Multispecific antibodies and their use

Abstract

Provided are multispecific antibodies or antigen-binding fragments thereof that can bind two or more antigens, or two or more epitopes of the same antigen; also provided are uses of the antibodies or antigen-binding fragments in the treatment, diagnosis, and prognosis of inflammatory diseases, autoimmune diseases, or spinal cord injuries.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to multispecific antibodies and their applications. Background Technology

[0002] Currently, powerful tools for cancer immunotherapy include monoclonal antibodies, tumor vaccines, immune checkpoint inhibitors, CAR-T cell immunotherapy, bispecific antibodies (BsAb), and multispecific antibodies. CAR-T and BsAb are receiving increasing attention as novel strategies for anti-tumor immunotherapy.

[0003] Bispecific antibodies (BsAbs) are engineered antibodies that can simultaneously bind to two specific epitopes or target proteins, enabling them to perform specific biological functions. Compared to combination therapy with two monoclonal antibody drugs, BsAbs improve antibody selectivity and functionality while reducing treatment costs. BsAbs are prepared primarily through methods such as chemical conjugation, two-hybridoma cell methods, and recombinant gene preparation.

[0004] However, there are difficulties in the preparation of bispecific antibodies, such as the generation of a large number of byproducts and the inhibition of biological activity during antibody preparation. Summary of the Invention

[0005] This invention provides multivalent and multispecific antibody or antigen-binding fragments and their applications. In some embodiments, this invention provides bispecific antibody or antigen-binding fragments. The antibody or antigen-binding fragments provided by this invention can bind to two or more antigens, or two or more epitopes of the same antigen, or two or more copies of the same epitope. The antibody or antigen-binding fragments provided by this invention are used to treat or improve inflammatory diseases, autoimmune diseases, cancer, or spinal cord injuries; the antibody or antigen-binding fragments provided by this invention are also used for the diagnosis and prognosis of related diseases.

[0006] In some embodiments, the antibody or antigen-binding fragment binds to two different antigenic epitopes: a first antigenic epitope x and a second antigenic epitope y. The antibody or antigen-binding fragment comprises at least two polypeptide chains. The first polypeptide chain, starting from the amino terminus, sequentially comprises VHa, CLa, VHb, and CH1, where VHa is the heavy chain variable region binding to the first antigenic epitope x, CLa is the first light chain constant region, VHb is the heavy chain variable region binding to the second antigenic epitope y, and CH1 is the first constant region of the heavy chain. In some embodiments, the first antigenic epitope x and the second antigenic epitope y are epitopes on different antigens, specifically a first antigen a and a second antigen b. In some embodiments, the first antigenic epitope x and the second antigenic epitope y are different epitopes on the same antigen a (or antigen b).

[0007] In some embodiments, the antibody or antigen-binding fragment binds to two different antigens, namely a first antigen a and a second antigen b, and the antibody or antigen-binding fragment comprises at least two polypeptide chains; wherein the first polypeptide chain sequentially comprises VHa, CLa, VHb and CH1 starting from the amino terminus, VHa is the heavy chain variable region that binds to the first antigen a, CLa is the first light chain constant region, VHb is the heavy chain variable region that binds to the second antigen b, and CH1 is the first constant region of the heavy chain.

[0008] In some implementations, VHa and CLa are covalently linked via linker L1, which contains 2 to 6 amino acids; and / or

[0009] CLa and VHb are covalently linked via linker L2; wherein L2 contains 10 to 30 amino acids, and at least 50% of the amino acids are glycine.

[0010] In some embodiments, VHa and CLa are covalently linked via linker L1, which contains 2 to 6 amino acids; CLa and VHb are covalently linked via linker L2, which contains 10 to 30 amino acids, and at least 50% of the amino acids are glycine. In some embodiments, L1 contains about 2, about 3, about 4, about 5, or about 6 amino acids. In some embodiments, L2 contains about 10, about 11, about 13, about 14, about 17, about 18, about 20, about 21, about 22, about 25, about 27, about 28, about 29, or about 30 amino acids, or a range between any two of these values ​​(including the endpoint) or any value therein. In some embodiments, L2 contains serine. In some implementations, L2 contains about 8, about 10, about 11, about 13, about 15, about 17, about 21, about 25 glycines, or a range between any two of these values ​​(including the endpoint) or any of these values.

[0011] In some embodiments, the second polypeptide chain sequentially comprises VLa, CH1, VLb, and CLb starting from the amino terminus, where VLa is a light chain variable region binding to the first epitope x, VLb is a light chain variable region binding to the second epitope y, and CLb is a second light chain constant region. In some embodiments, the first antigenic epitope x and the second antigenic epitope y are epitopes on different antigens, specifically a first antigen a and a second antigen b. In some embodiments, the first antigenic epitope x and the second antigenic epitope y are different epitopes on the same antigen a (or antigen b).

[0012] In some embodiments, the second polypeptide chain sequentially comprises VLa, CH1, VLb, and CLb starting from the amino terminus; wherein VLa is a light chain variable region that binds to the first antigen a, VLb is a light chain variable region that binds to the second antigen b, and CLb is a second light chain constant region.

[0013] In some implementations, VLa and CH1 are covalently linked via linker L3, which contains 2 to 6 amino acids; and / or

[0014] CH1 and VLb are covalently linked via linker L4; wherein L4 contains 10 to 30 amino acids, and at least 50% of the amino acids are glycine.

[0015] In some embodiments, VLa and CH1 are covalently linked via linker L3, which contains 2 to 6 amino acids; CH1 and VLb are covalently linked via linker L4, wherein L4 contains 10 to 30 amino acids, and at least 50% of the amino acids are glycine. In some embodiments, L3 contains about 2, about 3, about 4, about 5, or about 6 amino acids. In some embodiments, L4 contains about 10, about 11, about 13, about 14, about 17, about 18, about 20, about 21, about 22, about 25, about 27, about 28, about 29, or about 30 amino acids, or a range between any two of these values ​​(including the endpoint) or any value therein. In some embodiments, L4 contains serine. In some implementations, L4 contains about 8, about 10, about 11, about 13, about 14, about 18, about 21, about 22 glycines, or a range between any two of these values ​​(including the endpoint) or any of these values.

[0016] In some embodiments, one of the two polypeptide chains further comprises an Fc, which includes a hinge region, a second constant region, and a third constant region of the heavy chain. In some embodiments, the Fc is a variant Fc region. In some embodiments, the variant Fc region has one or more amino acid modifications, such as substitution, deletion, or insertion, relative to the parental Fc region. In some embodiments, the amino acid modifications of the Fc region alter the effector functional activity relative to the activity of the parental Fc region. In some embodiments, the variant Fc region may have altered (i.e., increased or decreased) antibody-dependent cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization, or cell binding. In some embodiments, the amino acid modifications of the Fc region relative to the parental Fc region can alter the affinity of the variant Fc region for FcγR (Fcγ receptor).

[0017] In some implementations, the first polypeptide chain contains Fc.

[0018] In some embodiments, the first polypeptide chain comprises the structure VHa-L1-CLa-L2-VHb-CH1, and the second polypeptide chain comprises the structure VLa-L3-CH1-L4-VLb-CLb. In some embodiments, the first polypeptide chain comprises the structure VHa-L1-CLa-L2-VHb-CH1-Fc, and the second polypeptide chain comprises the structure VLa-L3-CH1-L4-VLb-CLb.

[0019] In some implementations, the CLa of the first polypeptide chain is connected to the CH1 of the second polypeptide chain via a disulfide bond, and the CH1 of the first polypeptide chain is connected to the CLb of the second polypeptide chain via a disulfide bond.

[0020] In some embodiments, antigens a and b are cytokines, cytokine receptors, chemokines, chemokine receptors, or cell surface proteins. In some embodiments, the antibody or antigen-binding fragment can specifically bind to cytokines. In some embodiments, cytokines include IL-1α (interleukin IL-1α), IL-1β (interleukin IL-1β), IL-13 (interleukin IL-13), IL-5 (interleukin IL-5), TNF-α (tumor necrosis factor α), TNF-β, and (tumor necrosis factor β), etc. In some embodiments, the antibody or antigen-binding fragment can specifically bind to immune checkpoint proteins. In some embodiments, the immune checkpoint proteins include TIM-3 (T cell immunoglobin domain and mucin domain-3), LAG-3 (lymphocyte activation gene-3 molecule), CTLA-4 (cytotoxic T lymphocyte-associated antigen), TIGIT (T cell Ig and ITIM domain), CD27 (differentiation cluster 27), OX40 (tumor necrosis factor receptor superfamily member 4), ICOS (inducible costimulator), BTLA (B and T lymphocyte attenuating factor), PD-1 (programmed death receptor 1), and CD137 (differentiation cluster 137), etc. In some embodiments, the antibody or antigen-binding fragment can specifically bind to cell surface proteins, such as tumor cell surface proteins PD-L1 (programmed death ligand 1), galactolectin 9, CD48 (differentiation cluster 48), CD40 (differentiation cluster 40), CD70 (differentiation cluster 70), B7H3 (CD276, differentiation cluster 276), and HVEM (Herpesvirus Entry Mediator), etc. In some embodiments, the antibody or antigen-binding fragment can bind to chemokines or chemokine receptors, such as CCL1, CCL3, CCL5, CCL7, CCL8, etc. in the CC chemokine subgroup.

[0021] In some implementations, antigens a and b are selected from the following groups: TIGIT and CTLA-4, OX40 and CTLA-4, TIGIT and PD-1, PD-L1 and CD47 (differentiation cluster 47), TIGIT and OX40, VEGF (vascular endothelial growth factor) and cMET (encoded by the c-met proto-oncogene), VEGF and DLL4 (delta-like ligand 4), VEGF and HGF (hepatocyte growth factor), VEGF and ANGPT2 (angiopoietin 2), TfR (transferrin receptor, CD71) and CD20 (differentiation cluster 20), PD-L1 and 4-1BB (CD137, a member of the tumor necrosis factor receptor superfamily), PSMA (prostate-specific membrane antigen) and CD28 (co-stimulatory molecule), PD-1 and PD-L1, HER2 (human epidermal growth factor receptor 2) and 4-1BB, PD-1 and TIM-3, PD CD47 and CD40 (differentiation cluster 47), GITR (glucocorticoid-induced tumor necrosis factor receptor) and CTLA-4, CD40 (differentiation cluster 40, tumor necrosis factor receptor superfamily member 5) and 4-1BB, OX40 and 4-1BB, LAG-3 and TIM-3, EGFR (epidermal growth factor receptor 1) and CTLA-4, CD19 (differentiation cluster 19) and CD22 (differentiation cluster 22), CD16 (differentiation cluster 16) and CD30 (differentiation cluster 30), CD3 (differentiation cluster 3) CD123 (differentiation cluster 123), BCMA (B cell maturation antigen) and CD47, MSLN (mesothelin) and CD47, EGFR and cMET, CD73 and TGFβ (transforming growth factor β), EGFR and TGFβ, CCR2 (CC chemokine receptor 2) and CSF1R (colony-stimulating factor 1 receptor), CD20 and CD3, CD19 and CD47, CDH17 (hepato-gut cadherin) and TRAILR2 (TRAIL receptor 2, TRAIL is a tumor marker). Tumor necrosis-associated apoptosis-inducing ligands (TALA), APLP2 (amyloid precursor protein 2) and HER2, IL-1α and IL-1β, IL-17 and IL-13, IL-4 and IL-13, BAFF (B cell activating factor) and IL-17A (interleukin-17A), CD3 and PD-1, IL-4Ra (interleukin-4 receptor subunit α) and IL-5, VEGF and IL-6 (interleukin IL-6), FGFR1 (fibroblast growth factor receptor 1) and KLB (klothobeta protein). In some embodiments, the antibody or antigen-binding fragment can simultaneously and specifically bind to antigen a and antigen b.

[0022] In some embodiments, antigen a is TIGIT and antigen b is CTLA-4. In some embodiments, antigen a is OX40 and antigen b is CTLA-4. In some embodiments, antigen a is OX40 and antigen b is TIGIT.

[0023] In some embodiments, antigen a is TIGIT and antigen b is CTLA-4, and the antibody or antigen-binding fragment comprises the following:

[0024] The VHa contains the heavy chain CDR or heavy chain variable region disclosed in US20190100591A1 or US20180169239A1;

[0025] The VHb contains the heavy chain CDR or heavy chain variable region disclosed in CN1404876A or US9963508; and / or

[0026] The VLa contains a light chain CDR or a light chain variable region disclosed in US20190100591A1 or US20180169239A1; and / or

[0027] The VLb contains the light chain CDR or light chain variable region disclosed in CN1404876A or US9963508.

[0028] The entire contents of US20190100591A1, US20180169239A1, CN1404876A, and US9963508 are incorporated herein by reference.

[0029] In some embodiments, antigen a is TIGIT and antigen b is CTLA-4, and the antibody or antigen-binding fragment comprises the following:

[0030] The VHa contains amino acids at positions 30-35 (VHaCDR1, SSYGMS) and / or positions 50-66 (VHaCDR2, TINSNGGSTYYPDSVKG) and / or positions 99-108 (VHaCDR3, LGTGTLGFAY) of the sequence shown in SEQ ID NO:1; and / or

[0031] The VHb contains amino acids 31-35 (VHbCDR1, SYTMH) and / or amino acids 50-66 (VHbCDR2, FISYDGNNKYYADSVKG) and / or amino acids 99-107 (VHbCDR3, TGWLGPFDY) in the sequence shown in SEQ ID NO:2; and / or

[0032] The VLa contains amino acids 24-34 (VLaCDR1, KASQDVKTAVS) and / or amino acids 50-56 (VLaCDR2, WASTRAT) and / or amino acids 89-97 (VLaCDR3, QQHYSTPWT) in the sequence shown in SEQ ID NO:3; and / or

[0033] The VLb contains amino acids 24-35 (VLbCDR1, RASQSVGSSYLA) and / or amino acids 51-57 (VLbCDR2, GAFSRAT) and / or amino acids 90-98 (VLbCDR3, QQYGSSPWT) in the sequence shown in SEQ ID NO:4.

[0034] In some embodiments, antigen a is TIGIT and antigen b is CTLA-4, and the antibody or antigen-binding fragment comprises the following:

[0035] The VHa contains the sequence shown in SEQ ID NO:1, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:1, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:1; and / or

[0036] The VHb contains the sequence shown in SEQ ID NO:2, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:2, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:2; and / or

[0037] The VLa contains the sequence shown in SEQ ID NO:3, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:3, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:3; and / or

[0038] The VLb contains the sequence shown in SEQ ID NO:4, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:4, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:4;

[0039] The CLa contains the sequence shown in SEQ ID NO:5, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:5, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:5;

[0040] The CLb contains the sequence shown in SEQ ID NO:6, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:6, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:6;

[0041] The CH1 contains the sequence shown in SEQ ID NO:7, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:7, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:7.

[0042] In some embodiments, antigen a is OX40 and antigen b is CTLA-4, and the antibody or antigen-binding fragment comprises the following:

[0043] The VHa contains the heavy chain CDR or heavy chain variable region disclosed in CN101331150A1 or US20150307617A1;

[0044] The VHb contains the heavy chain CDR or heavy chain variable region disclosed in CN1404876A or US9963508; and / or

[0045] The VLa contains the light chain CDR or light chain variable region disclosed in CN101331150A1 or US20150307617A1; and / or

[0046] The VLb contains the light chain CDR or light chain variable region disclosed in CN1404876A or US99635.

[0047] The entire contents of CN101331150A1, US20150307617A1, CN1404876A, and US9963508 are incorporated herein by reference.

