WO2022007807A1

WO2022007807A1 - Bispecific antibody and use thereof

Info

Publication number: WO2022007807A1
Application number: PCT/CN2021/104810
Authority: WO
Inventors: 梁世忠; 黄俊杰; 梁炳辉; 徐振前; 俞金泉; 李胜峰
Original assignee: 百奥泰生物制药股份有限公司
Priority date: 2020-07-07
Filing date: 2021-07-06
Publication date: 2022-01-13
Also published as: CN115776898A

Abstract

Provided are a bispecific antibody and use thereof. The bispecific antibody or antigen binding fragment can bind to two antigens or two epitopes of the same antigen. The antibody or antigen binding fragment can be used for treating various diseases, such as inflammatory diseases, autoimmune diseases, cancer or spinal cord injury, and can also be used for the diagnosis and prognosis of related diseases.

Description

Bispecific antibodies and their applications

technical field

The invention belongs to the field of biomedicine, and particularly relates to bispecific antibodies and applications thereof.

Background technique

Current powerful tools for cancer immunotherapy include monoclonal antibodies, tumor vaccines, immune checkpoint inhibitors, CAR-T cell immunotherapy, bispecific antibodies (BsAbs), and multispecific antibodies. CAR-T and BsAb have received increasing attention as new strategies for anti-tumor immunotherapy.

Bispecific antibodies are artificially engineered antibodies that can simultaneously bind two specific epitopes or target proteins, and can play some special biological functions. Compared with the combined treatment of two monoclonal antibody drugs, BsAb improves the antibody selectivity and functionality, and reduces the treatment cost. The preparation of BsAb mainly includes chemical coupling, double-hybridoma cell method, recombinant gene preparation and other methods.

However, there are difficulties in the preparation of bispecific antibodies, such as the production of a large number of by-products and the inhibition of biological activity.

SUMMARY OF THE INVENTION

The present invention provides multivalent and multispecific antibodies or antigen-binding fragments and uses thereof. In some embodiments, the present invention provides bispecific antibodies or antigen-binding fragments. The antibodies or antigen-binding fragments provided herein can bind two or more antigens, or two or more epitopes of the same antigen, or two or more copies of the same epitope. The antibodies or antigen-binding fragments provided by the present invention can be used to treat or improve inflammatory diseases, autoimmune diseases, cancer or spinal cord injury; the antibodies or antigen-binding fragments provided by the present invention can also be used for the diagnosis and prognosis of related diseases.

In some embodiments, the antibody or antigen-binding fragment binds two different epitopes, a first epitope x and a second epitope y, comprising the first epitope x and the second epitope y, respectively The combined first antigen a binding part and the second antigen b binding part, and the antibody or antigen binding fragment comprises at least 3 polypeptide chains; wherein, the first polypeptide chain from the amino terminus sequentially comprises VHa, CH1a, VHb and CLb, VHa is the heavy chain variable region of the first antigen a binding portion, CH1a is the heavy chain first constant region of the first antigen a binding portion, VHb is the heavy chain variable region of the second antigen b binding portion, CLb is The light chain constant region of the second antigen b binding portion. In some embodiments, the first epitope x and second epitope y are epitopes on different antigens (first antigen a and second antigen b), respectively. In some embodiments, the first epitope x and the second epitope y are different epitopes on the same antigen a (or antigen b).

In some embodiments, the antibody or antigen-binding fragment comprises a first antigen a-binding portion and a second antigen-b binding portion that bind to two different antigens, a first antigen a and a second antigen b, respectively, and the antibody or The antigen-binding fragment comprises at least 3 polypeptide chains; wherein, the first polypeptide chain comprises VHa, CH1a, VHb and CLb in sequence from the amino terminus to the carboxyl terminus, where VHa is the heavy chain variable region of the first antigen a binding portion, and CH1a is the The heavy chain first constant region of the first antigen a binding portion, VHb is the heavy chain variable region of the second antigen b binding portion, and CLb is the light chain constant region of the second antigen b binding portion.

In some embodiments, CH1a and VHb are covalently linked through linker L1; wherein L1 contains 5 to 33 amino acids, and at least 50% of the amino acids are glycines; and/or

VHb and CLb are covalently linked through linker L2, which contains 2 to 6 amino acids.

In some embodiments, CH1a and VHb are covalently linked through linker L1, L1 contains 5 to 33 amino acids, and at least 50% of the amino acids are glycine; VHb and CLb are covalently linked through linker L2, L2 contains 2 to 6 amino acids.

In some embodiments, L1 contains about 5, about 6, about 9, 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, about 31, about 33 amino acids, or a range (including endpoints) between or between any two of these values any value. In some embodiments, L1 contains serine. In some embodiments, L1 contains about 4, about 8, about 10, about 12, about 15, about 16, about 20, about 24 glycines, or any two of these values range between (including the endpoints) or any value therein. In some embodiments, L2 contains about 2, about 3, about 4, about 5, or about 6 amino acids.

In some embodiments, the second polypeptide chain comprises VLa, CLa in sequence from the amino terminus to the carboxy terminus; wherein VLa is the light chain variable region of the first antigen a binding moiety and CLa is the first antigen a binding moiety. part of the light chain constant region.

In some embodiments, the third polypeptide chain comprises, from amino terminus to carboxy terminus, VLb, CH1b; wherein VLb is the light chain variable region of the second antigen b binding portion, and CH1b is the second antigen b binding portion heavy chain constant region.

In some embodiments, VLb and CH1b are covalently linked through linker L3, which contains 2 to 6 amino acids. In some embodiments, L3 contains about 2, about 3, about 4, about 5, or about 6 amino acids.

In some embodiments, the first polypeptide chain or the third polypeptide chain further comprises an Fc, which is a hinge region comprising a heavy chain, a second constant region, and a third constant region. 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 substitutions, deletions or insertions, relative to the parent Fc region. In some embodiments, the amino acid modification of the Fc region alters effector function activity relative to the activity of the parental Fc region. In some embodiments, variant Fc regions may have altered (ie, increased or decreased) antibody-dependent cellular cytotoxicity (ADCC), complement-mediated cytotoxicity (CDC), phagocytosis, opsonization, or cell binding . In some embodiments, the Fc region amino acid modifications can alter the affinity of the variant Fc region for FcγRs (Fcγ receptors) relative to the parent Fc region. In some embodiments, the Fc region is derived from IgGl or IgG4.

In some embodiments, the third polypeptide chain comprises Fc.

In some embodiments, the first polypeptide chain comprises the structure VHa-CH1a-L1-VHb-L2-CLb, the second polypeptide chain comprises the structure VLa-CLa, and the third polypeptide chain comprises the structure VLb-L3-CH1b; or

The first polypeptide chain comprises the structure VHa-CH1a-L1-VHb-L2-CLb, the second polypeptide chain comprises the structure VLa-CLa, and the third polypeptide chain comprises the structure VLb-L3-CH1b-Fc.

In some embodiments, CH1a of the first polypeptide chain is connected to CLa of the second polypeptide chain by a disulfide bond, and CLb of the first polypeptide chain is connected to CH1b of the third polypeptide chain by a disulfide bond.

In some embodiments, the antigens a, b are cytokines, cytokine receptors, chemokines, chemokine receptors or cell surface proteins. In some embodiments, the antibody or antigen-binding fragment specifically binds a cytokine. 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 - alpha (tumor necrosis factor alpha), TNF-beta and (tumor necrosis factor beta) etc. In some embodiments, the antibody or antigen-binding fragment can specifically bind to an immune checkpoint protein. In some embodiments, the immune checkpoint proteins include TIM-3 (T cell immunoglobin domain and mucin domain-3), LAG3 (lymphocyte activation gene-3 molecule), CTLA-4 (cytotoxic T lymphocyte associated antigen) ), TIGIT (T cell Ig and ITIM domain), CD27 (cluster of differentiation 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 (cluster of differentiation 137). 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), galectin 9, CD48 (cluster of differentiation 48), CD40 (cluster of differentiation 40), CD70 (cluster of differentiation 70), B7H3 (CD276, cluster of differentiation 276) and HVEM (Herpesvirus Entry Mediator) and so on. In some embodiments, the antibody or antigen-binding fragment is capable of binding a chemokine or chemokine receptor, such as CCL1, CCL3, CCL5, CCL7, CCL8, etc., in the CC chemokine subset.

In some embodiments, antigens a, b are each selected from the group consisting of TIGIT and CTLA-4, OX40 and CTLA-4, TIGIT and PD-1, PD-L1 and CD47 (cluster of differentiation 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 (cluster of differentiation 20), PD-L1 and 4-1BB (CD137, a member of the tumor necrosis factor receptor superfamily), PSMA (prostate-specific membrane antigen) and CD28 (costimulatory molecules), PD-1 and PD-L1, HER2 (human epidermal growth factor receptor 2) and 4-1BB, PD-1 and TIM-3, PD-1 and CD47 (cluster of differentiation 47), GITR (glucocorticoid-induced tumor necrosis factor receptor) and CTLA-4, CD40 (cluster of differentiation 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) and CTLA-4, CD19 (Cluster of Differentiation 19) and CD22 (Cluster of Differentiation 22), CD16 (Cluster of Differentiation 16) and CD30 (Cluster of Differentiation 30), CD3 (Cluster of Differentiation 3) and CD123 (cluster of differentiation 123), BCMA (B cell maturation antigen) and CD47, MSLN (mesothelin) and CD47, EGFR and cMET, CD73 and TGFβ (transforming growth factor beta), EGFR and TGFβ, CCR2 (CC chemotaxis) factor receptor 2) and CSF1R (colony stimulating factor 1 receptor), CD20 and CD3, CD19 and CD47, CDH17 (hepatic and intestinal cadherin) and TRAILR2 (TRAIL receptor 2, TRAIL is a tumor necrosis-related apoptosis-inducing ligand) , APLP2 (amyloid peptide precursor-like 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 alpha) and IL-5, VEGF and IL-6 (interleukin IL-6), FGFR1 (fibroblast growth factor receptor 1) ) and KLB (klotho beta protein). In some embodiments, the antibody or antigen-binding fragment is capable of specifically binding both antigen a and antigen b.

In some embodiments, the antigen a is OX40 and the antigen b is CTLA-4. In some embodiments, the antigen a is OX40 and the antigen b is TIGIT. In some embodiments, the antigen a is TIGIT and the antigen b is OX40. In some embodiments, antigen a is TIGIT and antigen b is PD-1.

In some embodiments, the antigen a is OX40 and the antigen b is TIGIT, and the antibody or antigen fragment comprises the following:

The VHa contains the heavy chain CDRs or heavy chain variable regions disclosed in CN101331150A1 or US20150307617A1; and/or

The VHb contains the heavy chain CDRs or heavy chain variable regions disclosed in US20190100591A1 or US20180169239A1; and/or

The VLa contains the light chain CDRs or light chain variable regions disclosed in CN101331150A1 or US20150307617A1; and/or

The VLb contains the light chain CDRs or light chain variable regions disclosed in US20190100591A1 or US20180169239A1.

The entire contents of CN101331150A1, US20150307617A1, US20190100591A1, US20180169239A1 are incorporated herein by reference.

Described VHa contains the 31st-35th amino acid (VHaCDR1, SYGMH) in the sequence shown in SEQ ID NO:1, and/or the 50th-66th amino acid (VHaCDR2, VISYDGSNQYYADSVKG), and/or the 99th-111th amino acid (VHaCDR3, DNQDSSPDVGIDY); and/or

The VHb contains amino acids at positions 30-35 in the sequence shown in SEQ ID NO: 3 (VHbCDR1, SSYGMS), and/or amino acids at positions 50-66 (VHbCDR2, TINSNGGSTYYPDSVKG), and/or amino acids at positions 99-108 (VHbCDR3, LGTGTLGFAY); and/or

The VLa contains amino acids at positions 24-34 in the sequence shown in SEQ ID NO: 4 (VLaCDR1, RASQNISPFLN), and/or amino acids at positions 50-56 (VLaCDR2, AASSLQS), and/or amino acids at positions 89-97 (VLaCDR3, QQYNSYPLT); and/or

The VLb contains amino acids 24-34 in the sequence shown in SEQ ID NO: 6 (VLbCDR1, KASQDVKTAVS), and/or amino acids 50-56 (VLbCDR2, WASTRAT), and/or amino acids 89-97 (VLbCDR3, QQHYSTPWT).

