQ ID NO: 136) to form the multifunctional fusion polypeptide complex; and a first polypeptide amino acid sequence (SEQ ID NO: 39) of a multifunctional fusion polypeptide complex in which a PD-L1 antibody Sugemalimab heavy chain C-terminal linker is covalently linked to a CD86 extracellular domain. NO: 40), the first polypeptide is covalently linked to the second polypeptide of the Sugemalimab light chain by the C-terminal linker of the LAG3 extracellular domain to form the multifunctional fusion polypeptide complex; the first polypeptide amino acid sequence of the multifunctional fusion polypeptide complex of the PD-L1 antibody Sugemalimab heavy chain C-terminal linker covalently linked to the IgV region of the CD86 extracellular domain (SEQ ID NO: 41), the first polypeptide is covalently linked to the second polypeptide of the Sugemalimab light chain by the C-terminal linker of the LAG3 extracellular domain to form the multifunctional fusion polypeptide complex.

[0666] Provided below are exemplary first polypeptide amino acid sequences of a multifunctional fusion polypeptide complex of a CD86 ectodomain C-terminal junction linker covalently linked to a heavy chain of an anti-PD-L1 antibody Sugemalimab, the first polypeptide covalently linked to a second polypeptide of a light chain of Sugemalimab to make the multifunctional fusion polypeptide complex; a CD86 ectodomain IgV region C-terminal junction linker covalently linked to a heavy chain of an anti-PD-L1 antibody Sugemalimab, the first polypeptide covalently linked to a second polypeptide of a light chain of Sugemalimab to make the multifunctional fusion polypeptide complex; a PD-L1 antibody Sugemalimab heavy chain C-terminal junction linker covalently linked to a CD86 ectodomain, the first polypeptide covalently linked to a second polypeptide of a light chain of Sugemalimab to make the multifunctional fusion polypeptide complex; a PD-L1 antibody Sugemalimab heavy chain C-terminal junction linker covalently linked to a CD86 ectodomain IgV region, the first polypeptide covalently linked to a second polypeptide of a light chain of Sugemalimab to make the multifunctional fusion polypeptide complex.

[0667] Provided below is an exemplary multifunctional fusion polypeptide complex first polypeptide amino acid sequence covalently linked to a LAG3 ectodomain D1 / D2 C-terminal junction linker covalently linked to a second polypeptide of a PD-L1 antibody Sugemalimab heavy chain consisting of the multifunctional fusion polypeptide complex; a multifunctional fusion polypeptide complex first polypeptide amino acid sequence covalently linked to a LAG3 ectodomain D1 / D2 C-terminal junction linker covalently linked to a second polypeptide of a PD-L1 antibody Sugemalimab heavy chain consisting of the multifunctional fusion polypeptide complex; a multifunctional fusion polypeptide complex first polypeptide amino acid sequence covalently linked to a LAG3 ectodomain D1 / D2 C-terminal junction linker covalently linked to a second polypeptide of a PD-L1 antibody Sugemalimab heavy chain consisting of the multifunctional fusion polypeptide complex; a multifunctional fusion polypeptide complex first polypeptide amino acid sequence covalently linked to a LAG3 ectodomain D1 / D2 C-terminal junction linker covalently linked to a second polypeptide of a PD-L1 antibody Sugemalimab heavy chain consisting of the multifunctional fusion polypeptide complex.

[0668] Provided below is an exemplary multifunctional fusion polypeptide complex first polypeptide amino acid sequence covalently linked to a LAG3 ectodomain D1 C-terminal junction linker covalently linked to a second polypeptide of a heavy chain of an anti-PD-L1 antibody Sugemalimab, the first polypeptide and the second polypeptide comprising the multifunctional fusion polypeptide complex; a multifunctional fusion polypeptide complex first polypeptide amino acid sequence covalently linked to a LAG3 ectodomain D1 C-terminal junction linker covalently linked to a second polypeptide of a heavy chain of an anti-PD-L1 antibody Sugemalimab, the first polypeptide and the second polypeptide comprising the multifunctional fusion polypeptide complex; a multifunctional fusion polypeptide complex first polypeptide amino acid sequence covalently linked to a LAG3 ectodomain D1 C-terminal junction linker covalently linked to a second polypeptide of a heavy chain of an anti-PD-L1 antibody Sugemalimab, the first polypeptide and the second polypeptide comprising the multifunctional fusion polypeptide complex; a multifunctional fusion polypeptide complex first polypeptide amino acid sequence covalently linked to a LAG3 ectodomain D1 C-terminal junction linker covalently linked to a second polypeptide of a heavy chain of an anti-PD-L1 antibody Sugemalimab, the first polypeptide and the second polypeptide comprising the multifunctional fusion polypeptide complex.

[0669] The following provides a first polypeptide amino acid sequence of a multifunctional fusion polypeptide complex of a CD86 extracellular domain C-terminal connecting linker covalently linked to a heavy chain of an anti-PD-L1 antibody Sugemalimab, the first polypeptide covalently linked to a second polypeptide of a LAG3 extracellular domain with a C-terminal connecting linker to a light chain of Sugemalimab, the multifunctional fusion polypeptide complex consisting of the first polypeptide and the second polypeptide; a first polypeptide amino acid sequence of a multifunctional fusion polypeptide complex of a CD86 extracellular domain IgV region C-terminal connecting linker covalently linked to a heavy chain of an anti-PD-L1 antibody Sugemalimab, the first polypeptide covalently linked to a second polypeptide of a LAG3 extracellular domain with a C-terminal connecting linker to a light chain of Sugemalimab, the multifunctional fusion polypeptide complex consisting of the first polypeptide and the second polypeptide; a first polypeptide amino acid sequence of a multifunctional fusion polypeptide complex of a PD-L1 antibody Sugemalimab heavy chain C-terminal connecting linker covalently linked to a CD86 extracellular domain, the first polypeptide covalently linked to a second polypeptide of a LAG3 extracellular domain with a C-terminal connecting linker to a light chain of Sugemalimab, the multifunctional fusion polypeptide complex consisting of the first polypeptide and the second polypeptide; a first polypeptide amino acid sequence of a multifunctional fusion polypeptide complex of a PD-L1 antibody Sugemalimab heavy chain C-terminal connecting linker covalently linked to a CD86 extracellular domain IgV region, the first polypeptide covalently linked to a second polypeptide of a LAG3 extracellular domain with a C-terminal connecting linker to a light chain of Sugemalimab, the multifunctional fusion polypeptide complex consisting of the first polypeptide and the second polypeptide.

[0670] Provided below are exemplary first polypeptide amino acid sequences of a multifunctional fusion polypeptide complex of a CD86 ectodomain C-terminal junction linker covalently linked to a heavy chain of an anti-PD-L1 antibody Sugemalimab, the first polypeptide covalently linked to a second polypeptide of a LAG3 ectodomain D1 / D2 / D3 with a C-terminal junction linker to a light chain of Sugemalimab to make the multifunctional fusion polypeptide complex; a CD86 ectodomain IgV region C-terminal junction linker covalently linked to a heavy chain of an anti-PD-L1 antibody Sugemalimab, the first polypeptide covalently linked to a second polypeptide of a LAG3 ectodomain D1 / D2 / D3 with a C-terminal junction linker to a light chain of Sugemalimab to make the multifunctional fusion polypeptide complex; a PD-L1 antibody Sugemalimab heavy chain C-terminal junction linker covalently linked to a CD86 ectodomain, the first polypeptide covalently linked to a second polypeptide of a LAG3 ectodomain D1 / D2 / D3 with a C-terminal junction linker to a light chain of Sugemalimab to make the multifunctional fusion polypeptide complex; a PD-L1 antibody Sugemalimab heavy chain C-terminal junction linker covalently linked to a CD86 ectodomain IgV region, the first polypeptide covalently linked to a second polypeptide of a LAG3 ectodomain D1 / D2 / D3 with a C-terminal junction linker to a light chain of Sugemalimab to make the multifunctional fusion polypeptide complex.