[0048] In some embodiments, antigen a is OX40 and antigen b is CTLA-4, and the antibody or antigen-binding fragment comprises the following:

[0049] The VHa contains amino acids 31-35 (VHaCDR1, SYGMH) and / or amino acids 50-66 (VHaCDR2, VIAEVGSNQYYADSVKG) and / or amino acids 99-111 (VHaCDR3, DNQDTSPDVGIDY) in the sequence shown in SEQ ID NO:8; and / or

[0050] The VHb contains amino acids 31-35 (VHbCDR1, SYTMH) and / or amino acids 50-66 (VHbCDR2, FISYDGNNKYYADSVKG) and / or amino acids 99-107 (VHbCDR3, TGWLGPFDY) in the sequence shown in SEQ ID NO:9; and / or

[0051] The VLa contains amino acids 24-34 (VLaCDR1, RASQNISPFLN) and / or amino acids 50-56 (VLaCDR2, AAVGLQS) and / or amino acids 89-97 (VLaCDR3, QQYTDYPLT) in the sequence shown in SEQ ID NO:10; and / or

[0052] The VLb contains amino acids 24-35 (VLbCDR1, RASQSVGSSYLA) and / or amino acids 51-57 (VLbCDR2, GAFSRAT) and / or amino acids 90-98 (VLbCDR3, QQYGSSPWT) in the sequence shown in SEQ ID NO:11.

[0053] In some embodiments, when antigen a is OX40 and antigen b is CTLA-4, the antibody or antigen-binding fragment comprises the following:

[0054] The VHa contains the sequence shown in SEQ ID NO:8, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:8, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:8; and / or

[0055] The VHb contains the sequence shown in SEQ ID NO:9, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:9, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:9; and / or

[0056] The VLa contains the sequence shown in SEQ ID NO:10, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:10, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:10; and / or

[0057] The VLb contains the sequence shown in SEQ ID NO:11, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:11, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:11.

[0058] The CLa contains the sequence shown in SEQ ID NO:12, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:12, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:12;

[0059] The CLb contains the sequence shown in SEQ ID NO:13, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:13, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:13;

[0060] The CH1 contains the sequence shown in SEQ ID NO:14, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:14, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:14.

[0061] In some embodiments, antigen a is OX40 and antigen b is TIGIT, and the antibody or antigen-binding fragment comprises the following:

[0062] The VHa contains the heavy chain CDR or heavy chain variable region disclosed in CN101331150A1 or US20150307617A1;

[0063] The VHb contains the heavy chain CDR or heavy chain variable region disclosed in US20190100591A1 or US20180169239A1; and / or

[0064] The VLa contains the light chain CDR or light chain variable region disclosed in CN101331150A1 or US20150307617A1; and / or

[0065] The VLb contains the light chain CDR or light chain variable region disclosed in US20190100591A1 or US20180169239A1.

[0066] In some embodiments, antigen a is OX40 and antigen b is TIGIT, and the antibody or antigen-binding fragment comprises the following:

[0067] The VHa contains amino acids 31-35 (VHaCDR1, SYGMH) and / or amino acids 50-66 (VHaCDR2, VIAEVGSNQYYADSVKG) and / or amino acids 99-111 (VHaCDR3, DNQDTSPDVGIDY) in the sequence shown in SEQ ID NO:15; and / or

[0068] The VHb contains amino acids at positions 30-35 (VHbCDR1, SSYGMS) and / or positions 50-66 (VHbCDR2, TINSNGGSTYYPDSVKG) and / or positions 99-108 (VHbCDR3, LGTGTLGFAY) of the sequence shown in SEQ ID NO:16; and / or

[0069] The VLa contains amino acids 24-34 (VLaCDR1, RASQNISPFLN) and / or amino acids 50-56 (VLaCDR2, AAVGLQS) and / or amino acids 89-97 (VLaCDR3, QQYTDYPLT) in the sequence shown in SEQ ID NO:17; and / or

[0070] The VLb contains amino acids 24-34 (VLbCDR1, KASQDVKTAVS) and / or amino acids 50-56 (VLbCDR2, WASTRAT) and / or amino acids 89-97 (VLbCDR3, QQHYSTPWT) in the sequence shown in SEQ ID NO:18.

[0071] In some embodiments, antigen a is OX40 and antigen b is TIGIT, and the antibody or antigen-binding fragment comprises the following:

[0072] The VHa contains the sequence shown in SEQ ID NO:15, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:15, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:15; and / or

[0073] The VHb contains the sequence shown in SEQ ID NO:16, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:16, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:16; and / or

[0074] The VLa contains the sequence shown in SEQ ID NO:17, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:17, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:17; and / or

[0075] The VLb contains the sequence shown in SEQ ID NO:18, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:18, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:18.

[0076] The CLa contains the sequence shown in SEQ ID NO:19, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:19, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:19;

[0077] The CLb contains the sequence shown in SEQ ID NO:20, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:20, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:20;

[0078] The CH1 contains the sequence shown in SEQ ID NO:21, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:21, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:21.

[0079] In some embodiments, the sequence with at least 80% identity is approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 90%, approximately 91%, approximately 92%, approximately 94%, approximately 95%, approximately 98%, approximately 99%, or a range between any two of these values ​​(including the endpoint) or any value therein. In some embodiments, one or more conserved amino acid substitutions are approximately 1, approximately 2, approximately 3, approximately 4, approximately 5, approximately 6, approximately 8, approximately 9, approximately 10, approximately 11, approximately 13, approximately 14, or approximately 15 conserved amino acid substitutions, or a range between any two of these values ​​(including the endpoint) or any value therein.

[0080] In some embodiments, L1 contains the sequence shown in SEQ ID NO:22;

[0081] The L2 contains a sequence selected from any one of SEQ ID NO:23-27, a sequence having at least 90% identity with a sequence shown in any one of SEQ ID NO:23-27, or an amino acid sequence having one or more conserved amino acid substitutions compared to a sequence shown in any one of SEQ ID NO:23-27; and / or

[0082] The L3 contains the sequence shown in SEQ ID NO:28; and / or

[0083] The L4 contains a sequence selected from any one of SEQ ID NO:29-33, a sequence having at least 90% identity with any one of the sequences shown in SEQ ID NO:29-33, or an amino acid sequence having one or more conserved amino acid substitutions compared to any one of the sequences shown in SEQ ID NO:29-33.

[0084] In some embodiments, L2 contains the sequence shown in SEQ ID NO:23, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:23, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:23; L4 contains the sequence shown in SEQ ID NO:29, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:29, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:29.

[0085] In some embodiments, L2 contains the sequence shown in SEQ ID NO:24, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:24, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:24; L4 contains the sequence shown in SEQ ID NO:30, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:30, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:30.

[0086] In some embodiments, L2 contains the sequence shown in SEQ ID NO:25, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:25, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:25; L4 contains the sequence shown in SEQ ID NO:31, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:31, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:31.

[0087] In some embodiments, L2 contains the sequence shown in SEQ ID NO:26, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:26, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:26; L4 contains the sequence shown in SEQ ID NO:32, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:32, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:32.

[0088] In some embodiments, L2 contains the sequence shown in SEQ ID NO:27, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:27, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:27; L4 contains the sequence shown in SEQ ID NO:33, a sequence having at least 90% identity with the sequence shown in SEQ ID NO:33, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:33.

[0089] In some embodiments, the sequence with at least 90% identity is about 90% identity, about 91% identity, about 92% identity, about 93% identity, about 95% identity, about 96% identity, about 97% identity, about 98% identity, about 99% identity, or a range between any two of these values ​​(including the endpoint) or any value therein. In some embodiments, one or more conserved amino acid substitutions are about 1, about 2, about 3, about 4, or about 5 conserved amino acid substitutions.

[0090] In some embodiments, L1 contains the sequence shown in SEQ ID NO:22, L2 contains the sequence shown in SEQ ID NO:23, L3 contains a sequence selected from the sequence shown in SEQ ID NO:28, and L4 contains the sequence shown in SEQ ID NO:29.

[0091] In some embodiments, L1 contains the sequence shown in SEQ ID NO:22, L2 contains the sequence shown in SEQ ID NO:24, L3 contains the sequence shown in SEQ ID NO:28, and L4 contains the sequence shown in SEQ ID NO:30.

[0092] In some embodiments, L1 contains the sequence shown in SEQ ID NO:22, L2 contains the sequence shown in SEQ ID NO:25, L3 contains the sequence shown in SEQ ID NO:28, and L4 contains the sequence shown in SEQ ID NO:31.

[0093] In some embodiments, L1 contains the sequence shown in SEQ ID NO:22, L2 contains the sequence shown in SEQ ID NO:26, L3 contains the sequence shown in SEQ ID NO:28, and L4 contains the sequence shown in SEQ ID NO:32.

[0094] In some embodiments, L1 contains the sequence shown in SEQ ID NO:22, L2 contains the sequence shown in SEQ ID NO:27, L3 contains the sequence shown in SEQ ID NO:28, and L4 contains the sequence shown in SEQ ID NO:33.

[0095] In some embodiments, the Fc contains the sequence shown in any one of SEQ ID NO:34-36, a sequence having at least 80% identity with the sequence shown in any one of SEQ ID NO:34-36, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in any one of SEQ ID NO:34-36. In some embodiments, the sequence with at least 80% identity is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or a range (including the endpoint) between any two of these values, or any value therein. In some embodiments, one or more conserved amino acid substitutions are about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, about 17, about 19, about 21, about 22, or about 25 conserved amino acid substitutions, or a range between any two of these values ​​(including the endpoint) or any of these values.

[0096] In some embodiments, the Fc contains the sequence shown in SEQ ID NO:34. In some embodiments, the Fc contains the sequence shown in SEQ ID NO:35. In some embodiments, the Fc contains the sequence shown in SEQ ID NO:36.

[0097] The present invention also provides an antibody or antigen-binding fragment that binds to two different antigens, TIGIT and CTLA-4; the antibody or antigen-binding fragment comprises:

[0098] Amino acids 30-35 (VHaCDR1, SSYGMS) and / or 50-66 (VHaCDR2, TINSNGGSTYYPDSVKG) and / or 99-108 (VHaCDR3, LGTGTLGFAY) in the sequence shown in SEQ ID NO:1; and

[0099] Amino acids 31-35 (VHbCDR1, SYTMH) and / or amino acids 50-66 (VHbCDR2, FISYDGNNKYYADSVKG) and / or amino acids 99-107 (VHbCDR3, TGWLGPFDY) in the sequence shown in SEQ ID NO:2; and / or

[0100] The sequence shown in SEQ ID NO:3 contains amino acids 24-34 (VLaCDR1, KASQDVKTAVS), and / or amino acids 50-56 (VLaCDR2, WASTRAT), and / or amino acids 89-97 (VLaCDR3, QQHYSTPWT); and

[0101] The amino acids at positions 24-35 (VLbCDR1, RASQSVGSSYLA), and / or positions 51-57 (VLbCDR2, GAFSRAT), and / or positions 90-98 (VLbCDR3, QQYGSSPWT) in the sequence shown in SEQ ID NO:4.

[0102] In some embodiments, the antibody or antigen-binding fragment comprises at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain.

[0103] The first polypeptide chain contains amino acids at positions 30-35 (VHaCDR1, SSYGMS) and / or positions 50-66 (VHaCDR2, TINSNGGSTYYPDSVKG) and / or positions 99-108 (VHaCDR3, LGTGTLGFAY) of the sequence shown in SEQ ID NO:1; and

[0104] Amino acids 31-35 (VHbCDR1, SYTMH) and / or amino acids 50-66 (VHbCDR2, FISYDGNNKYYADSVKG) and / or amino acids 99-107 (VHbCDR3, TGWLGPFDY) in the sequence shown in SEQ ID NO:2; and / or

[0105] The second polypeptide chain contains amino acids 24-34 (VLaCDR1, KASQDVKTAVS) and / or amino acids 50-56 (VLaCDR2, WASTRAT) and / or amino acids 89-97 (VLaCDR3, QQHYSTPWT) in the sequence shown in SEQ ID NO:3; and

[0106] The amino acids at positions 24-35 (VLbCDR1, RASQSVGSSYLA), and / or positions 51-57 (VLbCDR2, GAFSRAT), and / or positions 90-98 (VLbCDR3, QQYGSSPWT) in the sequence shown in SEQ ID NO:4.

[0107] This invention also provides an antibody or antigen-binding fragment that binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is TIGIT and the second antigen b is CTLA-4; the antibody or antigen-binding fragment comprises at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO:37, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:37, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:37; the second polypeptide chain contains the sequence shown in SEQ ID NO:38, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:38, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:38; or

[0108] The first polypeptide chain contains the sequence shown in SEQ ID NO:39, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:39, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:39; the second polypeptide chain contains the sequence shown in SEQ ID NO:40, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:40, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:40; or

[0109] The first polypeptide chain contains the sequence shown in SEQ ID NO:41, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:41, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:41; the second polypeptide chain contains the sequence shown in SEQ ID NO:42, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:42, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:42; or

[0110] The first polypeptide chain contains the sequence shown in SEQ ID NO:43, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:43, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:43; the second polypeptide chain contains the sequence shown in SEQ ID NO:44, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:44, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:44.

[0111] In some implementations, the sequence with at least 80% identity is approximately 80% identity, approximately 81% identity, approximately 82% identity, approximately 83% identity, approximately 85% identity, approximately 86% identity, approximately 87% identity, approximately 88% identity, approximately 90% identity, approximately 91% identity, approximately 92% identity, approximately 94% identity, approximately 95% identity, approximately 98% identity, approximately 99% identity, or a range (including the endpoint) between any two of these values ​​or any value therein. In some embodiments, one or more conserved amino acid substitutions are about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, about 17, about 19, about 21, about 22, about 25, about 27, about 29, about 31, about 33, about 35, about 38, about 41, about 42, about 47, about 49 conserved amino acid substitutions, or a range between any two of these values ​​(including the endpoint) or any value therein.

[0112] In some embodiments, the antibody or antigen-binding fragment binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is TIGIT and the second antigen b is CTLA-4, and the antibody or antigen-binding fragment comprises at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO:37, and the second polypeptide chain contains the sequence shown in SEQ ID NO:38.

[0113] In some embodiments, the antibody or antigen-binding fragment binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is TIGIT and the second antigen b is CTLA-4, and the antibody or antigen-binding fragment comprises at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO:39, and the second polypeptide chain contains the sequence shown in SEQ ID NO:40.

[0114] In some embodiments, the antibody or antigen-binding fragment binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is TIGIT and the second antigen b is CTLA-4, and the antibody or antigen-binding fragment comprises at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO:41, and the second polypeptide chain contains the sequence shown in SEQ ID NO:42.

[0115] In some embodiments, the antibody or antigen-binding fragment binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is TIGIT and the second antigen b is CTLA-4, and the antibody or antigen-binding fragment comprises at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO:43, and the second polypeptide chain contains the sequence shown in SEQ ID NO:44.

[0116] The present invention also provides an antibody or antigen-binding fragment that binds to two different antigens, OX40 and CTLA-4; the antibody or antigen-binding fragment comprises:

[0117] Amino acids 31-35 (VHaCDR1, SYGMH) and / or amino acids 50-66 (VHaCDR2, VIAEVGSNQYYADSVKG) and / or amino acids 99-111 (VHaCDR3, DNQDTSPDVGIDY) in the sequence shown in SEQ ID NO:8; and

[0118] Amino acids 31-35 (VHbCDR1, SYTMH) and / or amino acids 50-66 (VHbCDR2, FISYDGNNKYYADSVKG) and / or amino acids 99-107 (VHbCDR3, TGWLGPFDY) in the sequence shown in SEQ ID NO:9; and / or

[0119] The sequence shown in SEQ ID NO:10 contains amino acids 24-34 (VLaCDR1, RASQNISPFLN), and / or amino acids 50-56 (VLaCDR2, AAVGLQS), and / or amino acids 89-97 (VLaCDR3, QQYTDYPLT); and

[0120] The amino acids at positions 24-35 (VLbCDR1, RASQSVGSSYLA), and / or positions 51-57 (VLbCDR2, GAFSRAT), and / or positions 90-98 (VLbCDR3, QQYGSSPWT) in the sequence shown in SEQ ID NO:11.

[0121] In some embodiments, the antibody or antigen-binding fragment comprises at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain.