The VHa contains the sequence shown in SEQ ID NO: 1, has at least 80% identity with the sequence shown in SEQ ID NO: 1, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 1 amino acid sequence of amino acid substitutions; and/or

The VHb contains the sequence shown in SEQ ID NO:3, has at least 80% identity with the sequence shown in SEQ ID NO:3, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO:3 amino acid sequence of amino acid substitutions; and/or

The VLa contains the sequence shown in SEQ ID NO: 4, has at least 80% identity with the sequence shown in SEQ ID NO: 4, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 4 amino acid sequence of amino acid substitutions; and/or

The VLb contains the sequence shown in SEQ ID NO:6, a sequence with at least 80% identity to the sequence shown in SEQ ID NO:6, or one or more conserved sequences compared with the sequence shown in SEQ ID NO:6 amino acid sequence of amino acid substitutions; and/or

The CLa contains the sequence shown in SEQ ID NO:7, a sequence with at least 80% identity to the sequence shown in SEQ ID NO:7, or one or more conserved sequences compared with the sequence shown in SEQ ID NO:7 amino acid sequence of amino acid substitutions; and/or

The CLb contains the sequence shown in SEQ ID NO: 8, has at least 80% identity with the sequence shown in SEQ ID NO: 8, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 8 amino acid sequence of amino acid substitutions; and/or

The CH1a contains the sequence shown in SEQ ID NO: 9, has at least 80% identity with the sequence shown in SEQ ID NO: 9, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 9 amino acid sequence of amino acid substitutions; and/or

The CH1b contains the sequence shown in SEQ ID NO: 9, has at least 80% identity with the sequence shown in SEQ ID NO: 9, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 9 Amino acid sequence of amino acid substitutions.

The VHa contains amino acids at positions 31-35 in the sequence shown in SEQ ID NO: 2 (VHaCDR1, SYGMH), and/or amino acids at positions 50-66 (VHaCDR2, VIAEVGSNQYYADSVKG), and/or amino acids at positions 99-111 (VHaCDR3, DNQDTSPDVGIDY); and/or

The VLa contains amino acids at positions 24-34 in the sequence shown in SEQ ID NO: 5 (VLaCDR1, RASQNISPFLN), and/or amino acids at positions 50-56 (VLaCDR2, AAVGLQS), and/or amino acids at positions 89-97 (VLaCDR3, QQYTDYPLT); and/or

The VHa contains the sequence shown in SEQ ID NO:2, has at least 80% identity with the sequence shown in SEQ ID NO:2, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO:2 amino acid sequence of amino acid substitutions; and/or

The VLa contains the sequence shown in SEQ ID NO:5, has at least 80% identity with the sequence shown in SEQ ID NO:5, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO:5 amino acid sequence of amino acid substitutions; and/or

In some embodiments, the antigen a is TIGIT and the antigen b is OX40, and the antibody or antigenic fragment comprises the following:

The VHa contains the heavy chain CDRs or heavy chain variable regions disclosed in US20190100591A1 or US20180169239A1; and/or

The VHb contains the heavy chain CDRs or heavy chain variable regions disclosed in CN101331150A1 or US20150307617A1; and/or

The VLa contains the light chain CDR or light chain variable region disclosed in US20190100591A1 or US20180169239A1; and/or the VLb contains the light chain CDR or light chain variable region disclosed in CN101331150A1 or US20150307617A1.

The VHa contains amino acids at positions 30-35 in the sequence shown in SEQ ID NO: 3 (VHaCDR1, SSYGMS), and/or amino acids at positions 50-66 (VHaCDR2, TINSNGGSTYYPDSVKG), and/or amino acids at positions 99-108 (VHaCDR3, LGTGTLGFAY); and/or

The VHb contains amino acids at positions 31-35 in the sequence shown in SEQ ID NO: 2 (VHbCDR1, SYGMH), and/or amino acids at positions 50-66 (VHbCDR2, VIAEVGSNQYYADSVKG), and/or amino acids at positions 99-111 (VHbCDR3, DNQDTSPDVGIDY); and/or

The VLa contains amino acids at positions 24-34 in the sequence shown in SEQ ID NO: 6 (VLaCDR1, KASQDVKTAVS), and/or amino acids at positions 50-56 (VLaCDR2, WASTRAT), and/or amino acids at positions 89-97 (VLaCDR3, QQHYSTPWT); and/or

The VLb contains amino acids at positions 24-34 in the sequence shown in SEQ ID NO: 5 (VLbCDR1, RASQNISPFLN), and/or amino acids at positions 50-56 (VLbCDR2, AAVGLQS), and/or amino acids at positions 89-97 (VLbCDR3, QQYTDYPLT).

The VHa contains the sequence shown in SEQ ID NO:3, has at least 80% identity with the sequence shown in SEQ ID NO:3, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO:3 amino acid sequence of amino acid substitutions; and/or

The VHb contains the sequence shown in SEQ ID NO: 2, has at least 80% identity with the sequence shown in SEQ ID NO: 2, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 2 amino acid sequence of amino acid substitutions; and/or

The VLa contains the sequence shown in SEQ ID NO: 6, has at least 80% identity with the sequence shown in SEQ ID NO: 6, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 6 amino acid sequence of amino acid substitutions; and/or

The VLb contains the sequence shown in SEQ ID NO:5, a sequence with at least 80% identity to the sequence shown in SEQ ID NO:5, or one or more conserved sequences compared with the sequence shown in SEQ ID NO:5 amino acid sequence of amino acid substitutions; and/or

In some embodiments, the antigen a is TIGIT and the antigen b is PD-1, and the antibody or antigen fragment comprises the following:

The VHb contains the heavy chain CDRs or heavy chain variable regions disclosed in CN1753912A or CN109485727A; and/or

The VLa contains the light chain CDRs or light chain variable regions disclosed in US20190100591A1 or US20180169239A1.

The VLb contains the light chain CDR or light chain variable region disclosed in CN1753912A or CN109485727A.

The entire contents of CN1753912A, CN109485727A are incorporated herein by reference.

The VHa contains amino acids at positions 30-35 in the sequence shown in SEQ ID NO: 10 (VHaCDR1, SSYGMS), and/or amino acids at positions 50-66 (VHaCDR2, TINSNGGSTYYPDSVKG), and/or amino acids at positions 99-108 (VHaCDR3, LGTGTLGFAY); and/or

The VHb contains amino acids at positions 31-36 in the sequence shown in SEQ ID NO: 11 (VHbCDR1, NYYMYW), and/or amino acids at positions 50-64 (VHbCDR2, GINPSNGGTNFNEKF), and/or amino acids at positions 98-109 (VHbCDR3, ARDYRLDMGFEF); and/or

The VLa contains amino acids at positions 24-34 in the sequence shown in SEQ ID NO: 12 (VLaCDR1, KASQDVKTAVS), and/or amino acids at positions 50-56 (VLaCDR2, WASTRAT), and/or amino acids at positions 89-97 (VLaCDR3, QQHYSTPWT); and/or

The VLb contains amino acids 25-38 in the sequence shown in SEQ ID NO: 13 (VLbCDR1, ASKGVSTSGYSYLH), and/or amino acids 54-59 (VLbCDR2, LASYLE), and/or amino acids 91-101 (VLbCDR3, YCQHAYDLPLT).

The VHa contains the sequence shown in SEQ ID NO: 10, has at least 80% identity with the sequence shown in SEQ ID NO: 10, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 10 amino acid sequence of amino acid substitutions; and/or

The VHb contains the sequence shown in SEQ ID NO: 11, has at least 80% identity with the sequence shown in SEQ ID NO: 11, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 11 amino acid sequence of amino acid substitutions; and/or

The VLa contains the sequence shown in SEQ ID NO: 12, has at least 80% identity with the sequence shown in SEQ ID NO: 12, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 12 amino acid sequence of amino acid substitutions; and/or

The VLb contains the sequence shown in SEQ ID NO: 13, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 13, or one or more conserved sequences compared with the sequence shown in SEQ ID NO: 13 amino acid sequence of amino acid substitutions; and/or

The CH1b contains the sequence shown in SEQ ID NO: 14, has at least 80% identity with the sequence shown in SEQ ID NO: 14, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 14 Amino acid sequence of amino acid substitutions.

In some embodiments, the L1 comprises a sequence selected from any one of SEQ ID NOs: 15-18 that is at least 90% identical to a sequence set forth in any one of SEQ ID NOs: 15-18 sequence, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 15-18; and/or

The L2 contains the sequence shown in SEQ ID NO: 19; and/or

The L3 contains the sequence shown in SEQ ID NO: 20.

In some embodiments, the L1 contains the sequence set forth in SEQ ID NO: 15, the L2 contains the sequence set forth in SEQ ID NO: 19, and the L3 contains the sequence set forth in SEQ ID NO: 20.

In some embodiments, the L1 contains the sequence set forth in SEQ ID NO: 16, the L2 contains the sequence set forth in SEQ ID NO: 19, and the L3 contains the sequence set forth in SEQ ID NO: 20.

In some embodiments, the L1 contains the sequence set forth in SEQ ID NO: 17, the L2 contains the sequence set forth in SEQ ID NO: 19, and the L3 contains the sequence set forth in SEQ ID NO: 20.

In some embodiments, the L1 contains the sequence set forth in SEQ ID NO: 18, the L2 contains the sequence set forth in SEQ ID NO: 19, and the L3 contains the sequence set forth in SEQ ID NO: 20.

In some embodiments, the Fc comprises a sequence set forth in SEQ ID NO: 21 or 22, a sequence at least 80% identical to a sequence set forth in SEQ ID NO: 21 or 22, or a sequence set forth in SEQ ID NO: 21 or 22 The sequences shown in 22 are compared to amino acid sequences with one or more conservative amino acid substitutions. In some embodiments, sequences that are at least 80% identical are about 80% identical, about 81% identical, about 82% identical, about 83% identical, about 85% identical, about 86% identical, 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 % identity, or the range (including endpoints) between any two of these numerical values or any value therein. In some embodiments, the one or more conservative 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 conservative amino acid substitutions, or between any two of these values the range (including the endpoints) or any value in it.

In some embodiments, the Fc comprises the sequence set forth in SEQ ID NO:21. In some embodiments, the Fc comprises the sequence set forth in SEQ ID NO:22.

The present invention also provides an antibody or antigen-binding fragment, the antibody or antigen-binding fragment binds two different antigens OX40 and TIGIT; the antibody or antigen-binding fragment at least comprises a first polypeptide chain, a second polypeptide chain and The third polypeptide chain 3 polypeptide chains;

The first polypeptide chain contains amino acids 31-35 in the sequence shown in SEQ ID NO: 1 (VHaCDR1, SYGMH), and/or amino acids 50-66 (VHaCDR2, VISYDGSNQYYADSVKG), and/or 99 - amino acid 111 (VHaCDR3, DNQDSSPDVGIDY); and

Amino acids 30-35 in the sequence shown in SEQ ID NO: 3 (VHbCDR1, SSYGMS), and/or amino acids 50-66 (VHbCDR2, TINSNGGSTYYPDSVKG), and/or amino acids 99-108 (VHbCDR3, LGTGTLGFAY );and / or

The second polypeptide chain contains amino acids 24-34 in the sequence shown in SEQ ID NO: 4 (VLaCDR1, RASQNISPFLN), and/or amino acids 50-56 (VLaCDR2, AASSLQS), and/or 89 - amino acid at position 97 (VLaCDR3, QQYNSYPLT); and/or

The third polypeptide chain contains amino acids 24-34 in the sequence shown in SEQ ID NO: 6 (VLbCDR1, KASQDVKTAVS), and/or amino acids 50-56 (VLbCDR2, WASTRAT), and/or 89 - Amino acid at position 97 (VLbCDR3, QQHYSTPWT).

In some embodiments, the first polypeptide chain comprises the sequence set forth in SEQ ID NO: 1; and the sequence set forth in SEQ ID NO: 3; and/or

The second polypeptide chain contains the sequence shown in SEQ ID NO: 4; and/or

The third polypeptide chain contains the sequence shown in SEQ ID NO:6.