[0671] Provided below are exemplary first polypeptide amino acid sequences of a multifunctional fusion polypeptide complex of a CD86 ectodomain C-terminal junction linker covalently linked to a heavy chain of an anti-PD-L1 antibody Sugemalimab, the first polypeptide covalently linked to a second polypeptide of a LAG3 ectodomain D1 / D2 with a C-terminal junction linker to a light chain of Sugemalimab comprising the multifunctional fusion polypeptide complex; a CD86 ectodomain IgV region C-terminal junction linker covalently linked to a heavy chain of an anti-PD-L1 antibody Sugemalimab, the first polypeptide covalently linked to a second polypeptide of a LAG3 ectodomain D1 / D2 with a C-terminal junction linker to a light chain of Sugemalimab comprising the multifunctional fusion polypeptide complex; a PD-L1 antibody Sugemalimab heavy chain C-terminal junction linker covalently linked to a CD86 ectodomain, the first polypeptide covalently linked to a second polypeptide of a LAG3 ectodomain D1 / D2 with a C-terminal junction linker to a light chain of Sugemalimab comprising the multifunctional fusion polypeptide complex; a PD-L1 antibody Sugemalimab heavy chain C-terminal junction linker covalently linked to a CD86 ectodomain IgV region, the first polypeptide covalently linked to a second polypeptide of a LAG3 ectodomain D1 / D2 with a C-terminal junction linker to a light chain of Sugemalimab comprising the multifunctional fusion polypeptide complex.

[0672] Provided below are exemplary first polypeptide amino acid sequences of a multifunctional fusion polypeptide complex of a CD86 ectodomain C-terminal junction linker covalently linked to a heavy chain of an anti-PD-L1 antibody Sugemalimab, the first polypeptide covalently linked to a second polypeptide of a LAG3 ectodomain D1 with a C-terminal junction linker to a light chain of Sugemalimab comprising the multifunctional fusion polypeptide complex; a CD86 ectodomain IgV region C-terminal junction linker covalently linked to a heavy chain of an anti-PD-L1 antibody Sugemalimab, the first polypeptide covalently linked to a second polypeptide of a LAG3 ectodomain D1 with a C-terminal junction linker to a light chain of Sugemalimab comprising the multifunctional fusion polypeptide complex; a PD-L1 antibody Sugemalimab heavy chain C-terminal junction linker covalently linked to a CD86 ectodomain, the first polypeptide covalently linked to a second polypeptide of a LAG3 ectodomain D1 with a C-terminal junction linker to a light chain of Sugemalimab comprising the multifunctional fusion polypeptide complex; a PD-L1 antibody Sugemalimab heavy chain C-terminal junction linker covalently linked to a CD86 ectodomain IgV region, the first polypeptide covalently linked to a second polypeptide of a LAG3 ectodomain D1 with a C-terminal junction linker to a light chain of Sugemalimab comprising the multifunctional fusion polypeptide complex.

[0673] Provided below are exemplary multifunctional fusion polypeptide complex second polypeptide amino acid sequences covalently linked to a CD86 ectodomain C-terminal junction linker covalently linked to a LAG3 ectodomain C-terminal junction linker covalently linked to a first polypeptide of a heavy chain of an anti-PD-L1 antibody, Sugemalimab (SEQ ID NO: 42), which, together with a first polypeptide of a heavy chain of Sugemalimab (SEQ ID NO: 144), comprises the multifunctional fusion polypeptide complex; multifunctional fusion polypeptide complex second polypeptide amino acid sequences covalently linked to a CD86 ectodomain IgV region C-terminal junction linker covalently linked to a LAG3 ectodomain C-terminal junction linker covalently linked to a first polypeptide of a heavy chain of an anti-PD-L1 antibody, Sugemalimab (SEQ ID NO: 43), which, together with a first polypeptide of a heavy chain of Sugemalimab, comprises the multifunctional fusion polypeptide complex; multifunctional fusion polypeptide complex second polypeptide amino acid sequences covalently linked to a CD86 ectodomain C-terminal junction linker covalently linked to a LAG3 ectodomain C-terminal junction linker covalently linked to a first polypeptide of a heavy chain of an anti-PD-L1 antibody, Sugemalimab (SEQ ID NO: 40), which, together with a first polypeptide of a heavy chain of Sugemalimab, comprises the multifunctional fusion polypeptide complex; multifunctional fusion polypeptide complex second polypeptide amino acid sequences covalently linked to a CD86 ectodomain IgV region C-terminal junction linker covalently linked to a LAG3 ectodomain C-terminal junction linker covalently linked to a first polypeptide of a heavy chain of an anti-PD-L1 antibody, Sugemalimab (SEQ ID NO: 41), which, together with a first polypeptide of a heavy chain of Sugemalimab, comprises the multifunctional fusion polypeptide complex.

[0674] Provided below are exemplary multifunctional fusion polypeptide complex second polypeptide amino acid sequences of a CD86 ectodomain C-terminal junction linker covalently linked to an anti-PD-L1 antibody Sugemalimab light chain that, together with a LAG3 ectodomain D1 / D2 / D3 C-terminal junction linker covalently linked to a Sugemalimab heavy chain first polypeptide, make up the multifunctional fusion polypeptide complex; CD86 ectodomain IgV region C-terminal junction linker covalently linked to an anti-PD-L1 antibody Sugemalimab light chain that, together with a LAG3 ectodomain D1 / D2 / D3 C-terminal junction linker covalently linked to a Sugemalimab heavy chain first polypeptide, make up the multifunctional fusion polypeptide complex; anti-PD-L1 antibody Sugemalimab light chain C-terminal junction linker covalently linked to a CD86 ectodomain that, together with a LAG3 ectodomain D1 / D2 / D3 C-terminal junction linker covalently linked to a Sugemalimab heavy chain first polypeptide, make up the multifunctional fusion polypeptide complex; anti-PD-L1 antibody Sugemalimab light chain C-terminal junction linker covalently linked to a CD86 ectodomain IgV region that, together with a LAG3 ectodomain D1 / D2 / D3 C-terminal junction linker covalently linked to a Sugemalimab heavy chain first polypeptide, make up the multifunctional fusion polypeptide complex.

[0675] Provided below are exemplary multifunctional fusion polypeptide complex second polypeptide amino acid sequences of a CD86 ectodomain C-terminal junction linker covalently linked to an anti-PD-L1 antibody Sugemalimab light chain that, together with a LAG3 ectodomain D1 / D2 C-terminal junction linker covalently linked to a Sugemalimab heavy chain first polypeptide, make up the multifunctional fusion polypeptide complex; CD86 ectodomain IgV region C-terminal junction linker covalently linked to an anti-PD-L1 antibody Sugemalimab light chain that, together with a LAG3 ectodomain D1 / D2 C-terminal junction linker covalently linked to a Sugemalimab heavy chain first polypeptide, make up the multifunctional fusion polypeptide complex; anti-PD-L1 antibody Sugemalimab light chain C-terminal junction linker covalently linked to a CD86 ectodomain that, together with a LAG3 ectodomain D1 / D2 C-terminal junction linker covalently linked to a Sugemalimab heavy chain first polypeptide, make up the multifunctional fusion polypeptide complex; anti-PD-L1 antibody Sugemalimab light chain C-terminal junction linker covalently linked to a CD86 ectodomain IgV region that, together with a LAG3 ectodomain D1 / D2 C-terminal junction linker covalently linked to a Sugemalimab heavy chain first polypeptide, make up the multifunctional fusion polypeptide complex.

[0676] Provided below are exemplary multifunctional fusion polypeptide complex second polypeptide amino acid sequences covalently linked to a CD86 ectodomain C-terminal junction linker to a light chain of an anti-PD-L1 antibody Sugemalimab covalently linked to a LAG3 ectodomain D1 C-terminal junction linker to a first polypeptide of a heavy chain of Sugemalimab; multifunctional fusion polypeptide complex second polypeptide amino acid sequences covalently linked to a CD86 ectodomain IgV region C-terminal junction linker to a light chain of an anti-PD-L1 antibody Sugemalimab covalently linked to a LAG3 ectodomain D1 C-terminal junction linker to a first polypeptide of a heavy chain of Sugemalimab; multifunctional fusion polypeptide complex second polypeptide amino acid sequences covalently linked to a CD86 ectodomain to a light chain of an anti-PD-L1 antibody Sugemalimab covalently linked to a LAG3 ectodomain D1 C-terminal junction linker to a first polypeptide of a heavy chain of Sugemalimab; multifunctional fusion polypeptide complex second polypeptide amino acid sequences covalently linked to a CD86 ectodomain IgV region to a light chain of an anti-PD-L1 antibody Sugemalimab covalently linked to a LAG3 ectodomain D1 C-terminal junction linker to a first polypeptide of a heavy chain of Sugemalimab.