[0122] The first polypeptide chain contains amino acids 31-35 (VHaCDR1, SYGMH) and / or amino acids 50-66 (VHaCDR2, VIAEVGSNQYYADSVKG) and / or amino acids 99-111 (VHaCDR3, DNQDTSPDVGIDY) in the sequence shown in SEQ ID NO:8; and

[0123] Amino acids 31-35 (VHbCDR1, SYTMH) and / or amino acids 50-66 (VHbCDR2, FISYDGNNKYYADSVKG) and / or amino acids 99-107 (VHbCDR3, TGWLGPFDY) in the sequence shown in SEQ ID NO:9; and / or

[0124] The second polypeptide chain contains amino acids 24-34 (VLaCDR1, RASQNISPFLN) and / or amino acids 50-56 (VLaCDR2, AAVGLQS) and / or amino acids 89-97 (VLaCDR3, QQYTDYPLT) in the sequence shown in SEQ ID NO:10; and

[0125] The amino acids at positions 24-35 (VLbCDR1, RASQSVGSSYLA), and / or positions 51-57 (VLbCDR2, GAFSRAT), and / or positions 90-98 (VLbCDR3, QQYGSSPWT) in the sequence shown in SEQ ID NO:11.

[0126] The present invention also provides an antibody or antigen-binding fragment that binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is OX40 and the second antigen b is CTLA-4; the antibody or antigen-binding fragment comprises at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO:45, having at least 80% identity with the sequence shown in SEQ ID NO:45, or having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:45; the second polypeptide chain contains the sequence shown in SEQ ID NO:46, having at least 80% identity with the sequence shown in SEQ ID NO:46, or having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:46. In some implementations, the sequence with at least 80% identity is approximately 80% identity, approximately 81% identity, approximately 82% identity, approximately 83% identity, approximately 85% identity, approximately 86% identity, approximately 87% identity, approximately 88% identity, approximately 90% identity, approximately 91% identity, approximately 92% identity, approximately 94% identity, approximately 95% identity, approximately 98% identity, approximately 99% identity, or a range (including the endpoint) between any two of these values ​​or any value therein. In some embodiments, one or more conserved amino acid substitutions are about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, about 17, about 19, about 21, about 22, about 25, about 27, about 29, about 31, about 33, about 35, about 38, about 41, about 42, about 47, about 49 conserved amino acid substitutions, or a range between any two of these values ​​(including the endpoint) or any value therein.

[0127] In some embodiments, the antibody or antigen-binding fragment binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is OX40 and the second antigen b is CTLA-4, and the antibody or antigen-binding fragment comprises at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO:45, and the second polypeptide chain contains the sequence shown in SEQ ID NO:46.

[0128] The present invention also provides an antibody or antigen-binding fragment that binds to two different antigens, OX40 and TIGIT; the antibody or antigen-binding fragment comprises:

[0129] Amino acids 31-35 (VHaCDR1, SYGMH) and / or amino acids 50-66 (VHaCDR2, VIAEVGSNQYYADSVKG) and / or amino acids 99-111 (VHaCDR3, DNQDTSPDVGIDY) in the sequence shown in SEQ ID NO:15; and

[0130] Amino acids 30-35 (VHbCDR1, SSYGMS) and / or 50-66 (VHbCDR2, TINSNGGSTYYPDSVKG) and / or 99-108 (VHbCDR3, LGTGTLGFAY) in the sequence shown in SEQ ID NO:16; and / or

[0131] The sequence shown in SEQ ID NO:17 contains amino acids 24-34 (VLaCDR1, RASQNISPFLN), and / or amino acids 50-56 (VLaCDR2, AAVGLQS), and / or amino acids 89-97 (VLaCDR3, QQYTDYPLT); and

[0132] The amino acids at positions 24-34 (VLbCDR1, KASQDVKTAVS), and / or positions 50-56 (VLbCDR2, WASTRAT), and / or positions 89-97 (VLbCDR3, QQHYSTPWT) in the sequence shown in SEQ ID NO:18.

[0133] In some embodiments, the antibody or antigen-binding fragment comprises at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain.

[0134] The first polypeptide chain contains amino acids 31-35 (VHaCDR1, SYGMH) and / or amino acids 50-66 (VHaCDR2, VIAEVGSNQYYADSVKG) and / or amino acids 99-111 (VHaCDR3, DNQDTSPDVGIDY) in the sequence shown in SEQ ID NO:15; and

[0135] Amino acids 30-35 (VHbCDR1, SSYGMS) and / or 50-66 (VHbCDR2, TINSNGGSTYYPDSVKG) and / or 99-108 (VHbCDR3, LGTGTLGFAY) in the sequence shown in SEQ ID NO:16; and / or

[0136] The second polypeptide chain contains amino acids 24-34 (VLaCDR1, RASQNISPFLN) and / or amino acids 50-56 (VLaCDR2, AAVGLQS) and / or amino acids 89-97 (VLaCDR3, QQYTDYPLT) in the sequence shown in SEQ ID NO:17; and

[0137] The amino acids at positions 24-34 (VLbCDR1, KASQDVKTAVS), and / or positions 50-56 (VLbCDR2, WASTRAT), and / or positions 89-97 (VLbCDR3, QQHYSTPWT) in the sequence shown in SEQ ID NO:18.

[0138] The present invention also provides an antibody or antigen-binding fragment that binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is OX40 and the second antigen b is TIGIT; the antibody or antigen-binding fragment comprises at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO:47 or 49, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:47 or 49, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:47 or 49; the second polypeptide chain contains the sequence shown in SEQ ID NO:48 or 50, a sequence having at least 80% identity with the sequence shown in SEQ ID NO:48 or 50, or an amino acid sequence having one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:48 or 50. In some implementations, the sequence with at least 80% identity is approximately 80% identity, approximately 81% identity, approximately 82% identity, approximately 83% identity, approximately 85% identity, approximately 86% identity, approximately 87% identity, approximately 88% identity, approximately 90% identity, approximately 91% identity, approximately 92% identity, approximately 94% identity, approximately 95% identity, approximately 98% identity, approximately 99% identity, or a range (including the endpoint) between any two of these values ​​or any value therein. In some embodiments, one or more conserved amino acid substitutions are about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, about 17, about 19, about 21, about 22, about 25, about 27, about 29, about 31, about 33, about 35, about 38, about 41, about 42, about 47, about 49 conserved amino acid substitutions, or a range between any two of these values ​​(including the endpoint) or any value therein.

[0139] In some embodiments, the antibody or antigen-binding fragment binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is OX40 and the second antigen b is TIGIT, and the antibody or antigen-binding fragment comprises at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO:47, and the second polypeptide chain contains the sequence shown in SEQ ID NO:48.

[0140] In some embodiments, the antibody or antigen-binding fragment binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is OX40 and the second antigen b is TIGIT, and the antibody or antigen-binding fragment comprises at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO:49, and the second polypeptide chain contains the sequence shown in SEQ ID NO:50.

[0141] In some embodiments, the antibody or antigen-binding fragment contains a first polypeptide chain and a third polypeptide chain with the same sequence, as well as a second polypeptide chain and a fourth polypeptide chain with the same sequence. In some embodiments, the antibody or antigen-binding fragment contains a first polypeptide chain and a third polypeptide chain with the same sequence, as well as a second polypeptide chain and a fourth polypeptide chain with the same sequence, wherein the Fc regions of the first and third polypeptide chains pair to form disulfide bonds.

[0142] In some embodiments, the antibody or antigen-binding fragment is a separated antibody or antigen-binding fragment.

[0143] The present invention also provides nucleic acid molecules encoding the aforementioned antibody or antigen-binding fragments. In some embodiments, the nucleic acid molecule is an isolated nucleic acid molecule.

[0144] The present invention also provides a vector comprising the aforementioned nucleic acid molecule. In some embodiments, the vector is an isolated vector.

[0145] The present invention also provides host cells comprising the nucleic acid molecule or vector. In some embodiments, the host cell is an isolated host cell. In some embodiments, the host cell is a CHO cell, a 293 cell, a Cos1 cell, a Cos7 cell, a CV1 cell, or a mouse L cell.

[0146] The present invention also provides pharmaceutical compositions comprising the above-described antibody or antigen-binding fragments, and pharmaceutically acceptable excipients.

[0147] This invention also provides treatment methods and uses. In some embodiments, methods for treating or improving various diseases (such as inflammatory diseases, autoimmune diseases, neurodegenerative diseases, cancer, or spinal cord injuries) are provided, the methods comprising administering an effective dose of the said antibody or antigen-binding fragment to a patient. In some embodiments, the use of the said antibody or antigen-binding fragment in medicaments for treating or improving various diseases (such as inflammatory diseases, autoimmune diseases, cancer, or spinal cord injuries) is provided. In some embodiments, the use of the said antibody or antigen-binding fragment in the preparation of medicaments for treating or improving various diseases (such as inflammatory diseases, autoimmune diseases, cancer, or spinal cord injuries) is provided.

[0148] In some embodiments, the autoimmune disease or inflammatory disease is selected from the group consisting of: Crohn's disease, psoriasis (including plaque psoriasis), arthritis (including rheumatoid arthritis, psoriatic arthritis, osteoarthritis, or juvenile idiopathic arthritis), multiple sclerosis, ankylosing spondylitis, and spondylosing arthritis. Arthropathy, systemic lupus erythematosus, uveitis, sepsis, neurodegenerative diseases, neuronal regeneration, spinal cord injury, primary and metastatic cancer, respiratory diseases, asthma, allergic and non-allergic asthma, asthma caused by infection, asthma caused by respiratory syncytial virus (RSV), chronic obstructive pulmonary disease (COPD), symptoms involving airway inflammation, eosinophilia, fibrosis and excessive mucus production, cystic fibrosis, pulmonary fibrosis, atopic diseases, atopic dermatitis, urticaria, eczema, allergic rhinitis, allergic gastroenteritis, inflammatory and / or autoimmune skin conditions, inflammatory and / or autoimmune gastrointestinal organ conditions, inflammatory bowel disease (IBD), ulcerative colitis, inflammatory and / or autoimmune liver diseases, cirrhosis, liver fibrosis, liver fibrosis caused by hepatitis B and / or hepatitis C virus, scleroderma. In some embodiments, the cancer is selected from the group consisting of hepatocellular carcinoma, glioblastoma, lymphoma, or Hodgkin's lymphoma. In some embodiments, the cancer is selected from the group consisting of melanoma (e.g., metastatic malignant melanoma), renal cell carcinoma (e.g., clear cell carcinoma), prostate cancer (e.g., hormone-refractory prostate adenocarcinoma), pancreatic cancer, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic malignancies. In some implementations, the cancer is selected from the group consisting of: Hodgkin's lymphoma, non-Hodgkin's lymphoma [NHL], precursor B-cell lymphoblastic leukemia / lymphoma, mature B-cell neoplasm, B-cell chronic lymphocytic leukemia / small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, follicular lymphoma, cutaneous follicular center lymphoma, marginal zone B-cell lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, plasmacytoma, plasmacytic myeloma, post-transplant lymphoproliferative disorder, Waldenstrom's macroglobulinemia, and anaplastic large cell lymphoma.

[0149] This invention also provides diagnostic methods and uses. In some embodiments, a method is provided for detecting the expression of antigen a and / or antigen b in a sample, by contacting the sample with the antibody or antigen-binding fragment, causing the antibody or antigen-binding fragment to bind to antigen a and / or antigen b, and detecting the binding, i.e., the content of antigen a and / or antigen b in the sample. In some embodiments, antigens a and b are cytokines, cytokine receptors, chemokines, chemokine receptors, or cell surface proteins. In some implementations, antigens a and b are selected from the following groups: TIGIT and CTLA-4, OX40 and CTLA-4, TIGIT and PD-1, PD-L1 and CD47, TIGIT and OX40, VEGF and cMET, VEGF and DLL4, VEGF and HGF, VEGF and ANGPT2, TfR and CD20, PD-L1 and 4-1BB, PSMA and CD28, PD-1 and PD-L1, HER2 and 4-1BB, PD-1 and TIM-3, PD-1 and CD47, GITR and CTLA-4, CD40 and 4-1BB, OX40 and 4-1BB, LAG-3 and T IM-3, EGFR and CTLA-4, CD19 and CD22, CD16 and CD30, CD3 and CD123, BCMA and CD47, MSLN and CD47, EGFR and cMET, CD73 and TGFβ, EGFR and TGFβ, CCR2 and CSF1R, CD20 and CD3, CD19 and CD47, CDH17 and TRAILR2, APLP2 and Her2, IL-1α and IL-1β, IL-17 and IL-13, IL-4 and IL-13, BAFF and IL-17A, CD3 and PD-1, IL-4Ra and IL-5, VEGF and IL-6, FGFR1 and KLB.

[0150] In some embodiments, the use of the said antibody or antigen-binding fragment in the preparation of kits for diagnosing inflammatory diseases, autoimmune diseases, neurodegenerative diseases, cancer, or spinal cord injuries is provided. In some embodiments, diagnostic kits comprising the said antibody or antigen-binding fragment are provided.

[0151] This invention provides multivalent and multispecific antibody or antigen-binding fragments and their applications. The multispecific antibody or antigen-binding fragments of this invention can bind to two or more antigens, or two or more epitopes of the same antigen. The antibody or antigen-binding fragments of this invention are used to treat or improve various diseases, such as inflammatory diseases, autoimmune diseases, cancer, or spinal cord injuries, and also for the diagnosis and prognosis of related diseases. Attached Figure Description

[0152] Figure 1This is a schematic diagram of the structure of the dual-characteristic antibody of the present invention in some embodiments.

[0153] Figure 2 The image shown is an SDS-PAGE pattern of antibody 1 in an embodiment of the present invention; wherein, lane 1 indicates that antibody 1 is in a non-reduced state, lane M indicates the maker, and lane 2 indicates that antibody 1 is in a reduced state.

[0154] Figure 3 The above are SDS-PAGE spectra of antibodies 2 and 3 in this embodiment of the invention; in the figure, CT-20BiAb represents antibody 2, CT-25BiAb represents antibody 3, lane 1 represents antibody 3 in a non-reduced state, lane 2 represents antibody 2 in a non-reduced state, lane 3 represents antibody 3 in a reduced state, lane 4 represents antibody 2 in a reduced state, and M represents maker.

[0155] Figure 4 The binding activity of antibodies 1, 2, and 3 to TIGIT-Fc is shown; where CT BiAb represents antibody 1, CT-20BiAb represents antibody 2, and CT-25BiAb represents antibody 3.

[0156] Figure 5 The binding activity of antibodies 1, 2, and 3 to CTLA-4-Fc is shown; where CT BiAb represents antibody 1, CT-20BiAb represents antibody 2, and CT-25BiAb represents antibody 3.

[0157] Figure 6 The biological activity of the antibody was detected using Promega's anti-CTLA-4 reporter gene assay system; where CTLA-4 / TIGIT BiAb represents antibody 1.

[0158] Figure 7 The biological activity of the antibody was detected using Promega's anti-CTLA-4 reporter gene assay system; CT-20BiAb represents antibody 2.

[0159] Figure 8 The biological activity of the antibody was detected using the anti-TIGIT reporter gene detection system; where CT BiAb represents antibody 1.

[0160] Figure 9 The biological activity of the antibody was detected using the anti-TIGIT reporter gene detection system; CT-20BiAb represents antibody 2.

[0161] Figure 10 The biological activity of the antibody was detected using the anti-TIGIT reporter gene detection system; CT-25BiAb represents antibody 3.

[0162] Figure 11 This is the SDS-PAGE pattern of antibody 5 (CTLA-4-OX40 bispecific antibody) in an embodiment of the present invention; where lane represents a swimming lane, and lane 1, lane 2 and lane 3 are arranged from left to right.

[0163] Figure 12 Antibody 5 and OX40 in the embodiments of the present invention + Cell binding curve; OX40 + The cells are those expressing OX40; the horizontal axis represents the logarithm of the antibody concentration, and the vertical axis represents the average fluorescence value of the two channels read on the flow cytometer; where O4 represents antibody 5.