The present invention also provides an antibody or antigen-binding fragment, the antibody or antigen-binding fragment binds 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 three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO: 23, which is the same as The sequence shown in SEQ ID NO: 23 has at least 80% identity, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 23; the second polypeptide chain contains SEQ ID NO: 23. The sequence shown in ID NO: 24, a sequence having at least 80% identity to the sequence shown in SEQ ID NO: 24, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 24 The third polypeptide chain contains the sequence shown in SEQ ID NO: 25, and has at least 80% identity with the sequence shown in SEQ ID NO: 25, or compared with the sequence shown in SEQ ID NO: 25. an amino acid sequence with one or more conservative amino acid substitutions; or

The first polypeptide chain contains the sequence shown in SEQ ID NO: 26, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 26, or a sequence compared with the sequence shown in SEQ ID NO: 26. or amino acid sequence of multiple conservative amino acid substitutions; the second polypeptide chain contains the sequence shown in SEQ ID NO: 24, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 24, or a sequence with SEQ ID NO: 24 Compared with the sequence shown in NO: 24, the amino acid sequence has one or more conservative amino acid substitutions; the third polypeptide chain contains the sequence shown in SEQ ID NO: 25, and has at least 80% of the sequence shown in SEQ ID NO: 25. A sequence of % identity, or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 25.

In some embodiments, the antibody or antigen-binding fragment comprises at least three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain comprises SEQ ID NO: 23 The sequence shown, the second polypeptide chain contains the sequence shown in SEQ ID NO: 24, and the third polypeptide chain contains the sequence shown in SEQ ID NO: 25.

In some embodiments, the antibody or antigen-binding fragment comprises at least three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain comprises SEQ ID NO: 26 The sequence shown, the second polypeptide chain contains the sequence shown in SEQ ID NO: 24, and the third polypeptide chain contains the sequence shown in SEQ ID NO: 25.

The first polypeptide chain contains amino acids 31-35 in the sequence shown in SEQ ID NO: 2 (VHaCDR1, SYGMH), and/or amino acids 50-66 (VHaCDR2, VIAEVGSNQYYADSVKG), and/or 99 - amino acid 111 (VHaCDR3, DNQDTSPDVGIDY); and

The second polypeptide chain contains amino acids 24-34 in the sequence shown in SEQ ID NO: 5 (VLaCDR1, RASQNISPFLN), and/or amino acids 50-56 (VLaCDR2, AAVGLQS), and/or 89 - amino acid at position 97 (VLaCDR3, QQYTDYPLT); and/or

In some embodiments, the first polypeptide chain comprises the sequence set forth in SEQ ID NO:2; and the sequence set forth in SEQ ID NO:3; and/or

The second polypeptide chain contains the sequence shown in SEQ ID NO: 5; and/or

The third polypeptide chain contains the sequence shown in SEQ ID NO:6.

The present invention also provides an antibody or antigen-binding fragment, the antibody or antigen-binding fragment binds 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 three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO: 27, which is the same as The sequence shown in SEQ ID NO: 27 has at least 80% identity, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 27; the second polypeptide chain contains SEQ ID NO: 27. The sequence shown in ID NO: 28, a sequence having at least 80% identity to the sequence shown in SEQ ID NO: 28, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 28 The third polypeptide chain contains the sequence shown in SEQ ID NO: 25, and has at least 80% identity with the sequence shown in SEQ ID NO: 25, or compared with the sequence shown in SEQ ID NO: 25. an amino acid sequence with one or more conservative amino acid substitutions; or

The first polypeptide chain contains the sequence shown in SEQ ID NO: 29, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 29, or a sequence compared with the sequence shown in SEQ ID NO: 29. or amino acid sequence of multiple conservative amino acid substitutions; the second polypeptide chain contains the sequence shown in SEQ ID NO: 28, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 28, or a sequence with SEQ ID NO: 28 Compared with the sequence shown in NO: 28, the amino acid sequence has one or more conservative amino acid substitutions; the third polypeptide chain contains the sequence shown in SEQ ID NO: 25, and has at least 80% of the sequence shown in SEQ ID NO: 25. A sequence of % identity, or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 25.

In some embodiments, the antibody or antigen-binding fragment comprises at least three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain comprises SEQ ID NO: 27 The sequence shown, the second polypeptide chain contains the sequence shown in SEQ ID NO: 28, and the third polypeptide chain contains the sequence shown in SEQ ID NO: 25.

In some embodiments, the antibody or antigen-binding fragment comprises at least three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain comprises SEQ ID NO: 29 The sequence shown, the second polypeptide chain contains the sequence shown in SEQ ID NO: 28, and the third polypeptide chain contains the sequence shown in SEQ ID NO: 25.

The present invention also provides an antibody or antigen-binding fragment, the antibody or antigen-binding fragment binds two different antigens TIGIT and OX40; the antibody or antigen-binding fragment at least comprises a first polypeptide chain, a second polypeptide chain and The third polypeptide chain 3 polypeptide chains;

The first polypeptide chain contains amino acids 30-35 in the sequence shown in SEQ ID NO: 3 (VHaCDR1, SSYGMS), and/or amino acids 50-66 (VHaCDR2, TINSNGGSTYYPDSVKG), and/or 99 - amino acid 108 (VHaCDR3, LGTGTLGFAY); and

Amino acids at positions 31-35 in the sequence shown in SEQ ID NO: 2 (VHbCDR1, SYGMH), and/or amino acids at positions 50-66 (VHbCDR2, VIAEVGSNQYYADSVKG), and/or amino acids at positions 99-111 (VHbCDR3, DNQDTSPDVGIDY) );and / or

The second polypeptide chain contains amino acids 24-34 in the sequence shown in SEQ ID NO: 6 (VLaCDR1, KASQDVKTAVS), and/or amino acids 50-56 (VLaCDR2, WASTRAT), and/or 89 - amino acid at position 97 (VLaCDR3, QQHYSTPWT); and/or

The third polypeptide chain contains amino acids 24-34 in the sequence shown in the sequence shown in SEQ ID NO: 5 (VLbCDR1, RASQNISPFLN), and/or amino acids 50-56 (VLbCDR2, AAVGLQS), and /or amino acids 89-97 (VLbCDR3, QQYTDYPLT).

In some embodiments, the first polypeptide chain comprises the sequence set forth in SEQ ID NO:3; and the sequence set forth in SEQ ID NO:2; and/or

The second polypeptide chain contains the sequence shown in SEQ ID NO: 6; and/or

The third polypeptide chain contains the sequence shown in SEQ ID NO:5.

The present invention also provides an antibody or antigen-binding fragment, the antibody or antigen-binding fragment binds 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 OX40; The antibody or antigen-binding fragment comprises at least three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO: 30, which is the same as The sequence shown in SEQ ID NO: 30 has at least 80% identity, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 30; the second polypeptide chain contains SEQ ID NO: 30. The sequence shown in ID NO: 31, the sequence having at least 80% identity to the sequence shown in SEQ ID NO: 31, or the amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 31 The third polypeptide chain contains the sequence shown in SEQ ID NO: 32, and has at least 80% identity with the sequence shown in SEQ ID NO: 32, or compared with the sequence shown in SEQ ID NO: 32. an amino acid sequence with one or more conservative amino acid substitutions; or

The first polypeptide chain contains the sequence shown in SEQ ID NO:33, a sequence with at least 80% identity to the sequence shown in SEQ ID NO:33, or a sequence compared with the sequence shown in SEQ ID NO:33. or amino acid sequence of multiple conservative amino acid substitutions; the second polypeptide chain contains the sequence shown in SEQ ID NO: 31, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 31, or a sequence with SEQ ID NO: 31 Compared with the sequence shown in NO: 31, the amino acid sequence has one or more conservative amino acid substitutions; the third polypeptide chain contains the sequence shown in SEQ ID NO: 32, and has at least 80% of the sequence shown in SEQ ID NO: 32. A sequence of % identity, or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 32.

In some embodiments, the antibody or antigen-binding fragment comprises at least three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain comprises SEQ ID NO: 30 The sequence shown, the second polypeptide chain contains the sequence shown in SEQ ID NO:31, and the third polypeptide chain contains the sequence shown in SEQ ID NO:32.

In some embodiments, the antibody or antigen-binding fragment comprises at least three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain comprises SEQ ID NO: 33 The sequence shown, the second polypeptide chain contains the sequence shown in SEQ ID NO: 31, and the third polypeptide chain contains the sequence shown in SEQ ID NO: 32.

The present invention also provides an antibody or antigen-binding fragment, the antibody or antigen-binding fragment binds two different antigens TIGIT and PD-1; the antibody or antigen-binding fragment at least comprises a first polypeptide chain, a second polypeptide chain and the third polypeptide chain 3 polypeptide chains;

The first polypeptide chain contains amino acids at positions 30-35 in the sequence shown in SEQ ID NO: 10 (VHaCDR1, SSYGMS), and/or amino acids at positions 50-66 (VHaCDR2, TINSNGGSTYYPDSVKG), and/or at positions 99 - amino acid 108 (VHaCDR3, LGTGTLGFAY); and

In the sequence shown in SEQ ID NO: 11, amino acids 31-36 (VHbCDR1, NYYMYW), and/or amino acids 50-64 (VHbCDR2, GINPSNGGTNFNEKF), and/or amino acids 98-109 (VHbCDR3, ARDYRLDMGFEF) );and / or

The second polypeptide chain contains amino acids 24-34 in the sequence shown in SEQ ID NO: 12 (VLaCDR1, KASQDVKTAVS), and/or amino acids 50-56 (VLaCDR2, WASTRAT), and/or 89 - amino acid at position 97 (VLaCDR3, QQHYSTPWT); and/or

The third polypeptide chain contains amino acids 25-38 in the sequence shown in the sequence shown in SEQ ID NO: 13 (VLbCDR1, ASKGVSTSGYSYLH), and/or amino acids 54-59 (VLbCDR2, LASYLE), and /or amino acids 91-101 (VLbCDR3, YCQHAYDLPLT).

In some embodiments, the first polypeptide chain comprises the sequence set forth in SEQ ID NO: 10; and the sequence set forth in SEQ ID NO: 11; and/or

The second polypeptide chain contains the sequence shown in SEQ ID NO: 12; and/or

The third polypeptide chain contains the sequence shown in SEQ ID NO: 13.

The present invention also provides an antibody or antigen-binding fragment that binds 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 PD- 1; the antibody or antigen-binding fragment comprises at least three polypeptide chains of a first polypeptide chain, a second polypeptide chain and a third polypeptide chain;

The first polypeptide chain contains the sequence shown in SEQ ID NO:34, a sequence with at least 80% identity to the sequence shown in SEQ ID NO:34, or a sequence compared with the sequence shown in SEQ ID NO:34. or amino acid sequence of multiple conservative amino acid substitutions; the second polypeptide chain contains the sequence shown in SEQ ID NO: 35, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 35, or a sequence with SEQ ID NO: 35 Compared with the sequence shown in NO: 35, the amino acid sequence has one or more conservative amino acid substitutions; the third polypeptide chain contains the sequence shown in SEQ ID NO: 36, and has at least 80% of the sequence shown in SEQ ID NO: 36. A sequence of % identity, or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:36.

In some embodiments, the antibody or antigen-binding fragment comprises at least three polypeptide chains: a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain;

The first polypeptide chain contains the sequence shown in SEQ ID NO:34, the second polypeptide chain contains the sequence shown in SEQ ID NO:35, and the third polypeptide chain contains the sequence shown in SEQ ID NO:36. the sequence shown.

In some embodiments, the antibody comprises a first polypeptide chain and a fourth polypeptide chain with the same sequence, a second polypeptide chain and a fifth polypeptide chain with the same sequence, and a third polypeptide chain with the same sequence and The sixth polypeptide chain. In some embodiments, the antibody comprises a first polypeptide chain and a fourth polypeptide chain with the same sequence, a second polypeptide chain and a fifth polypeptide chain with the same sequence, and a third polypeptide chain with the same sequence and The sixth polypeptide chain, the third polypeptide chain and the Fc region of the sixth polypeptide chain are paired to form a disulfide bond.