[0677] Provided below are exemplary multifunctional fusion polypeptide complex second polypeptide amino acid sequences of a CD86 ectodomain C-terminal junction linker covalently linked to an anti-PD-L1 antibody Sugemalimab light chain, the second polypeptide covalently linked to a LAG3 ectodomain first polypeptide and a Sugemalimab heavy chain C-terminal junction linker to make up the multifunctional fusion polypeptide complex; CD86 ectodomain IgV region C-terminal junction linker covalently linked to an anti-PD-L1 antibody Sugemalimab light chain, the second polypeptide covalently linked to a LAG3 ectodomain first polypeptide and a Sugemalimab heavy chain C-terminal junction linker to make up the multifunctional fusion polypeptide complex; anti-PD-L1 antibody Sugemalimab light chain C-terminal junction linker covalently linked to a CD86 ectodomain, the second polypeptide covalently linked to a LAG3 ectodomain first polypeptide and a Sugemalimab heavy chain C-terminal junction linker to make up the multifunctional fusion polypeptide complex; anti-PD-L1 antibody Sugemalimab light chain C-terminal junction linker covalently linked to a CD86 ectodomain IgV region, the second polypeptide covalently linked to a LAG3 ectodomain first polypeptide and a Sugemalimab heavy chain C-terminal junction linker to make up the multifunctional fusion polypeptide complex.

[0678] Provided below are exemplary multifunctional fusion polypeptide complex second polypeptide amino acid sequences of a CD86 ectodomain C-terminal junction linker covalently linked to an anti-PD-L1 antibody Sugemalimab light chain that, together with a Sugemalimab heavy chain C-terminal junction linker covalently linked to a LAG3 ectodomain D1 / D2 / D3 first polypeptide, make up the multifunctional fusion polypeptide complex; CD86 ectodomain IgV region C-terminal junction linker covalently linked to an anti-PD-L1 antibody Sugemalimab light chain that, together with a Sugemalimab heavy chain C-terminal junction linker covalently linked to a LAG3 ectodomain D1 / D2 / D3 first polypeptide, make up the multifunctional fusion polypeptide complex; anti-PD-L1 antibody Sugemalimab light chain C-terminal junction linker covalently linked to a CD86 ectodomain that, together with a Sugemalimab heavy chain C-terminal junction linker covalently linked to a LAG3 ectodomain D1 / D2 / D3 first polypeptide, make up the multifunctional fusion polypeptide complex; anti-PD-L1 antibody Sugemalimab light chain C-terminal junction linker covalently linked to a CD86 ectodomain IgV region that, together with a Sugemalimab heavy chain C-terminal junction linker covalently linked to a LAG3 ectodomain D1 / D2 / D3 first polypeptide, make up the multifunctional fusion polypeptide complex.

[0679] Provided below are exemplary multifunctional fusion polypeptide complex second polypeptide amino acid sequences covalently linked to a CD86 ectodomain C-terminal junction linker covalently linked to an anti-PD-L1 antibody Sugemalimab light chain covalently linked to a LAG3 ectodomain D1 / D2 first polypeptide consisting of the multifunctional fusion polypeptide complex; CD86 ectodomain IgV region C-terminal junction linker covalently linked to an anti-PD-L1 antibody Sugemalimab light chain covalently linked to a LAG3 ectodomain D1 / D2 first polypeptide consisting of the multifunctional fusion polypeptide complex; anti-PD-L1 antibody Sugemalimab light chain C-terminal junction linker covalently linked to a CD86 ectodomain covalently linked to a LAG3 ectodomain D1 / D2 first polypeptide consisting of the multifunctional fusion polypeptide complex; and anti-PD-L1 antibody Sugemalimab light chain C-terminal junction linker covalently linked to a CD86 ectodomain IgV region covalently linked to a LAG3 ectodomain D1 / D2 first polypeptide consisting of the multifunctional fusion polypeptide complex.

[0680] Provided below are exemplary second polypeptide amino acid sequences of a multifunctional fusion polypeptide complex of a CD86 ectodomain C-terminal junction linker covalently linked to a light chain of an anti-PD-L1 antibody Sugemalimab, the second polypeptide covalently linked to a LAG3 ectodomain D1 first polypeptide to make up the multifunctional fusion polypeptide complex with a C-terminal junction linker covalently linked to a heavy chain of Sugemalimab; a CD86 ectodomain IgV region C-terminal junction linker covalently linked to a light chain of an anti-PD-L1 antibody Sugemalimab, the second polypeptide covalently linked to a LAG3 ectodomain D1 first polypeptide to make up the multifunctional fusion polypeptide complex; a CD86 ectodomain C-terminal junction linker covalently linked to a light chain of an anti-PD-L1 antibody Sugemalimab, the second polypeptide covalently linked to a LAG3 ectodomain D1 first polypeptide to make up the multifunctional fusion polypeptide complex; a CD86 ectodomain IgV region C-terminal junction linker covalently linked to a light chain of an anti-PD-L1 antibody Sugemalimab, the second polypeptide covalently linked to a LAG3 ectodomain D1 first polypeptide to make up the multifunctional fusion polypeptide complex.

[0681] For example, the antibody AN_Bif_Abs_0016 can be made up of a Sugemalimab heavy chain antibody AN_Bif_Abs_0016 first polypeptide (SEQ ID NO: 4) and a Sugemalimab light chain second polypeptide (SEQ ID NO: 5) by expressing the AN_Bif_Abs_0016 first polypeptide polynucleotide sequence (SEQ ID NO: 152) vector and the AN_Bif_Abs_0016 second polypeptide polynucleotide sequence (SEQ ID NO: 153) vector into a host cell to express the AN_Bif_Abs_0016 antibody.

[0682] For example, bifunctional antibody AN_Bif_Abs_0220 can be formed by covalently connecting CD86 extracellular domain C-terminal connection linker peptide (SEQ ID NO: 34) to the N-terminal of Sugemalimab heavy chain to form chimeric antibody AN_Bif_Abs_0220 first polypeptide (SEQ ID NO: 38) and Sugemalimab light chain second polypeptide (SEQ ID NO: 5), and the assembly of AN_Bif_Abs_0220 bifunctional antibody is realized by introducing the expression of AN_Bif_Abs_0220 first polypeptide polynucleotide sequence (SEQ ID NO: 154) vector and AN_Bif_Abs_0220 second polypeptide polynucleotide sequence (SEQ ID NO: 153) vector into host cells for expression.

[0683] For example, bifunctional antibody AN_Bif_Abs_0222 can be formed by covalently connecting Sugemalimab heavy chain Fc domain connection linker peptide (SEQ ID NO: 34) to the N-terminal of CD86 extracellular domain to form chimeric antibody AN_Bif_Abs_0222 first polypeptide (SEQ ID NO: 36) and Sugemalimab light chain second polypeptide (SEQ ID NO: 5), and the assembly of AN_Bif_Abs_0222 bifunctional antibody is realized by introducing the expression of AN_Bif_Abs_0222 first polypeptide polynucleotide sequence (SEQ ID NO: 155) vector and AN_Bif_Abs_0222 second polypeptide polynucleotide sequence (SEQ ID NO: 153) vector into host cells for expression.

[0684] For example, bifunctional antibody AN_Bif_Abs_0703 can be formed by covalently connecting Sugemalimab heavy chain Fc domain connection linker peptide (SEQ ID NO: 34) to the IgV region of CD86 extracellular domain to form chimeric antibody AN_Bif_Abs_0703 first polypeptide (SEQ ID NO: 37) and Sugemalimab light chain second polypeptide (SEQ ID NO: 5), and the assembly of AN_Bif_Abs_0703 bifunctional antibody is realized by introducing the expression of AN_Bif_Abs_0703 first polypeptide polynucleotide sequence (SEQ ID NO: 156) vector and AN_Bif_Abs_0703 second polypeptide polynucleotide sequence (SEQ ID NO: 153) vector into host cells for expression.