[0164] Figure 13 This is the affinity kinetic fitting curve of antibody 5 binding to OX40 antigen and CTLA-4 antigen in an embodiment of the present invention, where O4 represents antibody 5.

[0165] Figure 14 The above are SDS-PAGE patterns of antibodies 6 and 7 in this embodiment of the invention; wherein, lane M represents the maker, lane 1 represents antibody 6 in a non-reduced state, lane 2 represents antibody 6 in a reduced state, lane 3 represents antibody 7 in a non-reduced state, and lane 4 represents antibody 7 in a reduced state; OT-4D-13aa antibody represents antibody 6, and OT-4D-30aa antibody represents antibody 7.

[0166] Figure 15 This is the affinity kinetic fitting curve of antibody 7 binding to OX40 antigen and TIGIT antigen in an embodiment of the present invention.

[0167] Figure 16 The figure shows the binding curve of antibody 7 to Jurkat-OX40 cells in an embodiment of the present invention; wherein, anti-OX40Ab represents OX40 monoclonal antibody (i.e., anti-OX40), and OT-4D-30a and 4D-30a both represent antibody 7; the unit of EC50 in the figure is nM.

[0168] Figure 17 The image shows the binding curve of antibody 7 to Jurkat-TIGIT cells in this embodiment of the invention; where anti-TigitAb represents the TIGIT monoclonal antibody (i.e., anti-TIGIT), and OT-4D-30a represents antibody 7.

[0169] the term

[0170] Unless otherwise stated, each of the following terms shall have the meaning described below.

[0171] definition

[0172] It should be noted that the term “a” entity refers to one or more of the same entity. For example, “an antibody” should be understood as one or more antibodies. Therefore, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably in this document.

[0173] As used herein, the terms “comprising” or “including” mean that compositions and methods include the listed elements, such as components or steps, but do not exclude others. “Substantially composed of” means that compositions and methods exclude other elements that have a fundamental effect on the characterization of the composition, but do not exclude elements that do not substantially affect the composition or method. “Composed of” means excluding elements not specifically listed.

[0174] The term "polypeptide" is intended to encompass both the singular and plural forms of "polypeptide" and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single or multiple chains of two or more amino acids and does not imply a specific length of the product. Therefore, the definition of "polypeptide" includes peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to two or more amino acid chains, and the term "polypeptide" can be used in place of any of the foregoing terms or interchangeably with any of the foregoing terms. The term "polypeptide" is also intended to refer to products modified after polypeptide expression, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications. Polypeptides can be derived from natural biological sources or produced through recombinant technologies, but they do not necessarily have to be translated from a specified nucleic acid sequence; they can be produced in any manner, including chemical synthesis.

[0175] An amino acid is an organic compound containing both an amino group and a carboxyl group, such as an α-amino acid, which can be encoded by nucleic acids directly or in its precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "degeneracy of the genetic code." Amino acids include both natural and non-natural amino acids. Natural amino acids include alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0176] "Conservative amino acid substitution" refers to the replacement of one amino acid residue with another amino acid residue containing a side chain (R group) with similar chemical properties (such as charge or hydrophobicity). Generally, conservative amino acid substitution does not substantially change the functional properties of a protein. Examples of amino acid classes containing chemically similar side chains include: 1) Aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) Aliphatic hydroxyl side chains: serine and threonine; 3) Amide-containing side chains: asparagine and glutamine; 4) Aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) Basic side chains: lysine, arginine, and histidine; 6) Acidic side chains: aspartic acid and glutamic acid.

[0177] The term "isolated" as used in this invention with respect to cells, nucleic acids, peptides, antibodies, etc., such as "isolated" DNA, RNA, peptides, and antibodies, refers to molecules isolated from one or more other components such as DNA or RNA, respectively, in the cellular natural environment. The term "isolated" as used in this invention also refers to nucleic acids or peptides that, when produced by recombinant DNA technology, are substantially free of cellular material, viral material, or cell culture medium, or chemical precursors or other chemicals used in chemical synthesis. Furthermore, "isolated nucleic acids" is intended to include nucleic acid fragments that are not present in their natural state and will not be present in their natural state. The term "isolated" is also used in this invention to refer to cells or peptides isolated from other cellular proteins or tissues. Isolated peptides are intended to include purified and recombinant peptides. Isolated peptides, antibodies, etc., are typically prepared by at least one purification step. In some embodiments, the purity of the isolated nucleic acids, peptides, antibodies, etc., is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range between any two of these values ​​(including the endpoint) or any value therein.

[0178] The term “recombination” refers to polypeptides or polynucleotides, meaning forms of polypeptides or polynucleotides that do not exist naturally. Unrestricted embodiments can be created by combining polynucleotides or polypeptides that do not normally exist.

[0179] "Homology," "identity," or "similarity" refers to the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing comparable positions in each sequence. Molecules are homologous at those positions when the positions in the compared sequences are occupied by the same bases or amino acids. The degree of homology between sequences is a function of the number of shared matching or homologous positions.

[0180] "Identity or sequence identity" of a polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) with another sequence at a certain percentage (e.g., 90%, 95%, 98%, or 99%) means that, when sequence alignment is performed, that percentage of bases (or amino acids) are identical in the two compared sequences. This alignment and identity percentage or sequence identity can be determined visually or using software programs known in the art, such as those described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. Alignment is preferably performed using default parameters. One alignment procedure is BLAST using default parameters, such as BLASTN and BLASTP, which use the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sortby=HIGHSCORE; Databases=non-redundant; GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are polynucleotides that have the above-specified percentages of identity and encode polypeptides with the same or similar biological activities.

[0181] Polynucleotides are specific sequences of four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T), or, when the polynucleotide is RNA, thymine replaced by uracil (U). The "polynucleotide sequence" can be represented by the letters of the polynucleotide molecule. This letter representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications, such as functional genomics and homology searches.

[0182] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Examples of unrestricted polynucleotides include: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonucleases, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If such modification is present, structural modifications to the nucleotides can be made before or after assembly of the polynucleotide. The sequence of the nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with labeled components. This term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiments of the polynucleotides disclosed herein include double-stranded forms and each of two complementary single-stranded forms known or predicted to constitute a double-stranded form.

[0183] When the term “encoding” is applied to polynucleotides, it refers to a polynucleotide called the “encoding” polypeptide that, in its natural state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce the polypeptide and / or fragments thereof.

[0184] "Antibody" or "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody, any antigen-binding fragment, or a single chain thereof. Therefore, the term "antibody" includes any protein or peptide containing at least a portion of an immunoglobulin molecule that has biological activity of binding to an antigen. Antibody and antigen-binding fragments include, but are not limited to, the complementarity-determining region (CDR), heavy chain variable region (VH), light chain variable region (VL), heavy chain constant region (CH), light chain constant region (CL), framework region (FR), or any portion thereof of the heavy chain or light chain or its ligand-binding moiety, or at least a portion of the binding protein. The CDR region includes the CDR regions of the light chain (VL CDR1-3) and the CDR regions of the heavy chain (VH CDR1-3). The antibody or antigen-binding fragment described in this embodiment of the invention is a bispecific antibody, which is a fusion of fragments that specifically bind to antigen a and antigen b: the first polypeptide chain contains VHa, CLa, VHb, and CH1, and the first polypeptide chain is similar to the heavy chain or heavy chain fragment of immunoglobulin; the second polypeptide chain contains the structures VLa, CH1, VLb, and CLa, and the second polypeptide chain is similar to the light chain of immunoglobulin.

[0185] The term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody. The antibody fragments of this invention may have a composition similar to F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc., in monospecific antibody fragments. Regardless of their structure, the antibody fragment binds to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, mirror isoforms, and bivalent antibodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

[0186] "Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In some respects, these regions are linked to short linker peptides of 10 to approximately 25 amino acids. The linker may be enriched with glycine to increase flexibility, and with serine or threonine to increase solubility, and may link the N-terminus of the VH to the C-terminus of the VL, and vice versa. Although the constant region of the protein has been removed and the linker has been introduced, it retains the specificity of the original immunoglobulin. ScFv molecules are generally known in the art, for example, as described in U.S. Patent 5,892,019.

[0187] The term "antibody" encompasses a wide range of polypeptides that can be distinguished biochemically. Those skilled in the art will understand that heavy chain classes include gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with further subclasses (e.g., γ1-γ4). The properties of this chain determine the "type" of the antibody, such as IgG, IgM, IgA, IgG, or IgE. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., have been well characterized and their assigned functional specificities are known. All immunoglobulin types are within the scope of protection disclosed in this invention. In some embodiments, the immunoglobulin molecule is of the IgG type. IgG typically comprises two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with a molecular weight of approximately 53,000-70,000 Daltons. These four chains are linked by disulfide bonds in a "Y" configuration, wherein the light chain begins at the "Y" port and continues to surround the heavy chain through a variable region.

[0188] The antibodies, antigen-binding fragments, or derivatives disclosed in this invention include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primate-like, chimeric antibodies, single-chain antibodies, epitope-binding fragments such as Fab-like, Fab'-like, and F(ab')2-like, and single-chain Fvs-like (scFv).

[0189] Light chains can be divided into kappa (κ) or lambda (λ). Each heavy chain can bind to either a κ or λ light chain. Generally, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, their light and heavy chains are covalently bonded, and the "tail" portions of the two heavy chains are linked by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the Y-configuration forked end to the C-terminus at the bottom of each chain. The variable region of the immunoglobulin κ light chain is Vκ; the variable region of the immunoglobulin λ light chain is V... λ .

[0190] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used according to function. The variable regions of the light chain (VL) and heavy chain (VH) determine antigen recognition and specificity. The constant regions of the light and heavy chains confer important biological properties such as secretion, transplacental migration, Fc receptor binding, complement binding, etc. By convention, the numbering of constant regions increases as they move further away from the antibody's antigen-binding site or N-terminus. The N-terminal region is the variable region, and the C-terminal region is the constant region; the CH3 and CL domains actually contain the carboxyl termini of the heavy and light chains, respectively.

[0191] In naturally occurring antibodies, assuming the antibody presents its three-dimensional conformation in an aqueous environment, the six "complementarity-determining regions" or "CDRs" present in each antigen-binding domain are short, discontinuous amino acid sequences that bind specifically to the antigen, forming the antigen-binding domain. The remaining amino acids in the framework region, known as the "framework region," exhibit less intermolecular variability. The framework region mostly adopts a β-sheet conformation, with CDRs forming linked loop structures, or in some cases, forming part of a β-sheet structure. Thus, the framework region positions the CDRs in the correct orientation by forming a scaffold through non-covalent interchain interactions. The antigen-binding domain with a CDR at a specific location forms a surface complementary to the epitope on the antigen, which facilitates the non-covalent binding of the antibody to its antigenic epitope. For a given heavy or light chain variable region, those skilled in the art can identify the amino acids containing the CDR and framework regions using known methods (see Kabat, E., et al., USDepartment of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0192] Where a term used and / or accepted in this field has two or more definitions, the definition of the term used herein includes all of those meanings unless explicitly stated otherwise. A concrete example is the use of the term “complementarity-determining region” (“CDR”) to describe a discontinuous antigen-binding site found within the variable region of heavy and light chain polypeptides. This specific region is described in Kabat et al., USDept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al. in J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entirety.

[0193] According to the definitions of Kabat and Chothia, a CDR includes overlaps or subsets of amino acid residues when compared with each other. Nevertheless, the application of either definition to refer to a CDR of an antibody or a variant thereof is within the scope of this invention. The exact residue numbering containing a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can typically determine which specific residues a CDR contains based on the amino acid sequence of the variable region of the antibody.

[0194] Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. Those skilled in the art can apply this "Kabat numbering" system to any variable region sequence without relying on experimental data other than the sequence itself. "Kabat numbering" refers to the numbering system proposed by Kabat et al., USDept. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1983). Antibodies can also use the EU numbering system.

[0195] The antibodies disclosed in this invention can be derived from any animal, including birds and mammals. In some embodiments, the antibodies are human, mouse, donkey, rabbit, goat, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region can be of condricthoid origin (e.g., from sharks).

[0196] The heavy chain constant region includes at least one of a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge regions), a CH2 domain, a CH3 domain, or a variant or fragment. The heavy chain constant region of an antibody can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of a polypeptide may include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another embodiment, the heavy chain constant region may include a hinge region partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In another embodiment, a portion of the heavy chain may include a chimeric hinge region partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0197] The “light chain constant region” comprises a portion of the amino acid sequence from the antibody light chain. Preferably, the light chain constant region contains at least one of a constant κ domain or a constant λ domain. A “light chain-heavy chain pair” refers to a set of light and heavy chains that can form dimers via disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0198] The “VH domain” includes the N-terminal variable domain of the immunoglobulin heavy chain, and the “CH1 domain” includes the first (mostly N-terminal) constant region of the immunoglobulin heavy chain. The CH2 domain does not pair tightly with other domains but inserts two N-linked branched carbohydrate chains between the two CH2 domains of the intact native IgG molecule. It has also been documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule, containing approximately 108 residues. The “hinge region” includes a portion of the heavy chain connecting the CH1 and CH2 domains. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be further subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

[0199] A disulfide bond is a covalent bond formed between two sulfur atoms. The thiol group of cysteine ​​can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by disulfide bonds.

[0200] A "chimeric antibody" is any antibody whose variable region is derived from or derived from a first species, while its constant region (which may be complete, partial, or modified) is derived from a second species. In some embodiments, the variable region is derived from a non-human source (e.g., a mouse or primate), while the constant region is derived from a human source.

[0201] "Specific binding" or "specific to..." generally refers to the formation of a relatively stable complex by which an antibody or antigen-binding fragment binds to a specific antigen through the complementarity of its antigen-binding domain with the epitope. "Specificity" can be expressed as the relative affinity of the antibody or antigen-binding fragment to a specific antigen or epitope. For example, if antibody "A" has a greater relative affinity to the same antigen than antibody "B," antibody "A" can be considered to have higher specificity for that antigen than antibody "B." Specific binding can be described using the equilibrium dissociation constant (KD), with a smaller KD indicating a tighter binding. Methods for determining whether two molecules bind specifically are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and optical interferometry of biofilm layers. Antibodies that "specifically bind" to antigen a include antibodies with an equilibrium dissociation constant KD of less than or equal to about 100 nM, less than or equal to about 10 nM, less than or equal to about 5 nM, or less than or equal to about 1 nM.

[0202] "Treatment" refers to therapeutic treatments and preventative or preventative measures aimed at preventing, mitigating, improving, or stopping adverse physiological changes or disorders, such as disease progression, including but not limited to the following, whether detectable or undetectable: symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement, mitigation, reduction, or disappearance of the disease state (whether partial or complete), and prolongation of expected survival without treatment. Patients requiring treatment include those already suffering from the condition or disorder, those susceptible to the condition or disorder, or those needing prevention of the condition or disorder, as well as those who can or are expected to benefit from the application of the antibody or pharmaceutical composition disclosed in this invention for detection, diagnostic procedures, and / or treatment.

[0203] "Patient" refers to any mammal that requires diagnosis, prognosis, or treatment, including humans, dogs, cats, rabbits, rats, horses, cattle, etc.

[0204] "Approximately" refers to a typical error range for a given value that is readily known to those skilled in the art. In some embodiments, "approximately" as used herein refers to the described value and its range of ±10%, ±5%, or ±1%.

[0205] EC 50 "That is, the half-maximal concentration (EC50)." 50 () refers to the concentration that can cause 50% of the maximum effect.

[0206] Bispecific antibodies

[0207] This invention provides, for example Figure 1The novel bispecific antibody or antigen binding fragment shown can regulate the binding activity of the bispecific antibody or antigen binding fragment to the two antigens it binds by adjusting the positions of the variable regions binding to the first antigen and the variable regions binding to the second antigen, as well as the linkers between them, thereby enhancing the antibody therapeutic effect and reducing side effects.