In some embodiments, the antibody or antigen-binding fragment is an isolated antibody or antigen-binding fragment.

The present invention also provides a nucleic acid molecule encoding the antibody or antigen-binding fragment. In some embodiments, the nucleic acid molecule is an isolated nucleic acid molecule.

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

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

The present invention also provides a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.

The present invention also provides methods and uses of treatment. In some embodiments, methods are provided for the treatment or amelioration of various diseases (eg, inflammatory diseases, autoimmune diseases, neurodegenerative diseases, cancer, or spinal cord injury) comprising administering to a patient an effective dose of the antibody or antigen-binding fragment. In some embodiments, use of the antibody or antigen-binding fragment in a medicament for the treatment or amelioration of various diseases, such as inflammatory diseases, autoimmune diseases, cancer, or spinal cord injury, is provided. In some embodiments, use of the antibody or antigen-binding fragment in the manufacture of a medicament for the treatment or amelioration of various diseases, such as inflammatory diseases, autoimmune diseases, cancer or spinal cord injury, is provided.

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 joints) inflammation, psoriatic arthritis, osteoarthritis or juvenile idiopathic arthritis), multiple sclerosis, ankylosing spondylitis, spondylosing arthropathy, systemic lupus erythematosus, uveitis, sepsis, neurodegeneration Sexual diseases, neuronal regeneration, spinal cord injury, primary and metastatic cancer, respiratory disorders, asthma, allergic and non-allergic asthma, asthma caused by infection, asthma caused by infection with respiratory syncytial virus (RSV), Chronic obstructive pulmonary disease (COPD), conditions involving airway inflammation, eosinophilia, fibrosis and excess mucus production, cystic fibrosis, pulmonary fibrosis, atopic disorders, atopic dermatitis, urticaria , eczema, allergic rhinitis, allergic gastroenteritis, inflammatory and/or autoimmune skin conditions, inflammatory and/or autoimmune gastrointestinal conditions, inflammatory bowel disease (IBD), ulcerative colitis, inflammation Sexual and/or autoimmune liver disease, cirrhosis, liver fibrosis, liver fibrosis caused by hepatitis B and/or C virus, scleroderma. In some embodiments, the cancer is selected from the group consisting of hepatocellular carcinoma, glioblastoma, lymphoma, Hodgkin's lymphoma. In some embodiments, the cancer is selected from the group consisting of melanoma (eg, metastatic malignant melanoma), kidney cancer (eg, clear cell carcinoma), prostate cancer (eg, hormone-refractory prostate) adenocarcinoma), pancreatic cancer, breast cancer, colon cancer, lung cancer (eg, non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glial stromal tumor, leukemia, lymphoma and other neoplastic malignant diseases. In some embodiments, the cancer is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma [NHL], precursor B-cell lymphoblastic leukemia/lymphoma tumor, mature B-cell neoplasia, 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, plasma cell myeloma, post-transplant lymphoproliferative disorder, transplant Waldenstrom's macroglobulinemia and anaplastic large cell lymphoma.

The present invention also provides diagnostic methods and uses. In some embodiments, methods are provided for detecting the expression of antigen a and/or antigen b in a sample, contacting the sample with the antibody or antigen-binding fragment such that the antibody or antigen-binding fragment binds antigen a and/or antigen b, and detect its binding, ie the amount of antigen a and/or antigen b in the sample. In some embodiments, the antigens a, b are cytokines, cytokine receptors, chemokines, chemokine receptors or cell surface proteins. In some embodiments, antigens a, b are selected from the group consisting of TIGIT and CTLA-4, OX40 and CTLA-4, TIGIT and PD-1, PD-L1 and CD47, TIGIT and OX40, VEGF and cMET, respectively , 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 TIM-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. In some embodiments, use of the antibody or antigen-binding fragment in the manufacture of a kit for diagnosing or prognosing inflammatory diseases, autoimmune diseases, neurodegenerative diseases, cancer, or spinal cord injury is provided. In some embodiments, a diagnostic or prognostic kit comprising the antibody or antigen-binding fragment is provided.

The present invention provides multivalent and multispecific antibodies or antigen-binding fragments that can bind two or more antigens, or two or more surfaces of the same antigen, and uses thereof. bit. The antibodies or antigen-binding fragments of the present invention can be used to treat or ameliorate various diseases, such as inflammatory diseases, autoimmune diseases, cancer or spinal cord injury, and can also be used for the diagnosis and prognosis of related diseases.

Description of drawings

Figure 1 is a schematic representation of the structure of bispecific antibodies of the invention in some embodiments.

Figure 2 is the SDS-PAGE map of antibodies 1-6 in Example 2 of the present invention; wherein, lane 1 indicates that antibodies 1-6 are in a non-reducing state, lane M indicates maker, and lane 2 indicates that antibodies 1-6 are in a reducing state; pOT -3c-15aa means Antibody 1, pOT-3c-30aa means Antibody 2, OT-3c-15aa means Antibody 3, OT-3c-30aa means Antibody 4, TO-3c-5aa means Antibody 5, TO-3c-30aa means Antibody 5 Antibody 6.

Figure 3 is the binding curve of antibodies 1-4 and antibody 6 to Jurkat-OX40 cells in Example 5 of the present invention; wherein, anti-OX40Ab is represented as OX40 monoclonal antibody (ie, anti-OX40).

FIG. 4 is the binding curve of antibody 5 in Example 5 of the present invention and Jurkat-OX40 cells.

Figure 5 is the binding curve of antibodies 1-4 and antibody 6 to Jurkat-Tigit cells in Example 5 of the present invention; wherein, anti-Tigit Ab is represented as Tigit monoclonal antibody (ie anti-Tigit).

Figure 6 is the binding curve of antibody 5 in Example 5 of the present invention and Jurkat-Tigit cells.

Figure 7 shows the in vitro activating activity of antibodies detected by the NFkB reporter gene system.

Figure 8 shows that the antibody in Example 7 of the present invention blocks the binding of PVR to Jurkat-Tigit cells.

Figure 9 shows the ability of the antibody in Example 8 of the present invention to relieve Tigit inhibitory activity.

Figure 10 shows that the antibody in Example 9 of the present invention stimulates human PBMC cells to secrete IL-2.

11 is the SDS-PAGE chart of antibody 7 in Example 11 of the present invention; wherein, TIGIT/PD-1 BiAb is represented as antibody 7.

Figure 12 shows the binding activity of antibody 7 to Tigit antigen in Example 12 of the present invention.

Figure 13 shows the binding activity of antibody 7 to PD-1 antigen in Example 12 of the present invention; wherein, anti-PD-1 is represented as PD-1 monoclonal antibody.

Fig. 14 is the affinity kinetic fitting curve of the binding of antibodies 1-6 to OX40 antigen and TIGIT antigen in the embodiment of the present invention; Fig. 14A is antibody 5, Fig. 14B is antibody 6, Fig. 14C is antibody 1, Fig. 14D is antibody 2 , Figure 14E is antibody 3, and Figure 14F is antibody 4.

the term

Unless otherwise specified, each of the following terms shall have the meaning set forth below.

definition

It should be noted that the term "an" entity refers to one or more of such entities, eg "an antibody" should be understood to mean one or more antibodies, thus the term "an" (or "an" ), "one or more" and "at least one" are used interchangeably herein.

The terms "comprising," "containing," or "including" as used herein mean that compositions, methods, and the like include recited elements, such as components or steps, but do not exclude others. "Consisting essentially of" means that the compositions and methods exclude other elements that have an essential effect on the characteristics of the combination, but do not exclude elements that have no essential effect on the compositions or methods. "Consisting of" means excluding elements not specifically recited.

"About" refers to the conventional error range of the corresponding numerical value readily known to those skilled in the relevant art. In some embodiments, references herein to "about" refer to the recited value and ranges of ±10%, ±5%, or ±1% thereof.

"EC _50" i.e., half maximal effective concentration (concentration for 50% of maximal effect , EC 50) means to cause a 50% maximal effect concentration.

The term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptide", and refers to a molecule composed of amino acid monomers linked linearly by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single chain or chains of two or more amino acids, and does not refer to a particular length of the product. Thus, the definition of "polypeptide" includes a 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" may Used in place of, or used interchangeably with, any of the above terms. The term "polypeptide" is also intended to refer to the product of post-expression modifications of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage or non-native Amino acid modifications that occur. A polypeptide may be derived from a natural biological source or produced by recombinant techniques, but it need not be translated from a given nucleic acid sequence, and it may be produced by any means including chemical synthesis.

"Amino acid" refers to an organic compound containing both an amino group and a carboxyl group, such as an alpha-amino acid, which can be encoded by a nucleic acid directly or in a 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 same amino acid is encoded by different codons called "degeneracy of the genetic code". Amino acids include natural amino acids and unnatural 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 Amino acid (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).

"Conservative amino acid substitution" refers to the replacement of one amino acid residue by another amino acid residue containing a side chain (R group) of similar chemical properties (eg, charge or hydrophobicity). In general, conservative amino acid substitutions are unlikely to substantially alter the functional properties of the 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.

The number of amino acids for "conservative amino acid substitutions of a linker" is about 1, about 2, about 3, about 4, or about 5 conservative amino acid substitutions. The number of amino acids for "conservative amino acid substitutions of VL, CL, VH, CH or Fc" is 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 conservative amino acid substitutions, or a range (including endpoints) between any two of these values, or any value therein. The number of amino acids for "conservative amino acid substitutions of the first polypeptide chain, the second polypeptide chain or the third polypeptide chain" is 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 conservative amino acid substitutions, or any two of these values A range between values (including endpoints) or any value therein.

The term "isolated" as used herein with reference to cells, nucleic acids, polypeptides, antibodies, etc., eg, "isolated" DNA, RNA, polypeptides, antibodies, refers to other components in the cell's natural environment, such as DNA or RNA, respectively of one or more of the isolated molecules. The term "isolated" as used herein also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material or cell culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" is intended to include nucleic acid fragments that do not, and would not exist in, their natural state. The term "isolated" is also used herein to refer to cells or polypeptides that are separated from other cellular proteins or tissues. Isolated polypeptides are intended to include purified and recombinant polypeptides. Isolated polypeptides, antibodies, etc. are typically prepared by at least one purification step. In some embodiments, the isolated nucleic acid, polypeptide, antibody, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99% pure, or any of these values The range between any two values of , including the endpoint, or any value therein.

The term "recombinant" refers to a polypeptide or polynucleotide and means a form of the polypeptide or polynucleotide that does not occur in nature, non-limiting examples may be combined to produce polynucleotides that do not normally exist or peptide.

"Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions within each sequence that can be aligned. A molecule is homologous when a position in the sequences being compared is occupied by the same base or amino acid. The degree of homology between sequences is a function of the number of matches or homologous positions shared by the sequences.

"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 these A range (including endpoints) between any two values in a numerical value or any value therein.

"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 (including endpoints) between any two of these values, or any value therein.

A polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) has a certain percentage (eg, 90%, 95%, 98% or 99%) "identity or sequence identity" to another sequence Refers to the percentage of bases (or amino acids) that are identical in the two sequences being compared when the sequences are aligned. The alignment and percent identity or sequence identity can be determined using visual inspection or software programs known in the art, such as those described by Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. Alignments are preferably performed using default parameters. One such alignment program is BLAST using default parameters, such as BLASTN and BLASTP, both of which use the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions =50sequences; 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 having the same or similar biological activity.

A polynucleotide is a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), or when polynucleotides For RNA, thymine is replaced by uracil (U). A "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, eg for functional genomics and homology searches.

The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (eg probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribose Somatic RNA, ribozyme, 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 modifications are present, structural modifications to the nucleotides can be made before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. The polynucleotide can be further modified after polymerization, for example by conjugation to a labeling component. The term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, embodiments of any polynucleotide of the present disclosure include the double-stranded form and each of the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

The term "encoding" when applied to a polynucleotide refers to a polynucleotide referred to as "encoding" a polypeptide, transcribed and/or in its native state or when manipulated by methods well known to those skilled in the art Or translation can yield the polypeptide and/or fragments thereof.