[0685] For example, bifunctional antibody AN_Bif_Abs_0158 can be formed by covalently linking CD86 extracellular domain IgV region C-terminal connecting linker peptide (SEQ ID NO: 34) to the N-terminal of Sugemalimab heavy chain to form chimeric antibody AN_Bif_Abs_0158 first polypeptide (SEQ ID NO: 39) and Sugemalimab light chain second polypeptide (SEQ ID NO: 5), which is achieved by introducing the vector expressing AN_Bif_Abs_0158 first polypeptide polynucleotide sequence (SEQ ID NO: 157) and the vector expressing AN_Bif_Abs_0158 second polypeptide polynucleotide sequence (SEQ ID NO: 153) into host cells to express.

[0686] For example, bifunctional antibody AN_Bif_Abs_0675 can be formed by covalently linking CD86 extracellular domain IgV region connecting linker peptide (SEQ ID NO: 34) to the N-terminal of Sugemalimab heavy chain to form chimeric antibody AN_Bif_Abs_0675 first polypeptide (SEQ ID NO: 43) and Sugemalimab light chain second polypeptide (SEQ ID NO: 5), which is achieved by introducing the vector expressing AN_Bif_Abs_0675 first polypeptide polynucleotide sequence (SEQ ID NO: 152) and the vector expressing AN_Bif_Abs_0675 second polypeptide polynucleotide sequence (SEQ ID NO: 158) into host cells to express.

[0687] For example, bifunctional antibody AN_Bif_Abs_0676 can be formed by covalently linking CD86 extracellular domain IgV region connecting linker peptide (SEQ ID NO: 34) to the N-terminal of Sugemalimab heavy chain to form chimeric antibody AN_Bif_Abs_0676 first polypeptide (SEQ ID NO: 43) and Sugemalimab light chain second polypeptide (SEQ ID NO: 5), which is achieved by introducing the vector expressing AN_Bif_Abs_0675 first polypeptide polynucleotide sequence (SEQ ID NO: 152) and the vector expressing AN_Bif_Abs_0675 second polypeptide polynucleotide sequence (SEQ ID NO: 158) into host cells to express.

[0688] For example, multifunctional antibody AN_Bif_Abs_0159 can be formed by a CD86 extracellular domain IgV region C-terminal connecting linker peptide (SEQ ID NO: 34) covalently connected to the N-terminal of the heavy chain of Sugemalimab to form a chimeric antibody AN_Bif_Abs_0159 first polypeptide (SEQ ID NO: 39) and a LAG3 D1 / D2 C-terminal connecting linker peptide (SEQ ID NO: 34) covalently connected to the second polypeptide of the light chain of Sugemalimab (SEQ ID NO: 138), and the assembly of AN_Bif_Abs_0159 multifunctional antibody is realized by introducing the vector expressing the polynucleotide sequence of the first polypeptide of AN_Bif_Abs_0159 (SEQ ID NO: 157) and the vector expressing the polynucleotide sequence of the second polypeptide of AN_Bif_Abs_0159 (SEQ ID NO: 160) into host cells for expression.

[0689] For example, multifunctional antibody AN_Bif_Abs_0160 can be formed by a CD86 extracellular domain IgV region C-terminal connecting linker peptide (SEQ ID NO: 34) covalently connected to the N-terminal of the heavy chain of Sugemalimab to form a chimeric antibody AN_Bif_Abs_0160 first polypeptide (SEQ ID NO: 39) and a LAG3 D1 / D2 C-terminal connecting linker peptide (SEQ ID NO: 34) covalently connected to the second polypeptide of the light chain of Sugemalimab (SEQ ID NO: 138), and the assembly of AN_Bif_Abs_0159 multifunctional antibody is realized by introducing the vector expressing the polynucleotide sequence of the first polypeptide of AN_Bif_Abs_0159 (SEQ ID NO: 157) and the vector expressing the polynucleotide sequence of the second polypeptide of AN_Bif_Abs_0159 (SEQ ID NO: 160) into host cells for expression.

[0690] For example, multifunctional antibody AN_Bif_Abs_0161 can be covalently linked by CD86 extracellular domain IgV region C-terminal connecting linker peptide (SEQ ID NO: 34) to the N-terminus of the heavy chain of Sugemalimab, and 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) consisting of, by introducing the vector expressing the AN_Bif_Abs_0161 first polypeptide polynucleotide sequence (SEQ ID NO: 162) and the vector expressing the AN_Bif_Abs_0161 second polypeptide polynucleotide sequence (SEQ ID NO: 153) into the host cell to express to realize the assembly into AN_Bif_Abs_0161 multifunctional antibody.

[0691] For example, multifunctional antibody AN_Bif_Abs_0644 can be covalently linked by CD86 extracellular domain C-terminal connecting linker peptide (SEQ ID NO: 34) to the N-terminus of the heavy chain of Sugemalimab, and Sugemalimab heavy chain C-terminal connecting 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) consisting of, by introducing the vector expressing the AN_Bif_Abs_0644 first polypeptide polynucleotide sequence (SEQ ID NO: 163) and the vector expressing the AN_Bif_Abs_0644 second polypeptide polynucleotide sequence (SEQ ID NO: 153) into the host cell to express to realize the assembly into AN_Bif_Abs_0644 multifunctional antibody.

[0692] For example, multifunctional antibody AN_Bif_Abs_0646 can be covalently linked by CD86 extracellular domain IgV region C-terminal connecting linker peptide (SEQ ID NO: 34) to the N-terminus of Sugemalimab heavy chain, and Sugemalimab heavy chain C-terminal connecting linker peptide (SEQ ID NO: 34) to LAG3 D1, to form chimeric antibody AN_Bif_Abs_0646 first polypeptide (SEQ ID NO: 115) and Sugemalimab light chain second polypeptide (SEQ ID NO: 5), which is composed of, by introducing the vector expressing AN_Bif_Abs_0646 first polypeptide polynucleotide sequence (SEQ ID NO: 164) and the vector expressing AN_Bif_Abs_0646 second polypeptide polynucleotide sequence (SEQ ID NO: 153) into host cells to express to realize the assembly of AN_Bif_Abs_0646 multifunctional antibody.

[0693] For example, multifunctional antibody AN_Bif_Abs_0162 can be covalently linked by CD86 extracellular domain IgV region C-terminal connecting linker peptide (SEQ ID NO: 34) to the N-terminus of Sugemalimab heavy chain, and Sugemalimab heavy chain C-terminal connecting linker peptide (SEQ ID NO: 34) to LAG3 extracellular domain, to form chimeric antibody AN_Bif_Abs_0162 first polypeptide (SEQ ID NO: 112) and Sugemalimab light chain second polypeptide (SEQ ID NO: 5), which is composed of, by introducing the vector expressing AN_Bif_Abs_0162 first polypeptide polynucleotide (SEQ ID NO: 165) and the vector expressing AN_Bif_Abs_0162 second polypeptide polynucleotide (SEQ ID NO: 153) into host cells to express to realize the assembly of AN_Bif_Abs_0162 multifunctional antibody.

[0694] For example, multifunctional antibody AN_Bif_Abs_0163 can be covalently linked by a CD86 extracellular domain C-terminal connecting linker peptide (SEQ ID NO: 34) to the N-terminus of a Sugemalimab heavy chain, a Sugemalimab heavy chain C-terminal connecting 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) constitute, by introducing the expression AN_Bif_Abs_0163 first polypeptide polynucleotide sequence (SEQ ID NO: 166) vector and the expression AN_Bif_Abs_0163 second polypeptide polynucleotide sequence (SEQ ID NO: 153) vector into the host cell to express to realize the assembly into AN_Bif_Abs_0163 multifunctional antibody.