[0208] 1) Antibodies targeting TIGIT and CTLA-4

[0209] This invention provides bispecific antibodies or antigen-binding fragments with high affinity for TIGIT and CTLA-4 proteins. The tested antibodies exhibit effective binding and biological activity and can be used for therapeutic and diagnostic purposes. For example, these antibody or antigen-binding fragments effectively block immune checkpoints and activate lymphocytes to release cytokines, enabling the treatment of various types of cancer, tumors, or related diseases.

[0210] Therefore, one embodiment of the present invention provides an antibody or antigen-binding fragment that targets TIGIT and CTLA-4, the antibody or antigen-binding fragment specifically binding to TIGIT and CTLA-4.

[0211] In some embodiments, the antibody or antigen-binding fragment comprises at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain contains the sequence shown in SEQ ID NO:37 and the second polypeptide chain contains the sequence shown in SEQ ID NO:38.

[0212] In some embodiments, the antibody or antigen-binding fragment comprises at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain. The first polypeptide chain contains the sequence shown in SEQ ID NO:37, excluding the Fc region sequence, and the second polypeptide chain contains the sequence shown in SEQ ID NO:38.

[0213] In some embodiments, the antibody or antigen-binding fragment comprises at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain contains the sequence shown in SEQ ID NO:39 and the second polypeptide chain contains the sequence shown in SEQ ID NO:40.

[0214] In some embodiments, the antibody or antigen-binding fragment comprises at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain. The first polypeptide chain contains the sequence shown in SEQ ID NO:39, excluding the Fc region sequence, and the second polypeptide chain contains the sequence shown in SEQ ID NO:40.

[0215] In some embodiments, the antibody or antigen-binding fragment comprises at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain contains the sequence shown in SEQ ID NO:41 and the second polypeptide chain contains the sequence shown in SEQ ID NO:42.

[0216] In some embodiments, the antibody or antigen-binding fragment comprises at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain. The first polypeptide chain contains the sequence shown in SEQ ID NO:41, excluding the Fc region sequence, and the second polypeptide chain contains the sequence shown in SEQ ID NO:42.

[0217] In some embodiments, the antibody or antigen-binding fragment comprises at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain contains the sequence shown in SEQ ID NO:43 and the second polypeptide chain contains the sequence shown in SEQ ID NO:44.

[0218] In some embodiments, the antibody or antigen-binding fragment comprises at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain. The first polypeptide chain contains the sequence shown in SEQ ID NO:43, excluding the Fc region sequence, and the second polypeptide chain contains the sequence shown in SEQ ID NO:44.

[0219] 2) Antibodies targeting OX40 and CTLA-4

[0220] This invention provides bispecific antibodies or antigen-binding fragments with high affinity for OX40 and CTLA-4 proteins. The tested antibodies exhibit effective binding and biological activity and can be used for therapeutic and diagnostic purposes. For example, these antibody or antigen-binding fragments effectively activate T cells and lymphocytes to release cytokines, enabling the treatment of various types of cancer, tumors, or related diseases.

[0221] Therefore, one embodiment of the present invention provides an antibody or antigen-binding fragment that targets OX40 and CTLA-4, the antibody or antigen-binding fragment specifically binding to OX40 and CTLA-4.

[0222] In some embodiments, the antibody or antigen-binding fragment comprises at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain contains the sequence shown in SEQ ID NO:45 and the second polypeptide chain contains the sequence shown in SEQ ID NO:46.

[0223] In some embodiments, the antibody or antigen-binding fragment comprises at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain. The first polypeptide chain contains the sequence in SEQ ID NO:45 excluding the Fc region sequence and the Fc region shown in SEQ ID NO:36, and the second polypeptide chain contains the sequence shown in SEQ ID NO:46.

[0224] 3) Antibodies targeting OX40 and TIGIT

[0225] This invention provides bispecific antibodies or antigen-binding fragments with high affinity for OX40 and TIGIT proteins. The tested antibodies exhibit effective binding and biological activity and can be used for therapeutic and diagnostic purposes. For example, these antibody or antigen-binding fragments effectively block immune checkpoints, activate T cells, and activate lymphocytes to release cytokines, for the treatment of various types of cancer, tumors, or related diseases.

[0226] Therefore, one embodiment of the present invention provides an antibody or antigen-binding fragment that targets OX40 and TIGIT, the antibody or antigen-binding fragment specifically binding to OX40 and TIGIT.

[0227] In some embodiments, the antibody or antigen-binding fragment comprises at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain contains the sequence shown in SEQ ID NO:47 and the second polypeptide chain contains the sequence shown in SEQ ID NO:48.

[0228] In some embodiments, the antibody or antigen-binding fragment comprises at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain. The first polypeptide chain contains the sequence shown in SEQ ID NO:47, excluding the Fc region sequence, and the second polypeptide chain contains the sequence shown in SEQ ID NO:48.

[0229] In some embodiments, the antibody or antigen-binding fragment comprises at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain contains the sequence shown in SEQ ID NO:49 and the second polypeptide chain contains the sequence shown in SEQ ID NO:50.

[0230] In some embodiments, the antibody or antigen-binding fragment comprises at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain. The first polypeptide chain contains the sequence shown in SEQ ID NO:49, excluding the Fc region sequence, and the second polypeptide chain contains the sequence shown in SEQ ID NO:50.

[0231] Those skilled in the art should also understand that the antibody or antigen-binding fragment sequence disclosed in this invention can be replaced, and the amino acid sequence after replacement differs from the naturally occurring amino acid sequence of the antibody. For example, the replaced amino acid sequence can be similar to the starting sequence, such as having a certain proportion of identity with the starting sequence. For example, its identity with the starting sequence can be about 80%, about 85%, about 90%, about 95%, about 98%, or about 99%, or a range between any two of these values ​​(including the endpoint) or any value therein.

[0232] In some embodiments, the amino acid-containing sequence of the antibody package has one or more modifying groups. For example, the bispecific antibodies disclosed in this invention (bispecific antibodies targeting TIGIT and CTLA-4, bispecific antibodies targeting OX40 and CTLA-4, and bispecific antibodies targeting OX40 and TIGIT) may contain resilient linker sequences or may be modified to add functional groups (e.g., PEG, drugs, toxins, or tags).

[0233] The antibody and antigen-binding fragments disclosed in this invention include modified derivatives, i.e., modified by covalent linking any type of molecule to the antibody, wherein the covalent linking does not prevent the antibody from binding to the epitope. Examples include, but are not limited to, glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization via known protecting / blocking groups, proteolytic cleavage, and linking to cellular ligands or other proteins. Any of the numerous chemical modifications can be performed using existing techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin.

[0234] In some implementations, antibodies may be conjugated with therapeutic agents, drug precursors, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceuticals, or PEG.

[0235] Antibodies can be conjugated or fused with therapeutic agents, which may include detectable markers (such as radioactive markers), immunomodulators, hormones, enzymes, oligonucleotides, photosensitizing agents, diagnostic agents, cytotoxic agents of drugs or toxins, ultrasound enhancers, non-radioactive markers and combinations thereof, and other such agents known in the art.

[0236] Antibodies can be detectably labeled by conjugating them to chemiluminescent compounds. The presence of the chemiluminescently labeled antibody is then determined by detecting the luminescence that occurs during the chemical reaction. Examples of chemiluminescently labeled compounds include luminol, isoluminol, aromatic acridine esters, imidazoles, acridine salts, and oxalates.

[0237] Preparation methods of antibodies and polynucleotides encoding antibodies

[0238] This invention also discloses polynucleotides or nucleic acid molecules encoding the antibodies, antigen-binding fragments, and derivatives thereof described herein. The polynucleotides disclosed herein may encode a first polypeptide (similar to the heavy chain of an immunoglobulin or a fragment thereof), a second polypeptide (similar to the light chain of an immunoglobulin), a heavy chain variable region, a light chain variable region, an Fc region, a partial heavy chain variable region, or a partial light chain variable region. Methods for preparing antibodies are well known in the art and are described herein. In some embodiments, the variable and constant regions of the antibodies and antigen-binding fragments disclosed herein are fully human. Fully human antibodies and antigen-binding fragments can be prepared using techniques disclosed in the art and techniques described herein. For example, fully human antibodies against a specific antigen can be prepared by administering the antigen to transgenic animals that have been modified to produce fully human antibodies in response to antigen challenge. Exemplary techniques that can be used to prepare such antibodies are found in U.S. Patents 6,458,592; 6,420,140, ​​the entire contents of which are incorporated herein by reference. The bispecific antibody described in this invention is a fusion of fragments that specifically bind to antigen a and antigen b. Some fragments in the bispecific antibody can be found in the preparation method of the antibody that binds to a single antigen described above.

[0239] In some embodiments, the prepared antibodies do not elicit a harmful immune response in the animal to be treated (e.g., human). In one embodiment, the antibodies, antigen-binding fragments, or derivatives disclosed in this invention are modified using techniques recognized in the art to reduce their immunogenicity. For example, the antibodies can be humanized, primate-like, deimmunized, or chimeric antibodies can be prepared. These types of antibodies are derived from non-human antibodies, typically murine or primate antibodies, which retain or substantially retain the antigen-binding properties of the parent antibody but have lower immunogenicity in humans. This can be achieved by a variety of methods, including (a) transplanting the entire non-human variable region into a human constant region to produce a chimeric antibody; (b) transplanting at least a portion of one or more non-human complementarity-determining regions (CDRs) into human framework and constant regions, with or without key framework residues; or (c) transplanting the entire non-human variable region but “hiding” them by replacing surface residues with human-like portions. Typically, framework residues in the human framework region are replaced by corresponding residues from the CDR donor antibody, such as residues that improve antigen binding. These framework substitutions can be identified using methods well-known in the art, such as simulating the interaction between CDRs and framework residues to identify framework residues that play an important role in antigen binding and using sequence comparison to identify aberrant framework residues at specific positions. (Refer to U.S. Patent 5,585,089; Riechmann et al., Nature 332:323 (1988); the entire contents of which are incorporated herein by reference). Antibodies can be humanized using a variety of techniques known in the art, such as CDR transplantation (EP 239,400; WO 91 / 09967; U.S. Patents 5,225,539,5,530,101 and 5,585,089), repair or surface rearrangement (EP592,106; EP519,596; Padlan, et al., Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska, et al., Proc. Natl. Sci. USA 91:969-973 (1994)), and chain rearrangement (U.S. Patent 5,565,332), the entire contents of which are incorporated herein by reference.

[0240] Deimmunization can also be used to reduce the immunogenicity of antibodies. In this invention, the term "deimmunization" includes altering antibodies to modify T-cell epitopes (see, for example, WO / 9852976A1 and WO / 0034317A2). For example, the heavy chain variable region sequence and light chain variable region sequence from the starting antibody are analyzed, and a "map" of human T-cell epitopes from each variable region is generated, showing the position of the epitope relative to complementarity-determining regions (CDRs) and other key residues within the sequence. Individual T-cell epitopes from the T-cell epitope map are analyzed to identify alternative amino acid substitutions with a lower risk of altering antibody activity. A series of alternative heavy chain variable region sequences and light chain variable region sequences containing combinations of amino acid substitutions are designed and subsequently incorporated into a series of binding peptides. Genes containing the modified variable regions and human constant regions of the complete heavy and light chains are then cloned into expression vectors, and plasmids are subsequently transformed into cell lines to produce complete antibodies. The antibodies are then compared using appropriate biochemical and biological experiments to identify the optimal antibody.

[0241] The binding specificity of the bispecific antibody or antigen-binding fragment disclosed in this invention can be detected by in vitro experiments, such as immunoprecipitation, radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).

[0242] Alternatively, the scFv in the bispecific antibody of this invention can be found in the techniques for producing single-chain units (US Patent 4,694,778; Bird, Science 242:423-442 (1988), Huston et al., Proc. Natl. Acad. Sci. USA 55:5879-5883 (1988), Ward et al., Nature 334:544-554 (1989), and Nie et al., Antibody Therapeutics 3(1):18-62 (2020)). Single-chain units are formed by bridging the heavy and light chain fragments of the Fv region with amino acids, resulting in single-chain fusion peptides. Alternatively, techniques for assembling functional Fv fragments in E. coli can be used (Skerra et al., Science 242:1038-1041 (1988)).

[0243] Examples of techniques that can be used to produce single-chain Fv (scFv) and antibodies include those described in U.S. Patents 4,946,778 and 5,258,498, as well as Huston et al., Methods in Enzymology 203:46-88 (1991), Shu et al., Proc. Natl. Sci. USA 90:1995-1999 (1993), and Skerra et al., Science 240:1038-1040 (1988). For certain applications, including the use of antibodies in humans and in vitro assays, chimeric antibodies, humanized antibodies, or fully human antibodies may be used. Chimeric antibodies are a class of molecules whose different portions are derived from different animal species, such as antibodies containing the variable region of a murine monoclonal antibody and the constant region of a human immunoglobulin. Methods for producing chimeric antibodies are known in the art, see Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gilles et al., J. Immunol. Methods 125:191-202 (1989); Neuberger et al., Nature 372:604-608 (1984); Takeda et al., Nature 314:452-454 (1985); and U.S. Patents 5,807,715, 4,816,567 and 4,816,397, the entire contents of which are incorporated herein by reference.

[0244] Furthermore, another efficient method for producing recombinant antibodies is disclosed in Newman, Biotechnology 10:1455-1460 (1992), specifically, this technique produces primate antibodies containing monkey variable region and human constant region sequences, the entire contents of which are incorporated herein by reference. This technique is also mentioned in commonly assigned U.S. Patents 5,658,570, 5,693,780, and 5,756,096, the entire contents of which are incorporated herein by reference.

[0245] Antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries derived from immunoglobulin sequences. Reference can also be made to U.S. Patents 4,444,887 and 4,716,111, and PCT publications WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO91 / 10741, the entire contents of which are incorporated herein by reference.

[0246] Fully human antibodies that recognize selective epitopes can be produced using a technique known as “guided selection.” In this method, a selected non-human monoclonal antibody (e.g., a mouse antibody) is used to guide the screening of fully human antibodies that recognize the same epitope (see U.S. Patent 5,565,332, the entire contents of which are incorporated herein by reference).

[0247] In another embodiment, DNA encoding the desired monoclonal antibody can be isolated and sequenced using conventional methods, such as oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies. Isolated and subclonal hybridoma cells can serve as sources of this DNA. Once isolated, the DNA can be placed in an expression vector and then transfected into prokaryotic or eukaryotic host cells such as *E. coli* cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce other immunoglobulins. The isolated DNA (which may be synthetic, as described herein) can also be used to prepare sequences of the constant and variable regions of the antibody, as described in U.S. Patent 5,658,570, the entire contents of which are incorporated herein by reference. This method involves extracting RNA from selected cells and converting it into cDNA, which is then amplified using Ig-specific primers via PCR. Suitable probes for this purpose are also mentioned in U.S. Patent 5,658,570.

[0248] Furthermore, using conventional recombinant DNA techniques, one or more CDRs of the antibodies of this invention can be inserted into the frame region, for example, into the human frame region, to construct humanized non-fully human antibodies. The frame region can be a naturally occurring or common frame region, preferably a human frame region (see Chothia et al., J. Mol. Biol. 278:457-479 (1998), which lists a series of human frame regions). Some polynucleotides can encode antibodies that specifically bind to at least one epitope of the target antigen, resulting from the combination of the frame region and the CDR. One or more amino acid substitutions can be made within the frame region, selectively choosing amino acid substitutions that improve the binding of the antibody to its antigen. Additionally, this method can be used to substitute or delete cysteine ​​residues in one or more variable regions involved in the formation of interchain disulfide bonds, thereby producing antibody molecules lacking one or more interchain disulfide bonds. Other modifications to polynucleotides within the scope of the art are also covered in this invention.

[0249] Cell lines for antibody production can be selected, constructed, and cultured using techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications. In this regard, the techniques described below suitable for use in this invention are referenced in Current Protocols in Immunology, Colligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), the entire contents of which, including supplements, are incorporated herein by reference.