"Antibody", "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. Antibodies can be whole antibodies and any antigen-binding fragments thereof or single chains thereof. The term "antibody" thus includes any protein or peptide in the molecule that contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Antibodies and antigen-binding fragments include, but are not limited to, the complementarity determining regions (CDRs), heavy chain variable regions (VH), light chain variable regions (VL), heavy chain constant regions of heavy or light chains or ligand binding portions thereof (CH), a light chain constant region (CL), a framework region (FR), or any portion thereof, or at least a portion of a binding protein. The CDR regions include 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 the embodiment of the present invention is a bispecific antibody, which is a fusion of antibody fragments that specifically bind to antigen a and antigen b: the first polypeptide chain contains the structure VHa-CH1a-L1-VHb-L2- CLb, the second polypeptide chain comprises the structure VLa-CLa, the third polypeptide chain comprises the structure VLb-L3-CH1b; or the first polypeptide chain comprises the structure VHa-CH1a-L1-VHb-L2-CLb, the second polypeptide The chain comprises the structure VLa-CLa, and the third polypeptide chain comprises the structure VLb-L3-CH1b-Fc; the first polypeptide chain, the second polypeptide chain and the third polypeptide chain constitute a structure similar to immunoglobulin.

The term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, and the composition of the antibody fragment of the invention may be similar to that of F(ab') ₂ , F(ab) ₂ , Fab', Fab in monospecific antibody fragments , Fv, scFv, etc. Regardless of their structure, antibody fragments bind to the same antigen that is recognized by the intact antibody. The term "antibody fragment" includes aptamers, Spiegelmers and diabodies. 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.

"Single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. In some aspects, these regions are linked to short linker peptides of 10 to about 25 amino acids. Linkers can be rich in glycine to increase flexibility, and serine or threonine to increase solubility, and can link the N-terminus of VH and the C-terminus of VL, and vice versa. Although the constant region has been removed and the linker introduced, the protein retains the specificity of the original immunoglobulin. ScFv molecules are generally known in the art and are described, for example, in US Pat. No. 5,892,019.

The term "antibody" includes a wide variety of biochemically distinguishable polypeptides. Those of skill in the art will appreciate that classes of heavy chains include gamma, mu, alpha, delta, or epsilon (gamma, mu, alpha, delta, epsilon), with some subclasses (eg, gamma1-gamma4). The nature of this chain determines the "class" of the antibody as IgG, IgM, IgA, IgG or IgE, respectively. Immunoglobulin subclasses (isotypes), eg, IgGl, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and the functional specificities conferred are known. All immunoglobulin species are within the scope of the present disclosure. In some embodiments, the immunoglobulin molecule is of the IgG class. The four chains are connected by disulfide bonds in a "Y" configuration, where the light chain begins at the "Y" mouth and continues through the variable region surrounding the heavy chain.

Antibodies, antigen-binding fragments or derivatives disclosed herein include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single chain antibodies, epitope binding Fragments (eg Fab, Fab' and F(ab') ₂ ), scFv.

Light chains can be classified as kappa (κ) or lambda (λ). Each heavy chain can bind to a kappa or lambda light chain. In general, when immunoglobulins are produced by hybridomas, B cells or genetically engineered host cells, their light and heavy chains are joined by covalent bonds, and the "tail" portion of the two heavy chains is joined by a covalent disulfide bond or non-covalent bond. In heavy chains, the amino acid sequence extends from the N-terminus of the forked terminus in the Y configuration to the C-terminus at the bottom of each chain. The variable region of immunoglobulin kappa light chain is Vκ; the variable region of immunoglobulin lambda light chain is _Vλ .

The variable regions of the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. The constant regions of the light and heavy chains confer important biological properties such as secretion, transplacental movement, Fc receptor binding, complement fixation, and the like. By convention, the numbering of constant regions increases as they become further from the antigen binding site or amino terminus of the antibody. The N-terminal portion is the variable region and the C-terminal portion is the constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chains, respectively.

In naturally occurring antibodies, the six "complementarity determining regions" or "CDRs" present in each antigen-binding domain are the short, A non-contiguous amino acid sequence that specifically binds an antigen. The remaining other amino acids in the antigen binding domain, referred to as the "framework" region, show less inter-molecular variability. The framework regions mostly adopt a β-sheet conformation, and the CDRs form loop structures to which they are attached, or in some cases form part of a β-sheet structure. Thus, the framework regions position the CDRs in the correct orientation by forming a scaffold through non-covalent interchain interactions. An antigen binding domain with CDRs at specific positions forms a surface complementary to an epitope on an antigen that facilitates non-covalent binding of the antibody to its antigenic epitope. For a given heavy or light chain variable region, one of ordinary skill in the art can identify amino acids comprising CDRs and framework regions by 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)).

Where there are two or more definitions of a term used and/or accepted in the art, the definition of the term used herein includes all such meanings unless explicitly stated to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. This particular 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), It is hereby incorporated by reference in its entirety.

CDRs as defined by Kabat and Chothia include overlaps or subsets of amino acid residues when compared to each other. Nonetheless, it is within the scope of the invention to apply either definition to refer to the CDRs of an antibody or variant thereof. The exact residue numbers encompassing a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can usually determine which specific residues the CDRs contain based on the amino acid sequence of the variable region of the antibody.

Kabat et al. also define a numbering system applicable to variable region sequences of any antibody. One of ordinary skill in the art can apply this "Kabat numbering" system to any variable region sequence independent of experimental data other than the sequence itself. "Kabat Numbering" means the numbering system proposed by Kabat et al., U.S. Dept. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1983). Antibodies may also use the EU or Chothia numbering system.

The antibodies disclosed herein can be derived from any animal, including birds and mammals. Preferably, the antibody is of human, murine, donkey, rabbit, goat, camel, llama, horse or chicken origin. In another embodiment, the variable regions may be of condricthoid origin (eg, from sharks).

The heavy chain constant region includes at least one of a CH1 domain, a hinge (eg, upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment. The heavy chain constant regions of antibodies can be derived from different immunoglobulin molecules. For example, heavy chain constant region polypeptide may comprise a CH1 domain derived from IgG ₁ molecule and a hinge region derived from IgG ₃ molecule. In another embodiment, the heavy chain constant region may comprise a hinge region _{derived in part from an IgG 1} molecule and in part from an IgG _{3 molecule.} In another embodiment, a part of the heavy chain may comprise a chimeric hinge region _{derived in part from an IgG 1} molecule and in part from an IgG _{4 molecule.}

A "light chain constant region" includes a portion of the amino acid sequence from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain. A "light chain-heavy chain pair" refers to a collection of light and heavy chains that can dimerize through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

The "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the "CH1 domain" includes the first (mostly amino-terminal) constant region of an immunoglobulin heavy chain. The CH2 domains are not tightly paired with other domains, but rather two N-linked branched carbohydrate chains are inserted between the two CH2 domains of the intact native IgG molecule. It is also documented that the CH3 domain extends from the CH2 domain to the C-terminus of an IgG molecule, and contains approximately 108 residues. The "hinge region" includes a portion of the heavy chain region connecting the CH1 domain and the CH2 domain. The hinge region comprises about 25 residues and is resilient, allowing the two N-terminal antigen binding regions to move independently. The hinge region can be subdivided into three distinct domains: upper, middle and lower hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

"Disulfide bond" refers to 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 a disulfide bond.

"Chimeric antibody" refers to any antibody whose variable regions are obtained or derived from a first species and whose constant regions (which may be intact, partial or modified) are derived from a second species. In certain embodiments, the variable regions are derived from a non-human source (eg, mouse or primate), and the constant regions are derived from a human source.

"Specifically binds" or "specifically for" generally refers to the formation of a relatively stable complex of an antibody or antigen-binding fragment with a specific antigen through complementary binding of its antigen-binding domain to an epitope. "Specificity" can be expressed in terms of the relative affinity with which an antibody or antigen-binding fragment binds to a particular antigen or epitope. For example, if antibody "A" has a greater relative affinity for the same antigen than antibody "B", antibody "A" can be considered to be more specific for that antigen than antibody "B". Specific binding can be described by the equilibrium dissociation constant (KD), a smaller KD means 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, optical interferometry of biological film layers, and the like. Antibodies that "specifically bind" antigen a include antibodies with an equilibrium dissociation constant KD less than or equal to about 100 nM, less than or equal to about 10 nM, less than or equal to about 5 nM, less than or equal to about 1 nM, or less than or equal to about 0.5 nM with antigen a .

"Treatment" means both therapeutic treatment and prophylactic or prophylactic measures, the purpose of which is to prevent, slow, ameliorate, or stop an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to the following whether detectable or undetectable As a result, remission of symptoms, reduction of disease severity, stabilization of disease state (ie, no worsening), delay or slowdown of disease progression, improvement, alleviation, alleviation or disappearance of disease state (whether in part or in whole), prolongation and Expected duration of survival when not receiving treatment, etc. Patients in need of treatment include patients already suffering from a condition or disorder, a patient susceptible to a condition or disorder, or a patient in need of prevention of such a condition or disorder, which may or may be expected from administration of the antibodies or pharmaceutical compositions disclosed herein for detection , patients who benefit from the diagnostic process and/or treatment.

"Patient" refers to any mammal in need of diagnosis, prognosis, or treatment, including humans, dogs, cats, rabbits, mice, horses, cows, and the like.

Antibodies targeting OX40 and TIGIT

The present invention provides bispecific antibodies or antigen-binding fragments with high affinity for OX40 and TIGIT proteins. The screened antibodies exhibit potent binding activity, biological activity, and can be used for therapeutic and diagnostic purposes. For example, these antibodies or antigen-binding fragments can effectively block inhibitory immune checkpoints and activate lymphocytes to release cytokines for the treatment of various types of cancers, tumors, or infections and other related diseases.

Accordingly, embodiments disclosed herein provide antibodies or antigen-binding fragments targeting OX40 and TIGIT that can specifically bind OX40 and TIGIT.

In some embodiments, the antibody or antigen-binding fragment comprises at least three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain comprises SEQ ID NO: 23 The sequence shown, the second polypeptide chain contains the sequence shown in SEQ ID NO: 24, and the third polypeptide chain contains the sequence other than Fc in SEQ ID NO: 25.

In some embodiments, the antibody or antigen-binding fragment comprises at least three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain comprises SEQ ID NO: 26 The sequence shown, the second polypeptide chain contains the sequence shown in SEQ ID NO: 24, and the third polypeptide chain contains the sequence other than Fc in SEQ ID NO: 25.

In some embodiments, the antibody or antigen-binding fragment comprises at least three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain comprises SEQ ID NO: 27 The sequence shown, the second polypeptide chain contains the sequence shown in SEQ ID NO: 28, and the third polypeptide chain contains the sequence shown in SEQ ID NO: 25 except the Fc sequence.

In some embodiments, the antibody or antigen-binding fragment comprises at least three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain comprises SEQ ID NO: 29 The sequence shown, the second polypeptide chain contains the sequence shown in SEQ ID NO: 28, and the third polypeptide chain contains the sequence shown in SEQ ID NO: 25 except the Fc sequence.

In some embodiments, the antibody or antigen-binding fragment comprises at least three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain comprises SEQ ID NO: 30 The sequence shown, the second polypeptide chain contains the sequence shown in SEQ ID NO: 31, and the third polypeptide chain contains the sequence other than Fc in SEQ ID NO: 32.

In some embodiments, the antibody or antigen-binding fragment comprises at least three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain comprises SEQ ID NO: 33 The sequence shown, the second polypeptide chain contains the sequence shown in SEQ ID NO:31, and the third polypeptide chain contains the sequence shown in SEQ ID NO:32.

In some embodiments, the antibody or antigen-binding fragment comprises at least three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain comprises SEQ ID NO: 33 The sequence shown, the second polypeptide chain contains the sequence shown in SEQ ID NO: 31, and the third polypeptide chain contains the sequence other than Fc in SEQ ID NO: 32.

Antibodies targeting TIGIT and PD-1

The present invention provides bispecific antibodies or antigen-binding fragments with high affinity for TIGIT and PD-1 proteins. The screened antibodies exhibit potent binding activity, biological activity, and can be used for therapeutic and diagnostic purposes. For example, these antibodies or antigen-binding fragments can effectively block inhibitory immune checkpoints and activate lymphocytes to release cytokines for the treatment of various types of cancers, tumors, or infections and other related diseases.