[0695] For example, multifunctional antibody AN_Bif_Abs_0164 can be covalently linked by a LAG3 extracellular domain C-terminal connecting linker peptide (SEQ ID NO: 34) to the N-terminus of a Sugemalimab heavy chain, a Sugemalimab 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_0164 first polypeptide (SEQ ID NO: 108) and a Sugemalimab light chain second polypeptide (SEQ ID NO: 5) constitute, by introducing the expression AN_Bif_Abs_0164 first polypeptide polynucleotide sequence (SEQ ID NO: 167) vector and the expression AN_Bif_Abs_0164 second polypeptide polynucleotide sequence (SEQ ID NO: 153) vector into the host cell to express to realize the assembly into AN_Bif_Abs_0164 multifunctional antibody.

[0696] For example, the multifunctional antibody AN_Bif_Abs_0166 can be formed by covalently connecting the LAG3 extracellular domain C-terminal connecting linker peptide (SEQ ID NO: 34) to the N-terminal of the Sugemalimab heavy chain, covalently connecting the Sugemalimab heavy chain C-terminal connecting linker peptide (SEQ ID NO: 34) 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), which is composed of, by introducing the expression AN_Bif_Abs_0166 first polypeptide polynucleotide (SEQ ID NO: 168) vector and the expression AN_Bif_Abs_0166 second polypeptide polynucleotide (SEQ ID NO: 153) vector into the host cell to express to realize the assembly into AN_Bif_Abs_0166 multifunctional antibody.

[0697] For example, the antibody AN_Bif_Abs_0031 can be formed by the Envafolimab to form the antibody AN_Bif_Abs_0031 first polypeptide (SEQ ID NO: 14), which is realized by introducing the expression AN_Bif_Abs_0031 first polypeptide polynucleotide (SEQ ID NO: 169) vector into the host cell to express to realize the assembly into AN_Bif_Abs_0031 antibody.

[0698] For example, the bifunctional antibody AN_Bif_Abs_0129 can be formed by covalently connecting the CD86 extracellular domain IgV region C-terminal connecting linker peptide (SEQ ID NO: 34) to the N-terminal of the Envafolimab to form a chimeric antibody AN_Bif_Abs_0129 first polypeptide (SEQ ID NO: 71), which is realized by introducing the expression AN_Bif_Abs_0129 first polypeptide polynucleotide (SEQ ID NO: 170) vector into the host cell to express to realize the assembly into AN_Bif_Abs_0129 bifunctional antibody.

[0699] For example, multifunctional antibody AN_Bif_Abs_0130 can be assembled by covalently linking CD86 ectodomain IgV region C-terminal junction peptide (SEQ ID NO:34) to the N-terminus of LAG3 D1 / D2, LAG3 D1 / D2 C-terminal junction peptide (SEQ ID NO:34) to the N-terminus of Envafolimab, forming chimeric antibody AN_Bif_Abs_0130 first polypeptide (SEQ ID NO: 171), which is expressed by introducing a vector expressing AN_Bif_Abs_0130 first polypeptide polynucleotide (SEQ ID NO: 172) into a host cell.

[0700] For example, multifunctional antibody AN_Bif_Abs_0131 can be assembled by covalently linking CD86 ectodomain IgV region C-terminal junction peptide (SEQ ID NO:34) to the N-terminus of Envafolimab, Envafolimab C-terminal junction peptide (SEQ ID NO:34) to the C-terminus of LAG3 D1 / D2, forming chimeric antibody AN_Bif_Abs_0131 first polypeptide (SEQ ID NO: 173), which is expressed by introducing a vector expressing AN_Bif_Abs_0131 first polypeptide polynucleotide (SEQ ID NO: 174) into a host cell.

[0701] For example, bifunctional antibody AN_Bif_Abs_0012 can be assembled by covalently linking Durvalumab heavy chain C-terminal junction peptide (SEQ ID NO:34) to the CD86 ectodomain, forming chimeric antibody AN_Bif_Abs_0012 first polypeptide (SEQ ID NO:44) and Durvalumab light chain second polypeptide (SEQ ID NO:9), which is expressed by introducing a vector expressing AN_Bif_Abs_0164 first polypeptide polynucleotide sequence (SEQ ID NO:175) and a vector expressing AN_Bif_Abs_0012 second polypeptide polynucleotide sequence (SEQ ID NO:176) into a host cell.

[0702] For example, antibody AN_Bif_Abs_0001 can be comprised of Durvalumab heavy chain antibody AN_Bif_Abs_0001 first polypeptide (SEQ ID NO: 8) and Durvalumab light chain second polypeptide (SEQ ID NO: 9) by expressing AN_Bif_Abs_0001 first polypeptide polynucleotide sequence (SEQ ID NO: 177) vector and AN_Bif_Abs_0001 second polypeptide polynucleotide sequence (SEQ ID NO: 176) vector into a host cell to express AN_Bif_Abs_0001 antibody.

[0703] In certain embodiments, the present application also provides a method for isolating a polypeptide complex.

[0704] In one aspect, the present application provides a method for producing a polypeptide complex, the method comprising introducing into a host cell a polynucleotide encoding the antibody polypeptide complex of the present application, 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, the stable dimer can be maintained by a native bond or / and a stable dimerization comprising at least one non-native inter-chain bond, the first polypeptide and the second polypeptide assemble into a stable polymer in the host cell, and form the stable complex having binding to the first cell target, the second cell target and / or the third cell target.

[0705] In other aspects, the present application provides a method for removing the inhibition of T cells by PD-L1 / PD-1, while providing stimulation of antigen presenting cells or / and T cell activation, the method comprising using the bifunctional and / or multifunctional fusion polypeptide of the present application to stimulate T cell activation, but not limited to the protocol used in the examples, which can be extended to the protocols that have been published or will be developed in the future for evaluating antigen presenting cells or / and T cell activation.

[0706] In another aspect, the bifunctional and / or multifunctional fusion polypeptide of the present application has the effect of blocking tumor growth, including but not limited to colon tumor, breast tumor, lung tumor, gastric tumor, melanoma, head and neck tumor, lymphoma, nasopharyngeal tumor, cervical tumor, esophageal tumor, renal tumor, skin squamous carcinoma, endometrial tumor, liver tumor, bladder tumor, urothelial tumor and / or skin tumor.

[0707] In another aspect, the present application provides an immunoconjugate, which can comprise the fusion polypeptide of the present application.

[0708] In another aspect, the present application provides a nucleic acid molecule, which can encode the fusion polypeptide of the present application.

[0709] In another aspect, the application provides a vector, which can comprise a nucleic acid molecule described herein.

[0710] In another aspect, the application provides a cell, which can comprise and / or express a fusion polypeptide described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, and / or a vector described herein.

[0711] In another aspect, the application provides a composition, which can comprise a fusion polypeptide described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, a vector described herein, and / or a cell described herein, and optionally a pharmaceutically acceptable carrier.

[0712] In another aspect, the application provides a method of making a fusion polypeptide described herein, which can comprise culturing a cell described herein under conditions such that the fusion polypeptide is expressed.

[0713] In another aspect, the application provides a method of blocking the interaction of a PD-L1 protein with a PD-1, which can comprise administering an effective amount of a fusion polypeptide described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein, and / or a composition described herein.

[0714] In another aspect, the application provides a method of stimulating antigen presenting cells and / or activating T cells, which can comprise administering an effective amount of a fusion polypeptide described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein, and / or a composition described herein.

[0715] According to the method described herein, the stimulating antigen presenting cells can comprise increasing the expression of a costimulatory molecule by an antigen presenting cell, maturing an antigen presenting cell by changing its morphology, increasing the secretion of a chemotactic factor by an antigen presenting cell, and / or increasing the phagocytic ability of an antigen presenting cell.

[0716] In another aspect, the application provides a method of inhibiting the growth and / or proliferation of a tumor or tumor cells, which can comprise administering an effective amount of a fusion polypeptide described herein, an immunoconjugate described herein, a nucleic acid molecule described herein, a vector described herein, a cell described herein, and / or a composition described herein.

[0717] In another aspect, the present application provides 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 for preventing, ameliorating and / or treating a tumor.

[0718] According to the use described herein, the tumor can comprise solid tumor and blood tumor.