[0250] In some implementations, the antibody expression vector includes at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include an enhancer, a Kozak sequence, and donor and recipient sites for RNA splicing flanking the insert sequence. Efficient transcription can be achieved using early and late promoters of SV40, early promoters of long terminal repeat sequences from retroviruses such as RSV, HTLV1, HIV, and cytomegalovirus, or other cellular promoters such as actin promoters. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI, and pCS2, etc. Commonly used mammalian cells include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells, and CHO cells.

[0251] In some implementations, the inserted gene fragment needs to contain selection markers. Common selection markers include dihydrofolate reductase, glutamine synthase, neomycin resistance, and hygromycin resistance genes to facilitate the selection and isolation of successfully transfected cells. The constructed plasmid is transfected into host cells lacking the aforementioned genes, and after being cultured in a selective medium, the successfully transfected cells grow in large numbers, producing the desired target protein.

[0252] Furthermore, mutations can be introduced into the nucleotide sequence encoding the antibody of the present invention using standard techniques known to those skilled in the art, including but not limited to site-directed mutagenesis leading to amino acid substitutions and PCR-mediated mutations. Variants (including derivatives) encode substitutions of fewer than 50 amino acids, fewer than 40 amino acids, fewer than 30 amino acids, fewer than 25 amino acids, fewer than 20 amino acids, fewer than 15 amino acids, fewer than 10 amino acids, fewer than 5 amino acids, fewer than 4 amino acids, fewer than 3 amino acids, or fewer than 2 amino acids relative to the original heavy chain variable regions VH CDR1, VH CDR2, VH CDR3 and light chain variable regions VL CDR1, VLCDR2, or VL CDR3. Alternatively, mutations can be randomly introduced along all or part of the coding sequence, for example, through saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.

[0253] In some implementations, the substitutions described herein are conserved amino acid substitutions.

[0254] Treatment

[0255] This invention also provides methods of treatment and uses. In some embodiments, a method is provided for treating or improving various types of cancer, tumors, or infections, said method comprising administering to a patient an effective dose of the said bispecific antibody: an antibody targeting TIGIT and CTLA-4, an antibody targeting OX40 and CTLA-4, or an antibody targeting OX40 and TIGIT. In some embodiments, the use of said bispecific antibody in treating or improving cancer, tumors, or infections is provided. In some embodiments, the use of said bispecific antibody in the preparation of a medicament for treating or improving cancer, tumors, or infections is provided.

[0256] The specific dosage and treatment regimen for any given patient will depend on a variety of factors, including the specific antibody or derivative used, the patient's age and weight, general health condition, sex and diet, as well as the timing of administration, frequency of excretion, drug combination, and the severity of the specific disease being treated. These factors will be determined by a healthcare professional, including those skilled in the art. The dosage will also depend on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined using principles of pharmacology and pharmacokinetics well known in the art.

[0257] Methods of administration for antibodies or derivatives include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural, and oral injection. Pharmaceutical compositions can be administered via any convenient route, such as by infusion or bolus injection, absorption through epithelial or mucosal membranes (e.g., oral mucosa, rectal and intestinal mucosa), and can be co-administered with other bioactive agents. Therefore, pharmaceutical compositions containing the antibodies of this invention can be administered orally, rectally, parenterally, intracerebrospinally, vaginally, intraperitoneally, topically (e.g., via powder, ointment, drops, or transdermal patch), orally, or via oral or nasal spray.

[0258] The term "parenteral" as used in this invention refers to administration methods including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous, and intra-articular injections and infusions.

[0259] The administration method can be systemic or local. Furthermore, it may be necessary to introduce the antibodies of the present invention into the central nervous system via any suitable route, including intraventricular and intrathecal injection; intraventricular injection can be assisted by connecting an intraventricular catheter to a reservoir such as an Ommaya reservoir. Administration can also be via the lungs, for example, by using an inhaler or nebulizer, and using nebulized formulations.

[0260] The antibodies of this invention can be applied topically to the area requiring treatment; this can be achieved, but is not limited to, by local infusion during surgery, such as topical application in conjunction with postoperative wound dressings, by injection, via catheter, by suppository, or by implantation of a porous, non-porous, or gel-like material, including membranes (e.g., silicone rubber membranes) or fibers. Preferably, when administering the proteins (including antibodies) of this invention, care must be taken to use materials that do not absorb proteins.

[0261] In some embodiments, the compositions of the present invention comprise nucleic acids or polynucleotides encoding antibodies, which can be administered in vivo to promote the expression of their encoded proteins by constructing them as part of a suitable nucleic acid expression vector, and then the aforementioned partial vector is administered to make it an intracellular part, for example by using a retroviral vector (see U.S. Patent 4,980,286), or by direct injection, or by using microparticle bombardment (e.g., gene gun; Biolistic, DuPont), or by coating with lipids or cell surface receptors or transfection reagents, or by administration by linking to a homeobox peptide known to enter the cell nucleus (see, for example, Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc. Optionally, the nucleic acid can be introduced into the cell and integrated into the host cell DNA for expression via homologous recombination.

[0262] In some embodiments, the antibody of the present invention is administered to a patient at a dose of 0.01 mg / kg to 100 mg / kg of patient body weight, or 0.1 mg / kg to 20 mg / kg of patient body weight. A second or more doses of the antibody or antigen-binding fragment may be subsequently administered after the initial dose, at a dose substantially the same as or less than the initial dose, wherein the subsequent doses may be spaced at least 1 to 3 days; or at least one week apart. Modifications such as lipolysis can enhance antibody uptake and tissue penetration (e.g., into the brain), thereby reducing the dose and frequency of administration of the antibody of the present invention.

[0263] Various known delivery systems can be used to administer the antibodies or derivatives of the present invention or the polynucleotides encoding them, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, receptor-mediated endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of nucleic acids as part of retroviruses or other vectors, etc.

[0264] combination therapy

[0265] In some embodiments, the antibodies of the present invention can be combined with other treatment or preventative regimens, including the administration of one or more of the antibodies of the present invention together with one or more other therapeutic agents or methods. For combination therapy, the antibodies can be administered simultaneously or separately from other therapeutic agents. When administered separately, the antibodies of the present invention can be administered before or after the administration of another therapeutic agent.

[0266] In some embodiments, the bispecific antibody of the present invention is administered in combination with a chemotherapeutic agent. In some embodiments, the bispecific antibody of the present invention is an antibody targeting TIGIT and CTLA-4, an antibody targeting OX40 and CTLA-4, or an antibody targeting OX40 and TIGIT. In some embodiments, chemotherapeutic agents that can be administered with the antibody of the present invention include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and actinomycin D), anti-estrogens (e.g., tamoxifen), antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, fluorouracil, interferon α-2b, glutamate, styromycin, mercaptopurine, and 6-thioguanine), and cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytarabine, cyclophosphamide, estradiol, hydroxyurea). Methylbenzylhydrazine, mitomycin, busulfan, cisplatin and vincristine sulfate), hormones (such as medroxyprogesterone, estradiol sodium phosphate, ethinylestradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorestradiol and testosterone), nitrogen mustard derivatives (such as melphalan, chlorambucil, dichloromethyldiethylammonium (nitrogen mustard) and thiotepa), steroids and combinations thereof (such as betamethasone sodium phosphate), and other compounds (such as dazometazidine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate and etoposide).

[0267] In some embodiments, the antibodies of the present invention are administered in combination with cytokines. Cytokines that can be administered with the antibodies of the present invention include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, and IL-15.

[0268] In some embodiments, the bispecific antibody of the present invention is administered in combination with a chemotherapeutic agent. Examples of chemotherapeutic agents include immunotherapeutic agents, including but not limited to therapeutic antibodies suitable for treating patients. Some examples of therapeutic antibodies include simtuzumab, abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, and bevacizumab. zumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab, cixutuzumab, cixutuzumab, clivatuzumab, conatumumab, daratumumab, dro Zitumab, Duligotuzumab, Dusigitumab, Detumomab, Dacetuzumab, Dalotuzumab, Ecomoxicillinab, Elotuzumab, Enctituximab, Ertumaxomab, Etaracizumab, Farletuzumab, Ficolacumab ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximabInotuzumab, intetumumab, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab Mitumomab, Moxetumomab, Narnatumab, Naptumomab, Negitumumab, Nimotuzumab, Nofetumomab, Ocaratuzumab, Ofatumumab, Olaratumab, Onartuzumab, Oportuzumab, Oregovomab, Per Panitumumab, Parsatuzumab, Patritumab, Pemtumomab, Pertuzumab, Pintumomab, Pritumumab, Racotumomab, Radretumab, Rilotumumab, Rituximab, Robatumumab, Satumomab, Sbutuzumab ibrotuzumab), siltuximab, solitumab, tacatuzumab, taplituzumab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumabVorsetuzumab, votumumab, and zalutumumab, among others.

[0269] In some embodiments, the antibodies of the present invention may be used in conjunction with immune checkpoint inhibitors. In some embodiments, the antibodies of the present invention are administered in combination with other treatment or preventative regimens, such as radiotherapy.

[0270] Pharmaceutical Composition

[0271] The present invention also provides pharmaceutical compositions. Such compositions comprise an effective dose of an antibody or antigen-binding fragment and an acceptable carrier. In some embodiments, the pharmaceutical composition further comprises an anticancer agent (e.g., an immune checkpoint inhibitor).

[0272] In some implementations, the term "pharmaceuticalally acceptable" refers to a substance approved by a government regulatory agency or listed in a recognized pharmacopoeia for use in animals, and particularly in humans. Furthermore, "pharmaceuticalally acceptable excipients" generally refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation aid.

[0273] The term "excipient" refers to a diluent, adjuvant, excipient, or carrier used to administer the active ingredient to a patient. Such pharmaceutical excipients can be sterile liquids, such as water and oils, including petroleum, animal, vegetable, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline, glucose, and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers such as acetates, citrates, or phosphates. Antimicrobial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as EDTA, and tonic agents such as sodium chloride or dextran are also foreseeable. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The composition can be formulated into suppositories using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in EW Martin's Remington's Pharmaceutical Sciences, which are incorporated herein by reference. Such compositions will contain a clinically effective dose of an antibody or antigen-binding fragment, preferably in a purified form, along with a suitable number of carriers to provide a dosage form suitable for the patient. The formulation should be suitable for the mode of administration. Parent formulations can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0274] In some embodiments, the composition is formulated into a pharmaceutical composition suitable for intravenous injection into the human body according to conventional procedures. Compositions for intravenous administration are typically solutions in sterile isotonic buffer solutions. The composition may also contain a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the active ingredient is supplied individually or in combination in unit doses, such as as a dry lyophilized powder or anhydrous concentrate in a sealed container (such as an ampoule or sachet) indicating the amount of active agent. When the composition is administered by infusion, it can be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, ampoules of sterile water or saline for injection can be used, allowing the active ingredient to be mixed prior to administration.

[0275] The compounds of the present invention can be formulated into neutral or salt forms. Pharmaceutically acceptable salts include salts derived from anions such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts derived from cations such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Detailed Implementation

[0276] The following specific embodiments further illustrate the technical solution of the present invention. These specific embodiments do not represent a limitation on the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the scope of protection of the present invention.

[0277] Unless otherwise specified, all materials and reagents used in the following examples are commercially available; for example, TIGIT-Fc antigen, CTLA-4-Fc antigen, and TIGIT-his antigen can be purchased from Shanghai Nearshore Technology Co., Ltd. or AcroBiosystems, and the anti-TIGIT reporter gene detection system can be purchased from Promega (catalog number JA1400), or can be prepared according to known methods.

[0278] Reference antibody

[0279] The anti-TIGIT antibody (reference antibody) was obtained after expression and purification in CHO cells. The heavy chain of the anti-TIGIT antibody is:

[0280]

[0281] The light chain of the anti-TIGIT antibody is:

[0282]

[0283] The anti-CTLA-4 antibody (reference antibody) was obtained after expression and purification in CHO cells. The heavy chain of the anti-CTLA-4 antibody is:

[0284]

[0285] The light chain of the anti-CTLA-4 antibody is:

[0286]

[0287]

[0288] The anti-OX40 antibody (the heavy chain sequence of the reference antibody is SEQ ID NO:75, and the light chain sequence is SEQ ID NO:74) was obtained after expression and purification in CHO cells. The heavy chain of anti-OX40 is:

[0289]

[0290] or

[0291]

[0292] The light chain of anti-OX40 is:

[0293]

[0294] Bispecific antibodies targeting TIGIT and CTLA-4

[0295] Example 1 Antibody Preparation Method

[0296] The structure of this bispecific antibody is as follows: Figure 1 As shown, the structure is similar to that of an immunoglobulin. The antibody contains a first polypeptide chain and a third polypeptide chain with the same sequence, as well as a second polypeptide chain and a fourth polypeptide chain with the same sequence. The first polypeptide chain contains the structure VHa-L1-CLa-L2-VHb-CH1-Fc, and the second polypeptide chain contains the structure VLa-L3-CH1-L4-VLb-CLb. Antigen a is TIGIT, and antigen b is CTLA-4.

[0297] The amino acid sequences of the first and second peptides of the antibody were optimized according to the codon bias characteristics of the host cell CHO (Chinese hamster ovary cells) to obtain the DNA sequences of the first and second peptides. To facilitate expression in CHO cells, a signal peptide and a Kozak sequence (a nucleic acid sequence located after the 5' cap structure of eukaryotic mRNA) were added to the sequences of both peptides, and stop codons were added to the ends of the sequences. Simultaneously, to facilitate cloning into the mammalian expression vector pCDNA3.1... TM (+)(Invitrogen, catalog number V79020), with restriction enzyme sites added to both ends of the sequence, Hind III at the 5' end and EcoRI at the 3' end.

[0298] The optimized and synthesized sequence clones were cloned into pCDNA3.1. TM (+) vector, then a large number of plasmids were extracted separately, and the first and second peptides were extracted using ExpiCHO at a plasmid molar ratio of 1:1. TMTransient expression was performed using the expression system (Gibco, catalog number A29133). The protein was harvested according to the instructions and purified using protein A.

[0299] The amino acid sequences associated with the bispecific antibodies targeting TIGIT and CTLA-4 are shown in Table 1, and the nucleic acid sequences associated with the bispecific antibodies targeting TIGIT and CTLA-4 are shown in Table 2. Linkers in Table 1 are indicated by bold italics and underlines; the first polypeptide of antibody 1 is shown in SEQ ID NO:37, and the second polypeptide of antibody 1 is shown in SEQ ID NO:38; the first polypeptide of antibody 2 is shown in SEQ ID NO:39, and the second polypeptide of antibody 2 is shown in SEQ ID NO:40; the first polypeptide of antibody 3 is shown in SEQ ID NO:41, and the second polypeptide of antibody 3 is shown in SEQ ID NO:42; the first polypeptide of antibody 4 is shown in SEQ ID NO:43, and the second polypeptide of antibody 4 is shown in SEQ ID NO:44. In Table 2, the italicized and underlined lowercase letters at the 5' end (from the 5' end to the 3' end) are, in order, the Hind III restriction site, the Kozak sequence, and the leader peptide sequence; the italicized and underlined lowercase letters at the 3' end (from the 5' end to the 3' end) are, in order, the stop codon and the EcoRI restriction site. Since antibodies 1, 2, 3, and 4 differ only in their linker sequences, Table 2 only lists the nucleic acid sequences of the first and second polypeptides of antibody 1, as well as the nucleic acid sequences of the linker.

[0300] Table 1. Amino acid sequences of bispecific antibodies targeting TIGIT and CTLA-4

[0301]

[0302]

[0303]

[0304]

[0305]

[0306] Table 2. Nucleic acid sequences encoding bispecific antibodies targeting TIGIT and CTLA-4.

[0307]

[0308]

[0309]

[0310]

[0311] Example 2 Antibody Purity Detection

[0312] The purified antibody was then analyzed by gel electrophoresis to determine its purity. Figure 2 As shown, antibody 1 has a high purity; Figure 3 As shown, antibodies 2 and 3 have high purity.