Accordingly, one embodiment of the present disclosure provides an antibody or antigen-binding fragment targeting TIGIT and PD-1, which antibody or antigen-binding fragment can specifically bind to TIGIT and PD-1.

In some embodiments, the antibody or antigen-binding fragment comprises at least three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain comprises SEQ ID NO: 34 The sequence shown, the second polypeptide chain contains the sequence shown in SEQ ID NO: 35, and the third polypeptide chain contains the sequence shown in SEQ ID NO: 36.

In some embodiments, the antibody or antigen-binding fragment comprises at least three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain comprises SEQ ID NO: 34 The sequence shown, the second polypeptide chain contains the sequence shown in SEQ ID NO: 35, and the third polypeptide chain contains the sequence other than Fc in SEQ ID NO: 36.

Those of ordinary skill in the art will also understand that the sequences of the antibodies or antigen-binding fragments disclosed in the present invention may be replaced, and the amino acid sequences thereof differ from the naturally occurring amino acid sequences of the antibody after replacement. For example, the substituted amino acid sequence can be similar to the starting sequence, such as having a certain proportion of identity with the starting sequence, such as it can be about 80%, about 85%, about 90% identical to the starting sequence , about 95%, about 98%, or about 99%, or a range between any two of these values (including the endpoint), or any value therein.

In certain embodiments, the antibody comprises an amino acid-containing sequence with one or more modifying groups. For example, the bispecific antibodies disclosed herein (bispecific antibodies targeting OX40 and TIGIT, bispecific antibodies targeting TIGIT and PD-1) may contain flexible linker sequences, or may be modified to add functionality Sexual groups (eg PEG, drugs, toxins or tags).

The antibodies and antigen-binding fragments disclosed herein include derivatives that are modified, ie, modified by covalent attachment of any type of molecule to the antibody, wherein the covalent attachment does not prevent the antibody from binding to the epitope. Including but not limited to the following examples, antibodies can be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytically cleaved, linked to cellular ligands or other proteins, etc. Any of a number of chemical modifications can be performed by existing techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.

In some embodiments, the antibody can be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, pharmaceutical agent, or PEG.

Antibodies can be conjugated or fused to therapeutic agents, which can include cells with detectable labels (eg, radiolabels), immunomodulators, hormones, enzymes, oligonucleotides, photosensitizing therapeutics, diagnostics, drugs, or toxins Toxic agents, ultrasound enhancers, non-radioactive labels and compositions thereof, and other such agents known in the art.

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

Antibodies and methods for producing antibody-encoding polynucleotides

The present invention also discloses polynucleotides or nucleic acid molecules encoding the antibodies, antigen-binding fragments, and derivatives thereof of the present invention. The polynucleotide disclosed in the present invention can encode the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, the variable region of the heavy chain, the variable region of the light chain, the Fc region, and part of the variable region of the heavy chain or part of the light chain variable region. Methods of making antibodies are well known in the art and described in the present invention. In certain embodiments, the antibodies and antigen-binding fragments of the present disclosure comprise variable and constant regions that are fully human. Fully human antibodies and antigen-binding fragments can be prepared using techniques disclosed in the art and described herein. For example, fully human antibodies directed against a particular antigen can be prepared by administering the antigen to transgenic animals that have been modified to produce fully human antibodies in response to challenge with the antigen. Exemplary techniques that can be used to prepare such antibodies are found in US Pat. Nos. 6,458,592; 6,420,140, the entire contents of which are incorporated herein by reference. The bispecific antibody in the present invention is a fusion of fragments that specifically bind to antigen a and antigen b. For some fragments of the bispecific antibody, please refer to the above-mentioned preparation method of an antibody that binds to a single antigen.

In certain embodiments, the antibodies produced do not elicit an adverse immune response in the animal (eg, human) to be treated. In one embodiment, the antibodies, antigen-binding fragments, or derivatives disclosed herein are modified to reduce their immunogenicity using art-recognized techniques. For example, antibodies can be humanized, primatized, deimmunized or chimeric antibodies can be prepared. These types of antibodies are derived from non-human antibodies, usually murine or primate antibodies, which retain or substantially retain the antigen-binding properties of the parent antibody but are less immunogenic in humans. This can be accomplished by a variety of methods, including (a) grafting entire non-human variable regions into human constant regions to generate chimeric antibodies; (b) grafting one or more non-human complementarity determining regions (CDRs) ) into human-derived framework and constant regions, with or without critical framework residues; or (c) grafted into the entire non-human variable region, but by replacing surface residues with human-like portions base thereby "hiding" them. Typically framework residues in the human framework regions will be replaced by corresponding residues from the CDR donor antibody, eg, residues that improve antigen binding. These framework substitutions can be identified by methods well known in the art, such as by modeling the interactions of CDRs and framework residues to identify framework residues that are important for antigen binding and by sequence alignment to identify framework residues that are aberrant at specific positions. (Refer to U.S. Patent 5,585,089; Riechmann et al., Nature 332:323 (1988); incorporated herein by reference in its entirety). Antibodies can be humanized using a variety of techniques known in the art, such as CDR grafting (EP 239,400; WO 91/09967; US Pat. Nos. 5,225,539, 5,530,101 and 5,585,089), repair or surface rearrangement (EP 592,106; EP 519,596; 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 Rearrangement of Chains (US Patent 5,565,332), the entire contents of which are incorporated herein by reference.

Deimmunization can also be used to reduce the immunogenicity of antibodies. In the present invention, the term "deimmunization" includes altering antibodies to modify T cell epitopes (see eg WO/9852976A1 and WO/0034317A2). For example, the heavy and light chain variable region sequences from the starting antibody are analyzed and a "map" of human T cell epitopes from each variable region is generated, showing the epitopes relative to complementarity determining regions (CDRs) and the positions of other key residues within the sequence. Individual T cell epitopes from T cell epitope maps were 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 comprising combinations of amino acid substitutions are designed, and these sequences are subsequently incorporated into a series of binding polypeptides. The genes containing the modified variable and human constant regions of the intact heavy and light chains are then cloned into expression vectors, and the plasmids are subsequently transformed into cell lines to produce intact antibodies. The optimal antibodies are then identified by comparing the antibodies in appropriate biochemical and biological experiments.

The binding specificity of the bispecific antibodies or antigen-binding fragments disclosed herein can be detected by in vitro assays such as co-immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

Alternatively, scFvs in the bispecific antibodies of the invention can be found in 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) and Ward et al., Nature 334:544-554 (1989) and Nie et al., Antibody Therapeutics 3(1):18-62 (2020)). The heavy and light chain fragments of the Fv region are bridged by amino acids to form a single-chain unit, resulting in a single-chain fusion peptide. Techniques for the assembly of functional Fv fragments in E. coli can also be used (Skerra et al., Science 242:1038-1041 (1988)).

Examples of techniques that can be used to produce single-chain Fv (scFv) and antibodies include, for example, U.S. Patent Nos. 4,946,778 and 5,258,498, and Huston et al., Methods in Enzymology 203:46-88 (1991), Shu et al., Proc. Natl. As described in Sci. USA 90: 1995-1999 (1993) and Skerra et al., Science 240: 1038-1040 (1988). For certain uses including the use of antibodies in humans and in vitro detection experiments, chimeric, humanized, or fully human antibodies may be used. Chimeric antibodies are a class of molecules in which different portions of the antibody are derived from different animal species, eg, antibodies having the variable regions of murine monoclonal antibodies and the constant regions of human immunoglobulins. Methods for producing chimeric antibodies are known in the art, see Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies 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 hereby incorporated by reference Incorporated herein.

In addition, in Newman, Biotechnology 10: 1455-1460 (1992), another highly efficient method of producing recombinant antibodies is disclosed, in particular, this technology can produce primate antibodies containing monkey variable region and human constant region sequences, The entire contents of this reference are incorporated herein by reference. In addition, this technology is mentioned in commonly assigned US Patents 5,658,570, 5,693,780 and 5,756,096, the entire contents of each of which are incorporated herein by reference.

Antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries from immunoglobulin sequences. See also U.S. Patent Nos. 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 WO 91/10741, each The entire contents of the patents are incorporated herein by reference.

In another embodiment, DNA encoding the desired monoclonal antibody can be isolated and subjected to Sequencing. Isolated and subcloned hybridoma cells can serve as a source of such 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 that does not produce other immunoglobulins in cells. Isolated DNA (which may be synthetic as described herein) can also be used to prepare the sequences of the constant and variable regions of antibodies, as described in US Pat. No. 5,658,570, which is incorporated herein by reference in its entirety. This method extracts RNA from selected cells and converts it into cDNA, which is then amplified by PCR techniques using Ig-specific primers. Suitable probes for this purpose are also mentioned in US Pat. No. 5,658,570.

Furthermore, using conventional recombinant DNA techniques, one or more CDRs of the antibodies of the invention can be inserted into framework regions, eg, into human framework regions, to construct humanized, non-fully human antibodies. Framework regions can be naturally occurring or consensus framework regions, preferably human framework regions (see Chothia et al., J. Mol. Biol. 278:457-479 (1998), which lists a list of human framework regions). Some polynucleotides may encode an antibody that specifically binds to at least one epitope of an antigen of interest resulting from a combination of framework regions and CDRs. One or more amino acid substitutions may be made within the framework regions, and amino acid substitutions may be selected to improve binding of the antibody to its antigen. Additionally, substitutions or deletions of cysteine residues in one or more variable regions involved in interchain disulfide bond formation can be performed using this method, resulting in antibody molecules lacking one or more interchain disulfide bonds. Other changes to polynucleotides that are within the skill in the art are also encompassed by the present invention.

Antibodies can be prepared using conventional recombinant DNA techniques. Antibody-producing vectors, cell lines, etc. 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, such as Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells, DLHacker, FMWurm, in Reference Module in Life Sciences, 2017, which in their entirety include Supplementary content is incorporated by reference in its entirety.

In some embodiments, DNA encoding the antibody can be designed and synthesized according to the amino acid sequences of the antibodies described herein according to conventional methods, placed in an expression vector, then transfected into host cells, and the transfected host cells are grown in culture to produce Monoclonal antibodies. In some embodiments, an antibody expression vector includes at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing flanking the inserted sequence. Efficient transcription can be obtained by the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV1, HIVI, and the early promoter of cytomegalovirus, and other cellular promoters such as muscle can be used. kinesin promoter. 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.

In some embodiments, the inserted gene fragment needs to contain a selectable marker. Common selectable markers include dihydrofolate reductase, glutamine synthase, neomycin resistance, hygromycin resistance and other selection genes, so as to facilitate transfection Screening isolation of successful cells. The constructed plasmid is transfected into host cells without the above-mentioned genes, and cultured in selective medium, the successfully transfected cells grow in large quantities to produce the desired target protein.

Furthermore, mutations can be introduced in the nucleotide sequences encoding the antibodies of the invention using standard techniques known to those of skill in the art, including but not limited to site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions. Variants (including derivatives) encode substitutions of less than 50 amino acids, less than Substitution of 40 amino acids, Substitution of less than 30 amino acids, Substitution of less than 25 amino acids, Substitution of less than 20 amino acids, Substitution of less than 15 amino acids, Substitution of less than 10 amino acids, Less than 5 amino acids Substitution of amino acids, substitution of less than 4 amino acids, substitution of less than 3 amino acids, or substitution of less than 2 amino acids. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, for example by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity.

In some embodiments, the substitutions described herein are conservative amino acid substitutions.

treatment method

The present invention also provides methods and uses of treatment. In some embodiments, there is provided a method for treating or ameliorating various types of cancer, tumor or infection related diseases, the method comprising administering to the patient an effective dose of the bispecific antibodies: targeting OX40 and Bispecific antibody for TIGIT, a bispecific antibody targeting TIGIT and PD-1. In some embodiments, the use of the bispecific antibody for the treatment or amelioration of related diseases such as cancer, tumor or infection is provided. In some embodiments, the use of the bispecific antibody in the preparation of a medicament for treating or ameliorating related diseases such as cancer, tumor or infection is provided.