[0719] According to the use described herein, the tumor can be selected from the group consisting of colon tumor, breast tumor, lung tumor, gastric tumor, melanoma, head and neck tumor, lymphoma, nasopharyngeal tumor, cervical tumor, esophageal tumor, renal tumor, endometrial tumor, liver tumor, bladder tumor, urothelial tumor and skin tumor. For example, the skin tumor can comprise skin squamous carcinoma.

[0720] In another aspect, 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 for use in preventing, ameliorating and / or treating a tumor.

[0721] In another aspect, 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 for use in preventing, ameliorating and / or treating a tumor, wherein the tumor comprises solid tumor and blood tumor.

[0722] In another aspect, 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 for use in preventing, ameliorating and / or treating a tumor, wherein the tumor can be selected from the group consisting of colon tumor, breast tumor, lung tumor, gastric tumor, melanoma, head and neck tumor, lymphoma, nasopharyngeal tumor, cervical tumor, esophageal tumor, renal tumor, endometrial tumor, liver tumor, bladder tumor, urothelial tumor and skin tumor. For example, the skin tumor can comprise skin squamous carcinoma.

[0723] In another aspect, the present application provides a method of preventing, ameliorating and / or treating a tumor, which can comprise administering to a subject in need thereof 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.

[0724] According to the method described herein, the tumor can comprise solid tumor and blood tumor.

[0725] According to the method described in the present application, the tumor can be 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, renal tumor, endometrial tumor, liver tumor, bladder tumor, urothelial tumor and skin tumor. For example, the skin tumor can include skin squamous carcinoma.

[0726] CD86 protein is a kind of immune-related molecule with immune response stimulation, which mainly realizes its function by regulating T cell immune response, including participating in immune defense, immune tolerance and immune tissue damage, etc. CD86 mainly plays an immune regulation role by combining with its ligands CD28 and CTLA4 to regulate immune response, and changing the affinity of CD86 with ligands can regulate the related functions mediated by CD86, especially enhancing the immune response function of the body to strengthen the control effect on diseases including tumors, infections, etc. CD86 is one of the member proteins of the B7 molecule family that co-stimulates activated T cells. The mature CD86 molecule is composed of extracellular domain (ECD), transmembrane domain and intracellular domain. The extracellular domain (ECD) is the key region of these molecules to bind to the corresponding receptors on T cells. The extracellular domain of CD86 contains immunoglobulin variable-like V (IgV) region and immunoglobulin constant-like C2 (IgC2) region, and the IgV domain is the key domain directly involved in receptor binding. The IgV domain of CD86 can bind to CD28 to induce T cell activation, proliferation and effector function. CTLA4 can also bind to the IgV region of CD86 to participate in immune suppression regulation. In the regulation of immune response, the activation of T cells is regulated by the binding of co-stimulatory signal receptors such as CD28 and the corresponding ligand CD86 on the surface of APC. However, in the tumor microenvironment, CD86 is usually not expressed or lowly expressed, which leads to the failure of T cells to be effectively activated, which is one of the key mechanisms of tumor immune escape. In addition, CTLA4 competes with CD28 to bind to CD86, and shows higher affinity characteristics, which leads to the failure of CD28 to effectively receive the immune stimulation signal mediated by CD86, so that T cells cannot be effectively activated or immunosuppressed.

[0727] Therefore, there is a need in the art for CD86 mutant polypeptides that improve the binding activity of CD86 to its ligand, which can effectively stimulate T cells and enhance immune response, so as to treat or prevent diseases caused by the inhibition of T cell function, such as infections, tumors, etc.

[0728] The CD86 mutant polypeptide formed by innovatively modifying the CD86 IgV domain can regulate the binding activity of CD86 and its ligand, so that the CD86 mutant polypeptide can effectively stimulate T cells to enhance immune response, thereby being capable of treating or preventing diseases caused by the inhibition of T cell function, such as infections, tumors and the like.

[0729] In one aspect, the present application provides an isolated CD86 variant polypeptide, which can comprise an amino acid substitution mutation of humanized CD86.

[0730] For example, the CD86 variant polypeptide can comprise an IgV domain (SEQ ID NO: 3) amino acid substitution mutation of humanized CD86.

[0731] For example, the CD86 mutant can comprise an IgV domain (SEQ ID NO: 3) amino acid substitution mutation of humanized CD86, which can comprise one or more of A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, H90F. For example, the CD86 IgV mutant polypeptide comprises a combination of 1, 2, 3, 4, 5 or more of the above amino acid site mutations.

[0732] For example, the IgV domain amino acid substitution mutant of CD86 can be selected from a combination of amino acid mutation sites set forth in 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, 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 / 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.

[0733] For example, the variant polypeptide can comprise an IgV domain amino acid substitution mutant of CD86 Q25I / F33L / H90I.

[0734] For example, the CD86 variant polypeptide can comprise an extracellular domain amino acid substitution mutant of humanized CD86.

[0735] For example, the CD86 variant polypeptide can comprise an extracellular domain (SEQ ID NO: 2) amino acid substitution mutant of humanized CD86, which can comprise one or more of A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, H90F. For example, the CD86 extracellular domain mutant polypeptide comprises a combination of 1, 2, 3, 4, 5, or more of the above amino acid site mutations.

[0736] The extracellular domain (SEQ ID NO: 2) amino acid substitution mutant of CD86 can be selected from a combination of amino acid mutation sites set forth in 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, 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 / 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.

[0737] For example, the CD86 variant polypeptide can comprise the extracellular domain amino acid substitution mutant Q25I / F33L / H90I of CD86.

[0738] For example, the CD86 variant polypeptide can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 178 to SEQ ID NO: 243.

[0739] In another aspect, the present application provides a fusion polypeptide, which can comprise a first domain and a second domain, the first domain can comprise a CD86 variant polypeptide, and the second domain can comprise an antibody or an antigen binding fragment thereof or other forms of polypeptides.

[0740] For example, the first domain can comprise the IgV domain amino acid substitution mutant of humanized CD86.

[0741] For example, the first domain may comprise an amino acid substitution mutant of the IgV domain (SEQ ID NO: 3) of humanized CD86, wherein the amino acid substitution mutation site may comprise one or more of the following: A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, and H90F. For example, the CD86 IgV mutant polypeptide may comprise a combination of one, two, three, four, five, or more of the above-mentioned amino acid site mutations.

[0742] For example, the first domain may comprise an amino acid substitution mutant of the IgV domain (SEQ ID NO: 3) of CD86, wherein the amino acid substitution mutant may be selected from a combination of 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 / F33V / H90V , Q25V / F33V / H90V, A13L / Q25I / F33L / H90I, A13I / Q25I / F33L / H90I, A13L / Q25V / F33L / H9 0I, A13I / Q25V / F33L / H90I, Q25I / F33L / I89L / H90I, Q25I / F33L / M60R / H90I, Q25V / F33L / I 89L / 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 / Q25I / F33 L / H90I, A13M / Q25I / F33L / H90I, Q25A / F33L / H90I, Q25M / F33L / H90I, Q25I / F33M / H90I, Q 25I / F33L / I89V / H90I, Q25I / F33L / I89F / H90I, Q25I / F33L / I89M / H90I and Q25I / F33L / H90M.

[0743] For example, the first domain may contain the CD86 IgV domain amino acid substitution mutant Q25I / F33L / H90I.

[0744] For example, the first domain can comprise an amino acid substitution mutant of the extracellular domain of humanized CD86 (SEQ ID NO: 2).

[0745] For example, the first domain can comprise an amino acid substitution mutant of the extracellular domain of humanized CD86 (SEQ ID NO: 2), which can comprise one or more of A13I, A13L, A13F, A13M, Q25A, Q25I, Q25V, Q25F, Q25M, F33A, F33L, F33V, F33M, M60R, I89A, I89L, I89V, I89F, I89M, H90I, H90V, H90M, H90F. For example, the CD86 extracellular domain mutant polypeptide comprises a combination of 1, 2, 3, 4, 5 or more of the above amino acid site mutations.

[0746] For example, the first domain can comprise an amino acid substitution mutant of the extracellular domain of humanized CD86 (SEQ ID NO: 2), which can comprise a combination of amino acid mutations 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, 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 / 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.