[0313] Example 3: Detection of antibody binding activity

[0314] 1) The binding activity of the purified bispecific antibodies was detected to determine whether they could bind normally to TIGIT or CTLA-4. The antibody-TIGIT binding assay was performed as follows: 100 μl of TIGIT-Fc antigen (1 μg / ml) was coated with PBS and incubated overnight at 2-8°C. The next day, 250 μl of PBS containing 3% BSA was added and the mixture was blocked at 37°C for 2 hours. The mixture was then washed twice with PBST, and serially diluted anti-TIGIT, antibody 1, antibody 2, and antibody 3 were added, starting at 3 μg / ml and diluted 1 / 3-fold for a total of 10 dilutions. 100 μl of the diluted antibody was added to each well and incubated at 37°C for 1 hour. The mixture was washed three times with PBST, and a 1:5000 dilution of enzyme-labeled secondary antibody (Sigma goat anti-human IgG) was added. 100 μl of kappa (IgG light chain conjugated with HRP, catalog number A7164-1ML) per well was incubated at 37°C for 1 hour. The mixture was washed 5 times with PBST, and TMB chromogenic solution was added for about 20 min of color development. Then, 1 M H2SO4 was added to terminate the reaction. The OD450 value was read within 30 min.

[0315] As attached Figure 4 As shown, the binding ability of antibodies 1 and 2 to human TIGIT-Fc is close to that of anti-TIGIT, while the binding ability of antibody 3 to human TIGIT-Fc is relatively low.

[0316] 2) The steps for the binding assay of the antibody of this invention to CTLA-4-Fc are similar to those described above. For example... Figure 5 As shown, antibodies 1, 2, and 3 can all bind to CTLA-4-Fc, with antibodies 1 and 2 exhibiting stronger binding affinity. Example 4: Detection of Antibody Affinity

[0317] 1) To determine the effect of linker lengths (L2 and L4) on the ability of bispecific antibodies to bind to hTIGIT-his protein (Shanghai Nearshore Technology Co., Ltd., CJ58), the affinity constants of anti-TIGIT, antibody 1, antibody 2 and antibody 3 to hTIGIT-his protein were measured using a Biacore T200 instrument. The specific experimental procedure is as follows: The hTIGIT-his antigen was serially diluted with HBS-EP+buffer in five steps, with an initial concentration of 50 nM, and then halved. The test proteins anti-TIGIT, antibody 1, antibody 2, and antibody 3 were diluted with HBS-EP+buffer to 2.5 μg / ml. The instrument used was a Biacore T200 with a Protein A chip (GE Healthcare, catalog number 29127556). The instrument settings were as follows: antibody capture binding time 25 s, flow rate 5 μl / min; sample binding time 120 s, dissociation time 300 s, flow rate 30 μl / min; regeneration conditions: Glycine-HCl (pH 1.5), regeneration time 60 s, flow rate 30 μl / min; detection channels (flow paths) were 2-1 and 4-3.

[0318] As shown in Table 3, compared with anti-TIGIT, the affinity of antibody 1 to hTIGIT-his was not reduced, and even increased to a certain extent; compared with anti-TIGIT, the affinity of antibodies 2 and 3 to hTIGIT-his was basically the same.

[0319] 2) The affinity constants of anti-CTLA-4, antibody 1, antibody 2 and antibody 3 with hCTLA-4-his protein (Shanghai Nearshore Technology Co., Ltd., CP33) were determined using a Biacore T200 instrument. The specific experimental procedure is as follows: The hCTLA-4-his antigen was serially diluted with HBS-EP+buffer in six steps, with an initial concentration of 100 nM, and then halved. The test proteins anti-CTLA-4, antibody 1, antibody 2, and antibody 3 were diluted with HBS-EP+buffer to 5 μg / ml. The instrument used was a Biacore T200 with a Protein A chip (GE Healthcare, catalog number: 29127556). The instrument settings were as follows: antibody capture binding time 15 s, flow rate 10 μl / min; sample binding time 180 s, dissociation time 300 s, flow rate 30 μl / min; regeneration conditions: Glycine-HCl (pH 1.5), regeneration time 60 s, flow rate 30 μl / min; detection channels (flow paths) were 2-1 and 4-3.

[0320] As shown in Table 4, compared with anti-CTLA-4, the affinity of antibody 1 and antibody 2 to hCTLA-4-his is basically the same; compared with anti-CTLA-4, antibody 3 has a lower affinity to hCTLA-4-his.

[0321] Table 3. Affinity of antibody to hTIGIT-his

[0322]

[0323] Table 4. Affinity of antibody to hCTLA-4-his

[0324]

[0325] Example 5: Detection of antibody biological activity

[0326] 1) To further identify the biological activity of the antibody, the biological activity of the antibody was detected using the anti-CTLA-4 reporter gene detection system (Promega, catalog number JA1400). The detection process was performed in accordance with the product instructions.

[0327] like Figure 6As shown, the control antibody anti-CTLA-4 exhibits good biological activity, activating the IL-2 promoter in the reporter system and thus activating downstream luciferase expression; antibody 1 shows relatively weak activation ability and relatively low biological activity. Figure 7 As shown, compared with anti-CTLA-4, antibody 2 has relatively weaker activation ability and relatively lower biological activity.

[0328] 2) The biological activity of the antibody was determined using the anti-TIGIT reporter gene detection system, and the detection process was similar to that of Promega's anti-CTLA-4 reporter gene detection system mentioned above.

[0329] like Figure 8-10 As shown, compared with the control antibody anti-TIGIT-4, antibodies 1, 2 and 3 did not show worse biological activity; in fact, antibodies 1 and 2 had relatively better activation ability.

[0330] Based on the above antibody biological activity tests, the increase in the length of linkers L2 and L4 did not significantly affect the function of the TIGIT target, but it did affect the function of the CTLA-4 target: the longer the linker length, the lower the biological activity against the CTLA-4 target; the antibody 1 of the present invention has superior biological activity.

[0331] Current clinical trials targeting TIGIT (Genentech MTIG-7129 clinical trial (NCT03563716) or OncoMed OMP-313M32 clinical trial (NCT03119428)) show that this target drug generally requires a high dose. The 2018 Society for Cancer Immunotherapy (SITC 2018) abstracts revealed that the TIGIT-targeted antibody OMP-313M32 did not experience any serious adverse events. However, for anti-CTLA-4 antibodies, clinical observations show that up to 60% of patients receiving the anti-CTLA-4 antibody Yervoy experience immune-related adverse events: 10%-30% are serious (grade 3-4) immune-related adverse events, and the risk of immune-related adverse events in patients receiving Yervoy is dose-dependent (Martins F, Sofiya L, Sykiotis GP, et al. Adverse effects of immune-checkpoint inhibitors: epidemiology, management and surveillance[J]. Nature Reviews Clinical). Oncology, 2019, 16(9):563-580.); The biological activity of antibody 1 against CTLA-4 in this invention is reduced to a certain extent compared with anti-CTLA-4. It is expected that it can both ensure the blocking of TIGIT and CTLA-4 targets and reduce or avoid the side effects of anti-CTLA-4 antibody.

[0332] Bispecific antibodies targeting OX40 and CTLA-4

[0333] Example 6 Antibody Preparation Method

[0334] The structure of this bispecific antibody is as follows: Figure 1 As shown, the structure is similar to that of an immunoglobulin. The antibody contains a first polypeptide chain and a third polypeptide chain with the same sequence, as well as a second polypeptide chain and a fourth polypeptide chain with the same sequence. The first polypeptide chain contains the structure VHa-L1-CLa-L2-VHb-CH1-Fc, and the second polypeptide chain contains the structure VLa-L3-CH1-L4-VLb-CLb. Antigen a is OX40, and antigen b is CTLA-4.

[0335] The sequence of the second polypeptide used to synthesize the antibody: the 5' end contains a HindIII restriction site and a leader peptide, and the 3' end contains a stop codon and an EcoRI restriction site; the sequence of the first polypeptide used to synthesize the antibody: the 5' end contains a HindIII restriction site and a leader peptide, and the 3' end contains a stop codon and an EcoRI restriction site.

[0336] Plasmids containing the nucleic acid sequences of the first and second polypeptides, respectively, were simultaneously and transiently transfected into 293F cells. 293F cells were cultured in CD 293 TGE Medium (BPM Cell Culture, catalog number: CM-1156). When cell viability reached above 95%, cells were passaged to 800,000-1,000,000 / mL using fresh medium. After 24 hours, when the cell density reached approximately 1,500,000-2,000,000 / mL, transfection was performed. The plasmid was first incubated at 65°C for 30 minutes. 0.5 μg of plasmid was used per 1,000,000 cells, and 3 μL of PEI (Polysciences, catalog number: 24765-2) was used per 1 μg of plasmid. The plasmid and PEI were diluted separately with culture medium (the combined volume of the two solutions was 5% of the total volume) and incubated at room temperature for 5 minutes. Then, the PEI solution was added to the plasmid solution, mixed well, and incubated at room temperature for 20 minutes. The complex solution was added to the cells, and after culturing in a shaker at 37°C for 24 hours, 10% (v / v) of CD Feed X Supplement (BPM Cell Culture, catalog number CF-1116) was added to the cells. The cell supernatant was harvested after 5 days and purified using Protein A to obtain bispecific antibody 5.

[0337] The amino acid sequences associated with bispecific antibodies 5 targeting OX40 and CTLA-4 are shown in Table 5, and the nucleic acid sequences associated with bispecific antibodies 5 targeting TIGIT and CTLA-4 are shown in Table 6. Linkers in Table 5 are indicated by bold italics and underlines.

[0338] Table 5. Amino acid sequences of bispecific antibodies targeting OX40 and CTLA-4

[0339]

[0340]

[0341] Table 6. Nucleic acid sequences encoding bispecific antibodies targeting OX40 and CTLA-4

[0342]

[0343]

[0344]

[0345] Example 7 Antibody Purity Detection

[0346] Antibody 5 was subjected to both reduction and non-reduction treatments, and then used for SDS-PAGE electrophoresis. Figure 11As shown, the bands for reduced and non-reduced antibodies are clear and free of impurities, indicating high sample purity: the non-reduced antibody 5 has a size greater than 250 kDa on the electrophoresis image; the first polypeptide of the reduced antibody 5 has a size of approximately 90 kDa, and the second polypeptide of the reduced antibody 5 has a size of approximately 50 kDa; these sizes are as expected. This demonstrates that the bispecific antibody 5 can be normally expressed in cells; the purified antibody 5 expressed in 293F cells can be used for further experiments.

[0347] Example 8: Detection of antibody binding activity

[0348] CTLA-4-Fc antigen and OX40-Fc antigen were coated onto ELISA plates and incubated overnight at 4°C. The next day, the plates were blocked with 5% BSA blocking buffer at 37°C for 2 hours. After washing, 5-fold serial dilutions of antibody 5, CTLA-4 monoclonal antibody (i.e., anti-CTLA-4), and OX40 monoclonal antibody (i.e., anti-OX40) were added, starting at 2 μg / mL, for a total of 12 concentrations (the last being a blank concentration of 0). Each concentration was used in duplicate, and the plates were incubated at 37°C for 2 hours. After washing, 1:10000 dilution of anti-human Kappa light chain secondary antibody was added, and the plates were incubated at 37°C for 0.5 hours. After washing, TMB single-component chromogenic buffer was added, and the plates were developed in the dark for 10-15 minutes. The reaction was terminated with 0.1M sulfuric acid, and the readings were taken at 450 nm. The EC50 was calculated from the ELISA binding curve. 50 The values ​​are shown in Table 7.

[0349] The preparation method of OX40-Fc antigen is as follows: The amino acid sequence of human OX40 (P43489) was found from the Uniprot protein database, where the amino acid sequence of the extracellular region of human OX40 consists of residues 1 to 216; the amino acid sequence of IgG1-Fc (P01857) from residues 104 to 330 was found from the Uniprot protein database; then, through artificial synthesis (General Electric), the nucleotide sequences corresponding to the extracellular region of OX40 and Fc were obtained, and after enzyme digestion and ligation, they were inserted into the pCDNA3.0 vector (Invitrogen) to obtain a recombinant plasmid; the above plasmid was then transiently transfected into HEK293 cells using PEI, and the supernatant was collected after 7 days of culture. Finally, OX40-Fc (i.e., hOX40-Fc) was obtained through purification.

[0350] As shown in Table 7, antibody 5 retains its ability to bind to both OX40 and CTLA-4 antigens; compared with monoclonal antibodies, antibody 5 has a better binding ability to OX40 and a poorer binding ability to CTLA-4.

[0351] Table 7. EC5 of antibody-antigen binding 50 value

[0352]

[0353] Example 9: Detection of Antibody Affinity

[0354] Biotin was conjugated to the OX40 monoclonal antibody, CTLA-4 monoclonal antibody, and antibody 5, respectively. The SA sensor was pre-wetted in PBS for 10 minutes. The biotin-labeled antibody was diluted to 10 μg / mL with PBST containing 0.5% BSA at pH 6.8 (hereinafter referred to as dilution buffer). The SA sensor was then immobilized in each of the three biotinylated antibodies until the signal reached approximately 4 nm. The CTLA-4-Fc antigen and hOX40-Fc antigen were diluted to 400 nM with dilution buffer, and a 2-fold serial dilution was performed using this as the starting concentration, preparing a total of 6 concentration points (400, 200, 100, 50, 25, 0 nM). Add dilution buffer, gradient concentration drug dilution buffer, regeneration buffer (1M MgCl2, pH 8.4), and neutralization buffer (PBST) sequentially to the corresponding columns of a 96-well plate, and run the following steps: ① Baseline: Detect baseline in dilution buffer for 60 seconds; ② Association: Bind in antibody gradient dilution buffer and sample blank (dilution buffer) for 300 seconds; ③ Dissociation: Dissociate in dilution buffer for 360 seconds; ④ Regeneration: Regenerate in regeneration buffer for 5 seconds; ⑤ Neutralization: Neutralize in neutralization buffer for 5 seconds; ⑥ Repeat the regeneration and neutralization cycles 3 times.

[0355] The data was processed and analyzed using ForteBio Data Analysis 8.2. The sample data was fitted after subtracting the reference (sample blank) signal to obtain the affinity constant K. D The affinity results of antibody 5 with CTLA-4-Fc antigen are shown in Table 8, and the affinity results of antibody 5 with OX40-Fc antigen are shown in Table 9.

[0356] Table 8. Kinetic parameters of antibody-CTLA-4 antigen affinity binding.

[0357]

[0358] Table 9 Kinetic parameters of antibody-OX40 antigen affinity binding

[0359]

[0360]

[0361] As shown in Table 8, antibody 5 has a higher affinity for the CTLA-4 antigen compared to monoclonal antibodies; as shown in Table 9, antibody 5 has a similar affinity for the OX40 antigen compared to monoclonal antibodies.

[0362] Example 10 Antibody and OX40 + Cell binding activity

[0363] Jurkat cells overexpressing human OX40 were generated by transfecting a pCMV vector (Invitrogen) carrying human OX40 cDNA. + Cells), OX40 were taken respectively + Cells were centrifuged to remove the culture medium, washed once with PBS, and the cell pellet was retained. OX40 monoclonal antibody and antibody 5 were diluted 3-fold starting at a concentration of 100 nM, for a total of 10 concentrations. The corresponding cell pellets (OX40) were resuspended in these antibody solutions at different concentrations. + Each concentration requires approximately 2 million cells. After incubating at 4°C for 2 hours, centrifuge to remove the supernatant and wash once with PBS. Add 1:1000 diluted anti-Fc-PE (Invitrogen, catalog number 12-4998-82) to each sample and incubate at 4°C in the dark for half an hour. Centrifuge to remove the supernatant and wash once with PBS. Resuspend each sample in PBS and then perform sample detection on a C6 flow cytometer, using channel 2 as the light source. The binding curve is shown below. Figure 12 The positive rate and fluorescence value statistics are shown in Table 10-11.

[0364] like Figure 12 And Table 10-11, compared with monoclonal antibodies, antibody 5 and OX40 + The binding activity of the cells is comparable.