The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the particular antibody or derivative used, the patient's age and weight, general health, 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 are left to the judgment of health care professionals, including those within the purview of those of ordinary skill in the art. The dosage will also depend on the individual patient to be treated, the route of administration, the type of formulation, the nature of the compound used, the severity of the disease, and the effect desired. The dose to be used can be determined by pharmacological and pharmacokinetic principles well known in the art. In some embodiments, the antibody of the invention is administered to a patient at a dose of 0.01 mg/kg to 100 mg/kg of the patient's body weight each time. In some embodiments, the administration is administered every 1 week, 2 weeks, 3 weeks, or monthly.

Methods of administration of the antibody or derivative include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural, and oral injection. The pharmaceutical compositions can be administered by any convenient route, such as by infusion or bolus injection, absorbed through epithelia or mucocutaneous (eg, oral mucosa, rectal and intestinal mucosa, etc.), and can be co-administered with other biologically active agents. Thus, pharmaceutical compositions containing the antibodies of the invention may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (eg, by powder, ointment, drops or transdermal patch), oral administration, or by oral or nasal spray.

The term "parenteral" as used herein refers to modes of administration including intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

Antibodies of the invention may be administered topically to the area in need of treatment; by, but not limited to, by local infusion during surgery, such as topical application in combination with post-surgical wound dressings, by injection, by catheter, by suppository, or by implant To achieve this, the implant is a porous, non-porous or gel-like material, including membranes (eg, silicone rubber membranes) or fibers. Preferably, when administering the proteins (including antibodies) of the invention, care must be taken to use materials that do not absorb the protein.

In some embodiments, the compositions of the invention comprise a nucleic acid or polynucleotide encoding an antibody, which can be administered in vivo to facilitate expression of the protein encoded thereby by constructing it as part of a suitable nucleic acid expression vector, Parts of the vectors described above are then administered to make them intracellular, for example, by the use of retroviral vectors (see US Pat. No. 4,980,286), or by direct injection, or by the use of particle bombardment (eg, a gene gun; Biolistic, Dupont) , or coated with lipids or cell surface receptors or transfection reagents, or administered by linkage to homeobox peptoids known to enter the nucleus (see e.g. Joliot et al., 1991, Proc.Natl.Acad.Sci .USA 88:1864-1868) and so on. Alternatively, the nucleic acid can be introduced into a cell by homologous recombination and integrated into the host cell DNA for expression.

The uptake and tissue penetration ability of the antibody or antigen-binding fragment can be enhanced by modifications such as lipidation, thereby reducing the dose and frequency of administration of the antibodies of the invention. Various known delivery systems can be used to administer the antibodies or derivatives of the invention or polynucleotides encoding them, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, receptor-mediated Endocytosis (see, eg, Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of nucleic acids as part of retroviral or other vectors, and the like.

combination therapy

In some embodiments, the antibodies of the invention may be used in conjunction with or in combination with other therapeutic or prophylactic regimens, including administration of one or more antibodies of the invention and one or more other therapeutic agents or methods. For combination therapy, the antibody and other therapeutic agents may be administered simultaneously or separately. When administered separately, the antibody of the invention may be administered before or after administration of the other other therapeutic agent.

In some embodiments, the bispecific antibodies of the invention are administered in combination with a chemotherapeutic agent. In some embodiments, the bispecific antibodies of the invention are bispecific antibodies targeting OX40 and TIGIT, or bispecific antibodies targeting TIGIT and PD-1. In some embodiments, chemotherapeutic agents that can be administered with the antibodies of the invention include, but are not limited to, antibiotic derivatives (eg, doxorubicin, bleomycin, daunorubicin, and actinomycin D), antiestrogens (eg, tamoxifen), antimetabolites (eg, fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamate, mithramycin, mercaptopurine, and 6-thiol Guanine), cytotoxic agents (such as carmustine, BCNU, lomustine, CCNU, cytarabine, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, Busulfan, cisplatin, and vincristine sulfate), hormones (eg, medroxyprogesterone, estramustine sodium phosphate, ethinylestradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorine diethylstilbestrol and testosterone), nitrogen mustard derivatives (e.g. melphalan, chlorambucil, dichloromethyldiethylammonium (chlorambucil), and thiotepa), steroids and combinations thereof (e.g. betadine) Metasone Sodium Phosphate), and other compounds such as dazolamide, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide.

In some embodiments, the antibodies of the invention are administered in combination with cytokines. Cytokines that can be administered with the antibodies of the 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, etc.

In some embodiments, the bispecific antibodies of the invention are administered in combination with a chemotherapeutic agent. Examples of chemotherapeutic agents include immunotherapeutic agents, including, but not limited to, therapeutic antibodies suitable for use in treating patients. Some examples of therapeutic antibodies include simtuzumab, abagovomab, adecatumumab, afutuzumab, alemtuzumab (alemtuzumab), altumomab, amatuximab, anatumomab, arcitumomab, bavituximab ), bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantor cantuzumab, catumaxomab, cetuximab, citatuzumab, cixutumumab, crivatuzumab clivatuzumab, conatumumab, daratumumab, drozitumab, duligotumab, duligotumab (dusigitumab), detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab ( elotuzumab), ensituximab, ertumaxomab, etaracizumab, farletuzumab, ficolatuzumab ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab ), girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab ), indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab, pull labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatum mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, moxetumumab (moxetumomab), narnatumab, naptumomab, necitumumab, nimotuzumab, nofetumomab , ocaratuzumab, ofatumumab, olaratumab, onartuzumab, oportuzumab, oregovomab, panitumumab, parsatuzumab, patritumab, pemtumomab, pertuzumab (pertuzumab), pintumomab, pritumumab, racotumomab, radretumab, rilotumumab ), rituximab, robatumumab, satumomab, sibrotuzumab, siltuximab, Solitomab, tacatuzumab, taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab veltuzumab, vorsetuzumab, votumumab and zalutumumab, etc.

In some embodiments, the antibodies of the invention may be used with immune checkpoint inhibitors. In some embodiments, the antibodies of the invention are administered in combination with other therapeutic or prophylactic regimens, such as radiotherapy.

pharmaceutical composition

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

In some embodiments, the term "pharmaceutically acceptable" refers to a substance approved by a regulatory agency of the government or listed in a recognized pharmacopeia for use in animals, particularly in humans. In addition, "pharmaceutically acceptable carrier" generally refers to any type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation aid and the like.

"Carrier" in the term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient or carrier with which the active ingredient can be administered to a patient. Such pharmaceutical carriers can be sterile liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is the preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerin, Propylene, ethylene glycol, water, ethanol, etc. The compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates, if desired. Antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and tonicity adjusting agents such as sodium chloride or dextrose are also contemplated. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated as a suppository with traditional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin, incorporated herein by reference. Such compositions will contain a clinically effective dose of the antibody or antigen-binding fragment, preferably in purified form, together with a suitable amount of carrier to provide a form suitable for administration to the patient. The formulation should be suitable for the mode of administration. The parental formulation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

In some embodiments, the compositions are formulated according to conventional procedures into pharmaceutical compositions suitable for intravenous injection into humans. Compositions for intravenous administration are usually solutions in sterile isotonic aqueous buffer. The composition may also contain a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the active ingredients are supplied in unit dosage form either individually or mixed together, eg, as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container (eg, an ampule or sachet) indicative of the amount of active agent. Where the composition is administered by infusion, the composition may be dispensed in an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of administering the composition by injection, ampoules of sterile water for injection or saline can be used, allowing the active ingredient to be mixed prior to administration.

The compounds of the present invention may be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions derived from, for example, hydrochloric, phosphoric, acetic, oxalic, tartaric, etc., and those derived from, for example, sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2- Salts with cations of ethylaminoethanol, histidine, procaine, etc.

detailed description

The technical solutions of the present invention are further described below through specific embodiments, which do not represent limitations on the protection scope of the present invention. Some non-essential modifications and adjustments made by others according to the concept of the present invention still belong to the protection scope of the present invention.

Materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified; for example, TIGIT-Fc antigen can be purchased from Shanghai Nearshore Technology Co., Ltd. (article number CS92), PD-1 his Antigen can be purchased from Shanghai Inshore Technology Co., Ltd. (Cat. No. CX91), TIGIT-his antigen can be purchased from Shanghai Inshore Technology Co., Ltd. or AcroBiosystems, and anti-TIGIT reporter gene detection system can be purchased from Promega (Cat. No. JA1400) , or can be prepared according to known methods.

The heavy chains of anti-TIGIT antibodies are:

(SEQ ID NO: 51);

The light chain of the anti-TIGIT antibody is:

Anti-TIGIT antibody was expressed and purified in CHO cells.

The heavy chain of the anti-OX40 antibody is:

The light chain of the anti-OX40 antibody is:

The anti-OX40 antibody was expressed and purified in CHO cells.

The heavy chains of anti-PD-1 antibodies are:

The light chains of anti-PD-1 antibodies are:

Anti-PD-1 antibody was expressed and purified in CHO cells.

Bispecific antibodies targeting OX40 and TIGIT

Example 1 Antibody preparation method

The structure of the bispecific antibody is shown in Figure 1, which is similar to immunoglobulin. The antibody contains the first polypeptide chain and the fourth polypeptide chain with the same sequence, the second polypeptide chain and the fourth polypeptide chain with the same sequence. Five polypeptide chains, as well as the third polypeptide chain and the sixth polypeptide chain with the same sequence, the Fc regions of the third polypeptide chain and the sixth polypeptide chain are paired to form a disulfide bond; the first polypeptide chain contains the structure VHa- CH1a-L1-VHb-L2-CLb, the second polypeptide chain comprises the structure VLa-CLa, and the third polypeptide chain comprises the structure VLb-L3-CH1b-Fc; wherein, antigen a is OX40 and antigen b is TIGIT, or antigen a is TIGIT and antigen b is OX40.

The nucleotide sequences corresponding to the first polypeptide chain, the second polypeptide chain and the third polypeptide chain in the bispecific antibody were obtained by artificial synthesis, and then the above nucleotides were respectively connected to the pCDNA3.0 vector by enzyme ligation. (purchased from Invitrogen Company), 3 recombinant plasmids for expressing full antibody were obtained. Using the Freedom CHO-S kit (purchased from Invitrogen) according to the manufacturer's instructions, the above plasmids were simultaneously transiently transfected into HEK293 cells by PEI, and the supernatant was collected after 7 days of culture, and finally the bispecific antibody protein sample was obtained by purification.

The amino acid sequences related to the bispecific antibodies targeting OX40 and TIGIT are shown in Table 1, the linker in Table 1 is represented by a single underline, and the Fc in Table 1 is represented by a double underline; nucleic acids related to the bispecific antibodies targeting OX40 and TIGIT See Table 2 for the sequence. Antibodies 1-4 bind to OX40 and TIGIT, and antibodies 5 and 6 bind to TIGIT and OX40; the amino acid sequence numbers of the polypeptide chains in antibodies 1-6 are shown in Table 3. In this application, the code name of Antibody 1 is pOT-3c-15aa, the code name of Antibody 2 is pOT-3c-30aa, the code name of Antibody 3 is OT-3c-15aa, the code name of Antibody 4 is OT-3c-30aa, and the code name of Antibody 5 is OT-3c-30aa. The code name is TO-3c-5aa, and the code name of antibody 6 is TO-3c-30aa.

Table 1 Amino acid sequences of bispecific antibodies targeting OX40 and TIGIT

Table 2 Nucleic acid sequences of bispecific antibodies targeting OX40 and TIGIT

Table 3 Composition of Antibody Polypeptide Chains

Example 2 Antibody purity detection

Gel electrophoresis detection of purified antibodies: to detect the composition and purity of bispecific antibodies under reducing and non-reducing conditions. As shown in Figure 2, under non-reducing conditions, bispecific antibodies migrate as a single band of about 250 KDa; under reducing conditions, OT-3c-15aa (antibody 3), TO-3c-5aa (antibody 5) and pOT -3c-15aa (antibody 1) each produced "two bands", one about 55KDa (the overlapping band of the first and third polypeptide chains) and the other about 25KDa (the second For OT-3c-30aa (antibody 4), TO-3c-30aa (antibody 6) and pOT-3c-30aa (antibody 2), each produced 3 bands under reducing conditions, two bands of about 55KDa Band (the first polypeptide chain and the third polypeptide chain), another band of about 25KDa (the second polypeptide chain). SDS-PAGE showed that the antibody 1-6 of the present application was a single substance, and the three polypeptide chains were effectively paired to form an IgG-like molecule; the size of the full length of the three polypeptide chains of the antibody 1-6 was consistent with the theoretical value.