[0747] For example, the first domain can comprise an extracellular domain amino acid substitution mutant Q25I / F33L / H90I of a humanized CD86 variant polypeptide.

[0748] For example, the first domain can comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 178 to SEQ ID NO: 243.

[0749] For example, the second domain can comprise an antibody or antigen binding fragment thereof.

[0750] For example, the second domain can bind one or more of the target selected from the group consisting of 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.

[0751] For example, the second domain can bind PD-L1 and / or PD-1.

[0752] For example, the second domain can comprise an HCDR3 of an antibody heavy chain, which can 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.

[0753] For example, the second domain can comprise an HCDR2 of an antibody heavy chain, which can 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.

[0754] For example, the second domain can comprise HCDR1 of an antibody heavy chain, which can 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.

[0755] For example, the second domain can comprise HCDR1 of an antibody heavy chain, which can 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.

[0756] For example, the second domain can comprise HCDR1 of an antibody heavy chain, which can 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.

[0757] For example, the second domain can comprise HCDR1 of an antibody heavy chain, which can 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.

[0758] For example, the second domain can comprise HCDR1 of an antibody heavy chain, which can 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.

[0759] For example, the second domain can comprise HCDR1 of an antibody heavy chain, which can 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.

[0760] For example, the second domain can comprise a light chain variable region VL of an antibody light chain, which can 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.

[0761] For example, the second domain can comprise an antibody light chain, which can 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.

[0762] For example, the second domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 4. For example, the antibody can be Sugemalimab.

[0763] For example, the second domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 5. For example, the antibody can be Sugemalimab.

[0764] For example, the second domain can comprise HCDR3, HCDR2 and HCDR1 of an antibody heavy chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 4, and the second domain can comprise LCDR3, LCDR2 and LCDR1 of an antibody light chain, which can comprise an amino acid sequence as set forth in SEQ ID NO: 5. For example, the antibody can be Sugemalimab.

[0765] For example, the second domain can comprise a heavy chain variable region ...

Claims

A LAG3 variant polypeptide comprising a truncation and / or mutation based on human LAG3. The fusion polypeptide or LAG3 variant polypeptide of claim 1, comprising a truncation based on wild-type human LAG3 extracellular region polypeptide. The LAG3 variant polypeptide of any of the preceding claims, comprising a truncation of 0-124 amino acids at the N-terminus and terminating at the C-terminus between amino acid positions 121-167 based on wild-type human LAG3 extracellular region polypeptide. The LAG3 variant polypeptide of any of the preceding claims, comprising a truncation of 0, 5, 21, 37, 45, 60, 71, 74, 79, 84, 89, 94, 99, 104, 109, or 114 amino acids at the N-terminus based on wild-type human LAG3 extracellular region polypeptide. The LAG3 variant polypeptide of any of the preceding claims, comprising a truncation of 0, 5, 21, 37, 45, 60, 71, 74, 81, or 99 amino acids at the N-terminus based on wild-type human LAG3 extracellular region polypeptide. The LAG3 variant polypeptide of any of the preceding claims, comprising a truncation of 0, 5, 21, 37, 45, 60, 71, or 74 amino acids at the N-terminus based on wild-type human LAG3 extracellular region polypeptide. The LAG3 variant polypeptide of any of the preceding claims, comprising a truncation terminating at the C-terminus at amino acid position 121, 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, or 167 based on wild-type human LAG3 extracellular region polypeptide. The LAG3 variant polypeptide of any of the preceding claims, comprising a truncation terminating at the C-terminus at amino acid position 121, 122, 129, 136, 146, 156, 161, or 167 based on wild-type human LAG3 extracellular region polypeptide. The LAG3 variant polypeptide of any of the preceding claims, comprising a truncation terminating at the C-terminus at amino acid position 121, 146, 156, 161, or 167 based on wild-type human LAG3 extracellular region polypeptide. The LAG3 variant polypeptide of any of the preceding claims, comprising a truncation terminating at the C-terminus at amino acid position 156, 161, or 167 based on wild-type human LAG3 extracellular region polypeptide. The LAG3 variant polypeptide of any of the preceding claims, which comprises 0, 5, 21, 37, 45, 60, 71, 74, 81, or 99 amino acid truncations at the N-terminus and terminates at the C-terminus at amino acid position 121, 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, or 167, based on the wild-type human LAG3 extracellular region polypeptide. The LAG3 variant polypeptide of any of the preceding claims, which comprises 0, 5, 21, 37, 45, 60, 71, or 74 amino acid truncations at the N-terminus and terminates at the C-terminus at amino acid position 122, 129, 136, 146, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, or 167, based on the wild-type human LAG3 extracellular region polypeptide. A LAG3 variant polypeptide, which comprises 125-145 amino acid truncations at the N-terminus and terminates at the C-terminus at a position of the wild-type LAG3 extracellular region polypeptide corresponding to any of amino acid positions 148-167 of the wild-type LAG3 extracellular region polypeptide, based on the wild-type human LAG3 extracellular region polypeptide. The LAG3 variant polypeptide of claim 13, which comprises 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, or 145 amino acid truncations at the N-terminus, based on the wild-type human LAG3 extracellular region polypeptide. The LAG3 variant polypeptide of any of claims 13-14, which terminates at the C-terminus at a position of the wild-type LAG3 extracellular region polypeptide corresponding to any of amino acid positions 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, of the wild-type LAG3 extracellular region polypeptide. The LAG3 variant polypeptide of any one of claims 13-15, which C-terminus terminates at a position corresponding to any one of amino acid positions 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, or 167 of the wild-type LAG3 extracellular region polypeptide. A LAG3 variant polypeptide comprising a truncation of 0-124 amino acids at the N- terminus based on the wild-type human LAG3 extracellular region polypeptide and a C- terminus that terminates at a position corresponding to any one of amino acid positions 1- 121 of the wild-type LAG3 extracellular region polypeptide. The LAG3 variant polypeptide of claim 17, which comprises a truncation of 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, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, or 124 amino acids at the N-terminus based on the wild-type human LAG3 extracellular region polypeptide. The LAG3 variant polypeptide of any of claims 17-18, which C-terminates at a position corresponding to any of amino acid positions 1-121, 2-121, 3-121, 4-121, 5-121, 6-121, 7-121, 8-121, 9-121, 10-121, 11-121, 12-121, 13-121, 14-121, 15-121, 16-121, 17-121, 18-121, 19-121, 20-121, 21-121, 22-121, 23-121, 24-121, 25-121, 26-121, 27-121, 28-121, 29-121, 30-121, 31-121, 32-121, 33-121, 34-121, 35-121, 36-121, 37-121, 38-121, 39-121, 40-121, 41-121, 42-121, 43-121, 44-121, 45-121, 46-121, 47-121, 48-121, 49-121, 50-121, 51-121, 52-121, 53-121, 54-121, 55-121, 56-121, 57-121, 58-121, 59-121, 60-121, 61-121, 62-121, 63-121, 64-121, 65-121, 66-121, 67-121, 68-121, 69-121, 70-121, 71-121, 72-121, 73-121, 74-121, 75-121, 76-121, 77-121, 78-121, 79-121, 80-121, 81-121, 82-121, 83-121, 84-121, 85-121, 86-121, 87-121, 88-121, 89-121, 90-121, 91-121, 92-121, 93-121, 94-121, 95-121, 96-121, 97-121, 98-121, 99-121, 100-121, 101-121, 102-121, 103-121, 104-121, 105-121, 106-121, 107-121, 108-121, 109-121, 110-121, 111-121, 112-121, 113-121, 114-121, 115-121, 116-121, 117-121, 118-121, 119-121, or 120-121 of the wild-type LAG3 extracellular region polypeptide. The LAG3 variant polypeptide of any one of claims 17-19, which terminates at the C-terminus at a position corresponding to amino acid position 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, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, or 121 of the wild-type LAG3 extracellular domain polypeptide. The LAG3 variant polypeptide of any one of the preceding claims, which comprises an amino acid mutation at an amino acid position. The LAG3 variant polypeptide of any one of the preceding claims, which comprises an amino acid mutation at an amino acid position, the amino acid mutation position being Arg at position 97. The LAG3 variant polypeptide of any one of the preceding claims, which comprises an amino acid mutation at an amino acid position, the amino acid mutation position being Arg at position 97 mutated to Glu. The LAG3 variant polypeptide of any one of the preceding claims, which is selected from the group consisting of the amino acid sequences set forth in SEQ ID NO: 244 to SEQ ID NO:

349. The LAG3 variant polypeptide of any one of the preceding claims, which comprises an amino acid mutation at an amino acid position, the amino acid mutation position being R110, R113, R119, R129, G130, and / or R141. The LAG3 variant polypeptide of any one of the preceding claims, which is truncated at the N-terminus by 81 amino acids, terminates at the C-terminus at amino acid position 121, and comprises a mutation at position R110, and / or R113, and / or R119. The LAG3 variant polypeptide of any one of the preceding claims, which is truncated at the N-terminus by 99 amino acids, terminates at the C-terminus at amino acid position 146, and comprises a mutation at position R129, and / or G130, and / or R141. The LAG3 variant polypeptide of any of the preceding claims, which is N-terminally truncated by 81 amino acids and C-terminally terminated at amino acid position 146, and comprises a mutation at the R110, and / or R113, and / or R119, and / or R129, and / or G130, and / or R141 site. The LAG3 variant polypeptide of any of the preceding claims, which comprises a mutation at the R110 site to K110. The LAG3 variant polypeptide of any of the preceding claims, which comprises a mutation at the R113 site to K113. The LAG3 variant polypeptide of any of the preceding claims, which comprises a mutation at the R119 site to K119. The LAG3 variant polypeptide of any of the preceding claims, which comprises a mutation at the R129 site to K129. The LAG3 variant polypeptide of any of the preceding claims, which comprises a mutation at the G130 site to P130, or A130, or T130, or Y130, or S130. The LAG3 variant polypeptide of any of the preceding claims, which comprises a mutation at the R141 site to K141. The LAG3 variant polypeptide of any of the preceding claims, which has an amino acid sequence selected from the group consisting of SEQ ID NO: 1285-SEQ ID NO: 1318. A fusion polypeptide comprising a first domain comprising the LAG3 variant polypeptide of any of the preceding claims and a second domain comprising an antibody or antigen binding fragment thereof or an immunoglobulin Fc domain. The fusion polypeptide of claim 36, wherein the second domain comprises an antibody or antigen binding fragment thereof. The fusion polypeptide of any of claims 36-37, 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. The fusion polypeptide of any of claims 37-38, wherein the antigen binding fragment is selected from one or more of the group consisting of a Fab, a Fab', a Fv fragment, a F(ab')2, a F(ab)2, a scFv, a di-scFv, a VHH, and a dAb. The fusion polypeptide of any one of claims 37-39, wherein the second domain is capable of binding one or more of the target selected from the group consisting of: 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. The fusion polypeptide of any one of claims 37-40, wherein the second domain is capable of binding PD-L1, PD-1, and / or PD-L2. The fusion polypeptide of any one of claims 37-41, wherein the indirect linkage comprises linkage via a linker. The fusion polypeptide of any one of claims 1-35, wherein the second domain comprises an immunoglobulin Fc domain. The fusion polypeptide of claim 43, wherein the immunoglobulin Fc domain comprises an IgG antibody Fc domain, the immunoglobulin IgG antibody comprising one or more selected from the group consisting of human IgG1, human IgG2, human IgG3, human IgG4, mouse IgG1, mouse IgG2a, mouse IgG2b, and mouse IgG3. The fusion polypeptide of any one of claims 43-44, wherein the second domain comprises an immunoglobulin Fc domain. The fusion polypeptide of any one of claims 43-45, wherein the second domain comprises an Fc domain of an immunoglobulin IgG antibody. The fusion polypeptide of any one of claims 43-46, wherein the second domain comprises an Fc domain of an immunoglobulin IgG antibody, the Fc domain of the immunoglobulin IgG antibody comprising 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. The fusion polypeptide of any one of claims 43-47, wherein the second domain comprises an Fc domain of a human immunoglobulin IgG4. The fusion polypeptide of any one of claims 36-48, wherein the first domain is directly or indirectly linked to the second domain. The fusion polypeptide of any one of claims 36-49, wherein the first domain is directly or indirectly linked to the N-terminus of the second domain. The fusion polypeptide of any one of claims 36-50, wherein the first domain is directly or indirectly linked to the C-terminus of the second domain. The fusion polypeptide of any one of claims 36-51, wherein the indirect linkage comprises linkage by a linker. The fusion polypeptide of claim 52, 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. The fusion polypeptide of any one of claims 36-53, further comprising a third domain that is an antibody or antigen-binding fragment or functional protein or active fragment thereof that is the same as or different from the second domain. The fusion polypeptide of claim 54, wherein the third domain is a functional protein or active fragment thereof that is capable of activating an innate immune response. The fusion polypeptide of any one of claims 54-55, wherein the third domain is capable of binding CD28 and / or CTLA4. The fusion polypeptide of any one of claims 54-56, wherein the third domain comprises CD80, CD86, or an active fragment thereof. An immunoconjugate comprising the LAG3 variant of any one of claims 1-35 and / or the fusion polypeptide of any one of claims 36-57. A nucleic acid molecule encoding the LAG3 variant of any one of claims 1-35 and / or the fusion polypeptide of any one of claims 36-57. A vector comprising the nucleic acid molecule of claim 59. A cell comprising and / or expressing the LAG3 variant of any one of claims 1-35, the fusion polypeptide of any one of claims 36-57, the immunoconjugate of claim 58, the nucleic acid molecule of claim 59, and / or the vector of claim 60. A composition comprising the LAG3 variant of any one of claims 1-35, the fusion polypeptide of any one of claims 36-57, the immunoconjugate of claim 58, the nucleic acid molecule of claim 59, and / or the vector of claim 60, and / or the cell of claim 61, and optionally a pharmaceutically acceptable carrier. A method of making the LAG3 variant of any one of claims 1-35, the fusion polypeptide of any one of claims 36-57, comprising culturing the cell of claim 61 under conditions such that the fusion polypeptide is expressed. A method of stimulating antigen presenting cells and / or activating T cell function, comprising the LAG3 variant of any one of claims 1-35. The method of claim 64, wherein stimulating antigen presenting cells comprises increasing antigen presenting cell co-stimulatory molecule expression, maturing antigen presenting cell morphology, increasing antigen presenting cell chemokine secretion, and / or increasing antigen presenting cell phagocytic capacity. The method of claim 64, wherein stimulating antigen presenting cells comprises increasing antigen presenting cell co-stimulatory molecule expression, maturing antigen presenting cell morphology, increasing antigen presenting cell chemokine secretion, and / or increasing antigen presenting cell phagocytic capacity. A method of inhibiting the growth and / or proliferation of a tumor or tumor cell, comprising administering an effective amount of the LAG3 variant of any one of claims 1-35, the fusion polypeptide of any one of claims 36-57, the immunoconjugate of claim 58, the nucleic acid molecule of claim 59, and / or the vector of claim 60, the cell of claim 61, and / or the composition of claim 62. Use of the LAG3 variant of any one of claims 1-35, the fusion polypeptide of any one of claims 36-57, the immunoconjugate of claim 58, the nucleic acid molecule of claim 59, and / or the vector of claim 60, the cell of claim 61, and / or the composition of claim 62 for the manufacture of a medicament, wherein the medicament is for preventing, ameliorating, and / or treating a tumor. The use according to claim 67, wherein the tumor comprises a solid tumor and / or a hematological tumor. The use according to any one of claims 67-68, wherein the tumor is selected from the group consisting of a colon tumor, a breast tumor, a lung tumor, a gastric tumor, a melanoma, a head and neck tumor, a lymphoma, a nasopharyngeal tumor, a cervical tumor, an esophageal tumor, a renal tumor, a cutaneous squamous carcinoma, an endometrial tumor, a hepatic tumor, a bladder tumor, a urothelial tumor, and a skin tumor.

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