[0365] Table 10 OX40 monoclonal antibody and OX40 + Cell binding positivity rate and fluorescence value

[0366]

[0367] Table 11 Antibody 5 and OX40 + Cell binding positivity rate and fluorescence value

[0368]

[0369] Example 11: Antibody simultaneously binds to OX40 antigen and CTLA-4 antigen

[0370] Biotin was conjugated to the OX40-Fc antigen, and the SA sensor was pre-wetted in PBS for 10 minutes before use. The biotinylated OX40-Fc antigen was diluted to 5 μg / mL with PBST containing 0.5% BSA at pH 6.8 (hereinafter referred to as dilution buffer). The SA sensor was then immobilized in the above-mentioned biotinylated OX40-Fc antigen solution until the signal was approximately 0.8 nM. The antibody to be tested, 5, was diluted to 50 nM with dilution buffer; the CTLA-4-Fc antigen was diluted to 200 nM with dilution buffer. Add dilution buffer, antibody 5-dilution buffer, CTLA-4-Fc antigen dilution buffer, regeneration buffer (1M MgCl2, pH 8.4), and neutralization buffer (PBST) sequentially to the corresponding columns of a 96-well plate, and run the following steps: ① Baseline: Detect baseline in dilution buffer for 60 seconds; ② Association: Bind in antibody gradient dilution buffer and sample blank (dilution buffer) for 300 seconds; ③ Baseline: Detect baseline in dilution buffer for 60 seconds; ④ Association: Bind in antibody gradient dilution buffer and sample blank (dilution buffer) for 300 seconds; ⑤ Dissociation: Dissociate in dilution buffer for 300 seconds; ⑥ Regeneration: Regenerate in regeneration buffer for 5 seconds; ⑦ Neutralization: Neutralize in neutralization buffer for 5 seconds; ⑧ Repeat the regeneration and neutralization cycles 3 times.

[0371] like Figure 13 As shown, the sensor binds to antibody 5 after immobilizing the OX40-Fc antigen, and there is a significant binding. When it binds to the CTLA-4-Fc antigen, there is still a certain binding signal value. Furthermore, no significant signal drop is observed during the dissociation step. This indicates that antibody 5 can bind to both the OX40 antigen and the CTLA-4 antigen simultaneously.

[0372] Bispecific antibodies targeting OX40 and TIGIT

[0373] Example 12: Method for preparing antibodies

[0374] The structure of this bispecific antibody is as follows: Figure 1 As shown, the structure is similar to that of an immunoglobulin. The antibody contains a first polypeptide chain and a third polypeptide chain with the same sequence, as well as a second polypeptide chain and a fourth polypeptide chain with the same sequence. The first polypeptide chain contains the structure VHa-L1-CLa-L2-VHb-CH1-Fc, and the second polypeptide chain contains the structure VLa-L3-CH1-L4-VLb-CLb. Antigen a is OX40, and antigen b is TIGIT.

[0375] The nucleic acid sequences corresponding to the first and second polypeptides of the bispecific antibody were artificially synthesized, and then ligated into the pCDNA3.0 vector (purchased from Invitrogen) by enzyme digestion and ligation, respectively, to obtain two recombinant plasmids for expressing the universal antibody. Following the manufacturer's instructions, the plasmids were transfected into HEK293 cells via PEI transiently according to the kit (purchased from Invitrogen). After 7 days of culture, the supernatant was collected, and finally, the bispecific antibody protein sample was obtained through purification.

[0376] The amino acid sequences associated with 6 and 7 of the bispecific antibodies targeting OX40 and TIGIT are shown in Table 12, and the nucleic acid sequences associated with 6 and 7 of the bispecific antibodies targeting TIGIT and TIGIT are shown in Table 13. Linkers in Table 12 are indicated by bold italics and underlines.

[0377] Table 12 Amino acid sequences of bispecific antibodies targeting OX40 and TIGIT

[0378]

[0379]

[0380]

[0381] Table 13 Nucleic acid sequences encoding bispecific antibodies targeting OX40 and TIGIT

[0382]

[0383]

[0384]

[0385]

[0386] Example 13 Antibody Purity Detection

[0387] The composition and purity of bispecific antibodies were analyzed by SDS-PAGE under both reducing and non-reducing conditions. Figure 14 As shown, under non-reducing conditions, antibodies 6 and 7 migrate with a single band; under reducing conditions, antibodies 6 and 7 each produce two bands; antibodies 6 and 7 are single substances, and these two polypeptide chains effectively pair to form an IgG-like protein.

[0388] Example 14 Detection of Antibody Affinity

[0389] ForteBio affinity assays can be performed using existing conventional methods (Estep Patricia, et al. High-throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013, 5(2): 270-8). The specific experimental procedure is as follows: the sensor is equilibrated offline in analytical buffer (such as PBS) for 20 minutes, and then the signal baseline is established by detection for 60 seconds. The purified antibodies 6 and 7 are loaded onto the corresponding sensors (ForteBio) and ForteBio affinity is measured. Antibodies 6 and 7 are adsorbed using protein sensors, and their binding and dissociation with OX40-his (AcroBiosystems) and Tigit-his (AcroBiosystems) are detected separately for about 5 minutes each. Finally, a 1:1 binding model is used for kinetic analysis.

[0390] Table 14 Affinity of antibodies to OX40-his and TIGIT-his

[0391]

[0392] As shown in Table 14, antibody 6 can bind to the OX40 antigen significantly, but its affinity is weaker than that of anti-OX40; antibody 7 can bind to the OX40 antigen significantly, and its affinity is comparable to that of anti-OX40; antibody 7 can also bind to the TIGIT antigen significantly, and its affinity is basically close to that of anti-TIGIT.

[0393] Example 15: Antibody simultaneously binds to OX40 antigen and TIGIT antigen

[0394] Similar to the Fortebio affinity assay described above, the ability of the bispecific antibody to simultaneously bind to both the OX40-his antigen (AcroBiosystems) and the TIGIT-his antigen (AcroBiosystems) was tested. A protein sensor was used to adsorb antibody 7 from the above examples; first, binding and dissociation with the OX40 antigen were detected, followed by binding and dissociation with the TIGIT antigen.

[0395] like Figure 15 As shown, antibody 7 can clearly bind to both OX40 and TIGIT antigens simultaneously without steric hindrance. Example 16: Binding activity of the antibody with Jurkat cells overexpressing OX40 or TIGIT.

[0396] 1) Jurkat-OX40 cells overexpressing human OX40 were generated by transfecting pCMV vector (Invirogen) containing human OX40 cDNA (i.e., the aforementioned OX40 cells). + Jurkat-hOX40 cells (0.5 × 10⁻⁶ cells) were used to prepare the cells. 6 Cells were incubated with different concentrations of antibody in PBS on ice for 40 minutes. The cells were then washed twice and incubated with secondary antibody in PBS (containing 0.1% BSA) on ice for 25 minutes. The cells were washed twice and analyzed by flow cytometry on an Accuri C6 system (BD Biosciences).

[0397] like Figure 16 As shown, antibody 7 (OT-4D-30a) can bind significantly to Jurkat-OX40 cells, and its binding affinity is close to that of anti-OX40.

[0398] 2) Jurkat cells overexpressing human TIGIT, known as Jurkat-Tigit cells, were generated by transfecting the pCMV vector carrying human TIGIT cDNA. The Jurkat-Tigit cells (0.5 × 10⁻⁶ cells) were then... 6 Cells were incubated with different concentrations of antibody in PBS containing 0.1% BSA on ice for 40 minutes. Cells were then washed twice and incubated with secondary antibody in PBS (containing 0.1% BSA) on ice for 25 minutes. Cells were washed twice and analyzed by flow cytometry on an Accuri C6 system (BD Biosciences).

[0399] like Figure 17 As shown, antibody 7 can bind significantly to Jurkat-Tigit cells, but its binding affinity is slightly weaker than that of anti-TIGIT.

Claims

1. An antibody or antigen-binding fragment, characterized in that, The antibody or antigen-binding fragment binds to two different antigens, a first antigen a and a second antigen b, and the antibody or antigen-binding fragment comprises at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain. The first polypeptide chain, starting from the amino terminus, sequentially comprises VHa, CLa, VHb, and CH1, where VHa is the heavy chain variable region binding to the first antigen a, CLa is the first light chain constant region, VHb is the heavy chain variable region binding to the second antigen b, and CH1 is the first constant region of the heavy chain. The second polypeptide chain, starting from the amino terminus, sequentially comprises VLa, CH1, VLb, and CLb, where VLa is the light chain variable region binding to the first antigen a, VLb is the light chain variable region binding to the second antigen b, and CLb is the second light chain constant region. The antigen a is TIGIT and the antigen b is CTLA-4, and the antibody or antigen-binding fragment comprises the following: The VHa comprises VHaCDR1-3, wherein VHaCDR1 is represented by amino acids 30-35 (SSYGMS) in the sequence shown in SEQ ID NO:1, VHaCDR2 is represented by amino acids 50-66 (TINSNGGSTYYPDSVKG) in the sequence shown in SEQ ID NO:1, and VHaCDR3 is represented by amino acids 99-108 (LGTGTLGFAY) in the sequence shown in SEQ ID NO:1; and The VHb comprises VHbCDR1-3, wherein VHbCDR1 is represented by amino acids 31-35 (SYTMH) in the sequence shown in SEQ ID NO:2, VHbCDR2 is represented by amino acids 50-66 (FISYDGNNKYYADSVKG) in the sequence shown in SEQ ID NO:2, and VHbCDR3 is represented by amino acids 99-107 (TGWLGPFDY) in the sequence shown in SEQ ID NO:2; and The VLa comprises VLaCDR1-3, wherein VLaCDR1 is represented by amino acids 24-34 (KASQDVKTAVS) in the sequence shown in SEQ ID NO:3, VLaCDR2 is represented by amino acids 50-56 (WASTRAT) in the sequence shown in SEQ ID NO:3, and VLaCDR3 is represented by amino acids 89-97 (QQHYSTPWT) in the sequence shown in SEQ ID NO:3; and The VLb comprises VLbCDR1-3, wherein VLbCDR1 is represented by amino acids 24-35 (RASQSVGSSYLA) in the sequence shown in SEQ ID NO:4, VLbCDR2 is represented by amino acids 51-57 (GAFSRAT) in the sequence shown in SEQ ID NO:4, and VLbCDR3 is represented by amino acids 90-98 (QQYGSSPWT) in the sequence shown in SEQ ID NO:

4.

2. The antibody or antigen-binding fragment as described in claim 1, characterized in that, VHa and CLa are covalently linked via linker L1, which contains 2 to 6 amino acids; and CLa and VHb are covalently linked via linker L2; wherein L2 contains 10 to 30 amino acids, and at least 50% of the amino acids are glycine.

3. The antibody or antigen-binding fragment as described in claim 1, characterized in that, VLa and CH1 are covalently linked via linker L3, which contains 2 to 6 amino acids; and / or CH1 and VLb are covalently linked via linker L4; wherein L4 contains 10 to 30 amino acids, and at least 50% of the amino acids are glycine.

4. The antibody or antigen-binding fragment as described in claim 1, characterized in that, The first polypeptide chain contains the structure VHa-L1-CLa-L2-VHb-CH1, and the second polypeptide chain contains the structure VLa-L3-CH1-L4-VLb-CLb.

5. The antibody or antigen-binding fragment as described in claim 4, characterized in that, The L1 contains the sequence shown in SEQ ID NO:22; and / or The L2 contains a sequence selected from any one of SEQ ID NO:23-27, or has at least 90% identity with any one of the sequences shown in SEQ ID NO:23-27, or has one or more conserved amino acid substitutions compared to any one of the sequences shown in SEQ ID NO:23-27; and / or The L3 contains the sequence shown in SEQ ID NO:28; and / or The L4 contains a sequence selected from any one of SEQ ID NO:29-33, or has at least 90% identity with any one of SEQ ID NO:29-33, or has one or more conserved amino acid substitutions compared to any one of SEQ ID NO:29-33.

6. The antibody or antigen-binding fragment according to any one of claims 1-5, characterized in that, One of the two polypeptide chains also includes an Fc, which contains the hinge region, the second constant region, and the third constant region of the heavy chain.

7. The antibody or antigen-binding fragment as described in claim 6, characterized in that, The first polypeptide chain contains the structure VHa-L1-CLa-L2-VHb-CH1-Fc, and the second polypeptide chain contains the structure VLa-L3-CH1-L4-VLb-CLb.

8. The antibody or antigen-binding fragment as described in claim 1, characterized in that, The antibody or antigen-binding fragment comprises the following: The VHa contains the sequence shown in SEQ ID NO:1, or has at least 80% identity with the sequence shown in SEQ ID NO:1, or has one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:1; and / or The VHb contains the sequence shown in SEQ ID NO:2, or has at least 90% identity with the sequence shown in SEQ ID NO:2, or has one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:2; and / or The VLa contains the sequence shown in SEQ ID NO:3, or has at least 90% identity with the sequence shown in SEQ ID NO:3, or has one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:3; and / or The VLb contains the sequence shown in SEQ ID NO:4, or a sequence that has at least 90% identity with the sequence shown in SEQ ID NO:4, or has one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:4; The CLa contains the sequence shown in SEQ ID NO:5, or has at least 90% identity with the sequence shown in SEQ ID NO:5, or has one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:5; The CLb contains the sequence shown in SEQ ID NO:6, or has at least 90% identity with the sequence shown in SEQ ID NO:6, or has one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:6; The CH1 contains the sequence shown in SEQ ID NO:7, or has at least 90% identity with the sequence shown in SEQ ID NO:7, or has one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:

7.

9. The antibody or antigen-binding fragment as described in claim 1, characterized in that, The antibody or antigen-binding fragment binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is TIGIT and the second antigen b is CTLA-4; the antibody or antigen-binding fragment comprises at least two polypeptide chains, a first polypeptide chain and a second polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO:37, or has at least 90% identity with the sequence shown in SEQ ID NO:37, or has one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:37; the second polypeptide chain contains the sequence shown in SEQ ID NO:38, or has at least 90% identity with the sequence shown in SEQ ID NO:38, or has one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:38; or The first polypeptide chain contains the sequence shown in SEQ ID NO:39, or has at least 90% identity with the sequence shown in SEQ ID NO:39, or has one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:39; the second polypeptide chain contains the sequence shown in SEQ ID NO:40, or has at least 90% identity with the sequence shown in SEQ ID NO:40, or has one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:40; or The first polypeptide chain contains the sequence shown in SEQ ID NO:41, or has at least 90% identity with the sequence shown in SEQ ID NO:41, or has one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:41; the second polypeptide chain contains the sequence shown in SEQ ID NO:42, or has at least 90% identity with the sequence shown in SEQ ID NO:42, or has one or more conserved amino acid substitutions compared to the sequence shown in SEQ ID NO:

42.

10. An antibody or antigen-binding fragment, characterized in that, The antibody or antigen-binding fragment comprises at least two polypeptide chains: a first polypeptide chain and a second polypeptide chain; the antibody or antigen-binding fragment binds to two different antigens: a first antigen a and a second antigen b, wherein the first antigen a is TIGIT and the second antigen b is CTLA-4. The first polypeptide chain is shown in SEQ ID NO:37, and the second polypeptide chain is shown in SEQ ID NO:38; or The first polypeptide chain is shown in SEQ ID NO:39, and the second polypeptide chain is shown in SEQ ID NO:40; or The first polypeptide chain is shown in SEQ ID NO:41, and the second polypeptide chain is shown in SEQ ID NO:

42.

11. A nucleic acid molecule encoding an antibody or antigen-binding fragment as described in any one of claims 1-10.

12. A carrier comprising the nucleic acid molecule as described in claim 11.

13. A host cell comprising the nucleic acid molecule of claim 11 or the vector of claim 12.

14. A pharmaceutical composition comprising an antibody or antigen-binding fragment as described in any one of claims 1-10.

15. A diagnostic kit comprising an antibody or antigen-binding fragment as described in any one of claims 1-10.

Images