Example 3 Detection of Antibody Binding Activity

ForteBio affinity was determined according to 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 process is as follows, the sensor is equilibrated in the analysis buffer (such as PBS) for 20 minutes under the midline, then the signal baseline is established for 60 seconds on the computer, and the purified antibodies 1-6 obtained as described above are loaded on the computer to the corresponding sensor ( ForteBio), the ForteBio affinity measurement was finally performed; the protein sensor was used to adsorb the above antibodies, and then the binding and dissociation with OX40-his antigen (AcroBiosystems) and TIgit-his antigen (AcroBiosystems) were detected for about 5 minutes each; finally, 1 :1 combined with the model to analyze the kinetics, and the specific results are shown in Table 4.

Table 4. Affinity parameters of antibodies binding to OX40-his and Tigit-his

As shown in Table 4, antibodies 1-6 can obviously bind to OX40-his antigen and Tigit-his antigen; OT-3c-15aa (antibody 3), OT-3c-30aa (antibody 4), TO-3c-5aa ( The affinity of antibody 5) and TO-3c-30aa (antibody 6) was not much different from that of anti-OX40Ab and anti-Tigit Ab.

Example 4 Antibodies simultaneously bind to OX40 antigen and TIGIT antigen

Similar to the above-mentioned Fortebio affinity determination method, it was tested whether the antibodies 1-6 could bind to OX40-his antigen (AcroBiosystems) and TIGIT-his antigen (AcroBiosystems) simultaneously. A protein sensor was used to adsorb the antibodies 1-6 in the above embodiments, and the binding and dissociation with the OX40 antigen was detected first, and then the binding and dissociation with the TIGIT antigen was detected.

The results showed (as shown in Figure 14 ) that all antibodies 1-6 could obviously bind to OX40 antigen and Tigit antigen without steric hindrance.

Example 5 Binding activity of antibody to Jurkat cells overexpressing OX40 or TIGIT

1) Jurkat-OX40 cells overexpressing human OX40 were generated by transfecting pCMV vector (Invirogen Company) with human OX40 cDNA, and Jurkat-hOX40 cells (0.5×10 ⁶ cells) were incubated with different concentrations of antibodies in PBS on ice 40 minutes. Cells were then washed twice and incubated with secondary antibody in PBS with 0.1% BSA on ice for 25 minutes. Cells were washed twice and analyzed by flow cytometry on the Accuri C6 system (BD Biosciences).

As shown in Figure 3 and Figure 4, antibodies 1-6 can obviously bind to Jurkat-OX40 cells, and their binding capacity is close to that of anti-OX40; among them, antibody 3 (OT-3c-15aa) and antibody 4 (OT-3c-30aa) ) is relatively good.

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

As shown in Figures 5 and 6, antibodies 1-6 clearly bind Jurkat-Tigit cells with a binding force close to that of anti-TIGIT.

Example 6 NFκB reporter gene system detects the in vitro activation activity of antibodies

In order to detect the biological activity of the bispecific antibody of the present invention in the presence of Jurkat-Tigit cells, activating the OX40-mediated signaling pathway; the NFκB reporter gene system was used to detect the activator activity of the antibody of the present application.

The experimental steps are as follows: the plasmid pGL6-NFkB-lufiferas-reporter (purchased from Biyuntian) was electroporated into Jurkat-hOX40 cells, and finally a stable monoclonal strain was obtained by pressurized screening with antibiotics (Hygromycin), which was named Jurkat-hOX40. hOX40-NFκB cells. Jurkat-hOX40-NFkB cells were revived, passaged three times, then ^{plated at 4×10 4} cells/well, 60 μl medium per well, bispecific antibody and 40,000 Jurkat-Tigit cells/well were added, and incubated for 4.5 hours , and then add 50 μl of fluorescent reagent (such as ONE-Glo ^™ Luciferase Assay System, purchased from promega company) to each well, and measure the fluorescence intensity.

As shown in Figure 7, in the presence of Tigit-expressing cells, antibodies 1-4 and antibody 6 significantly activated the NFkB signaling pathway downstream of OX40, achieving the effect of activating the immune system.

Example 7 Antibodies block the binding of PVR to Jurkat cells overexpressing Tigit

In order to verify whether the bispecific antibody of the present invention can block the binding of PVR (poliovirus receptor, poliovirus receptor) to Jurkat cells overexpressing Tigit, flow cytometry is used in this example, and the test process is as follows: using PVR- Fc bio (purchased from ACROBiosystems) was prepared with antibody diluent, and the concentration of PVR-Fc bio was 200nM, and then the anti-bispecific antibody was serially diluted. The initial concentration of antibody in 100μl system was 15nM, with a total of 10 concentration points; Jurakt-Tigit cells were taken. 1 million/tube, discard the medium after centrifugation, wash once with 200 μl PBS, and then add 150 μl of a series of different concentrations of PVR-FC bio+ bispecific antibody to the washed cells. Incubate at 4°C for 1.5 hours; wash once with 200μl PBS, add 100μl of secondary antibody PE-Streptavidin (1:1000 dilution), and incubate at 4°C for 30min; finally wash once with 400μl PBS, resuspend in 200μl PBS and load on the flow analyzer .

As shown in Figure 8, OT-3c-15aa (antibody 3), pOT-3c-15aa (antibody 1) and TO-3c-30aa (antibody 6) can effectively block the binding of PVR to Jurakt-Tigit cells, and prevent the The inhibitory activity is slightly weaker than that of anti-Tigit.

The ability of the antibody of Example 8 to relieve Tigit inhibitory activity

The biological activity of the bispecific antibody was determined by the anti-TIGIT reporter gene detection system. For the detection method, please refer to the product specification and patent CN107106608A.

As shown in Fig. 9, OT-3c-15aa (antibody 3), OT-3c-30aa (antibody 4)a and pOT-3c-15aa (antibody 1) could relieve the inhibitory activity of Tigit at the cellular level; among them pOT The effect of -3c-15aa to relieve the inhibitory activity of Tigit is close to that of anti-Tigit.

Example 9 Antibodies stimulate the secretion of IL-2 from human PBMC cells

The agonist activity of the bispecific antibodies of the invention was assessed by measuring inflammatory cytokines released by T cells following T cell activation. After taking PBMCs (peripheral blood mononuclear cells), they were plated at 200,000 cells/well, 200 μl of medium was plated into 96-well plates, and 80 μg/ml of SEB (Staphylococcal Enterotoxin B) was added to activate PBMCs, and 2 days later, they were collected PBMC. After washing, 110,000 cells/well were plated, 100 μl of medium was added, and 20 μl of antibodies diluted 3-fold starting from 100 nM were added. After 3 days, the level of IL-2 in the medium was detected by ELISA kit.

As shown in Figure 10, pOT-3c-15aa (antibody 1) and OT-3c-15aa (antibody 3) could significantly activate PBMC to secrete IL-2, but weaker than anti-OX40.

Bispecific antibodies targeting TIGIT and PD-1

Example 10 Antibody preparation method

The structure of the bispecific antibody is shown in Figure 1, which is similar to immunoglobulin. The antibody contains the first polypeptide chain and the fourth polypeptide chain with the same sequence, the second polypeptide chain and the fourth polypeptide chain with the same sequence. Five polypeptide chains, as well as the third polypeptide chain and the sixth polypeptide chain with the same sequence, the Fc regions of the third polypeptide chain and the sixth polypeptide chain are paired to form a disulfide bond; the first polypeptide chain contains the structure VHa-CH1a -L1-VHb-L2-CLb, the second polypeptide chain comprises the structure VLa-CLa, and the third polypeptide chain comprises the structure VLb-L3-CH1b-Fc; wherein, antigen a is TIGIT and antigen b is PD-1.

The amino acid sequences of the first polypeptide chain, the second polypeptide chain and the third polypeptide chain of the antibody were sequence optimized according to the codon preference characteristics of the host cell CHO (Chinese hamster ovary cells) to obtain the first polypeptide chain, the third polypeptide chain, and the The DNA sequences corresponding to the second polypeptide chain and the third polypeptide chain. In order to facilitate expression in CHO cells, a signal peptide and Kozak sequence were added to the first polypeptide chain, the second polypeptide chain and the third polypeptide chain respectively (Kozak sequence is located behind the cap structure at the 5' end of eukaryotic mRNA A nucleic acid sequence), with a stop codon added to the end of the sequence. At the same time, in order to facilitate cloning into the mammalian expression vector pCDNA3.1 ^TM (+) (Invitrogen, Cat. No. V79020), enzyme cleavage sites are also added to both ends of the sequence, and the 5' end is Hind III and the 3' end is EcoR I .

The optimized and synthesized nucleic acid sequence clones were cloned into pCDNA3.1 ^TM (+) vector respectively, and then a large number of plasmids were extracted respectively. The first polypeptide chain, the second polypeptide chain and the third polypeptide chain were in a plasmid molar ratio of 1. :1:1 ^{Transient expression was performed using the ExpiCHO™} Expression System (Gibco, Cat. No. A29133). The protein was harvested and purified with protein A according to the instructions.

The amino acid sequences related to the bispecific antibodies targeting TIGIT and PD-1 are shown in Table 5, and the nucleic acid sequences related to the bispecific antibodies targeting TIGIT and PD-1 are shown in Table 6. In Table 5, the linker is represented by single underline, and Fc is represented by double underline; to the 3' end) followed by a stop codon and an EcoR I restriction site. The first polypeptide chain of antibody 7 is shown in SEQ ID NO:34, the second polypeptide chain of antibody 7 is shown in SEQ ID NO:35, and the third polypeptide chain of antibody 7 is shown in SEQ ID NO:36 ; Antibody 7 is codenamed TIGIT/PD-1 BiAb.

Table 5 Amino acid sequences of bispecific antibodies targeting TIGIT and PD-1

Table 6 Nucleic acid sequences of bispecific antibodies targeting TIGIT and PD-1

Example 11 Antibody Purity Detection

Perform gel electrophoresis detection on the purified antibody: Check the purity of the purified antibody. As shown in Fig. 11, the purity of Antibody 7 was high.

Example 12 Detection of Antibody Binding Activity

1) The binding activity of the purified dual-specific specific antibody is detected to detect whether the antibody can normally bind to TIGIT or PD-1, respectively. The binding assay process was as follows: 1 μg/ml of TIGIT-Fc antigen was coated with PBS, 100 μl per well, and then placed in a refrigerator at 2-8 °C for overnight coating; the next day, 250 μl of PBS containing 3% BSA was added and placed in a 37 °C incubator Blocked for 2 hours; then washed twice with PBST, and then added gradient dilutions of anti-TIGIT and TIGIT/PD1 BiAb (antibody 7), starting at a concentration of 3 μg/ml, 3-fold dilution, a total of 10 dilution gradients; in the corresponding wells Add 100 μl of diluted antibody to each well, incubate in a 37°C incubator for 1 hour; wash 3 times with PBST, add 1:5000-fold diluted enzyme-labeled secondary antibody (Sigma goat anti-human IgG kappa light chain-conjugated HRP, Item No.: A7164-1ML) 100 μl per well, incubate for 1 hour in a 37°C incubator; wash 5 times with PBST, add TMB chromogenic solution for color development for about 20 minutes, add 1M H ₂ SO ₄ to stop, read OD450 within 30 minutes value.

As shown in Figure 12, TIGIT/PD-1 BiAb (antibody 7) can bind well to TIGIT-Fc, and the binding ability is comparable to the control antibody anti-TIGIT.

2) The test process of binding to PD-1 his antigen is basically similar to the above test process. As shown in Figure 13, TIGIT/PD-1 BiAb (antibody 7) can bind to PD-1 his, and the binding ability is weaker than that of anti-PD-1.

Claims

An antibody or antigen-binding fragment, characterized in that the antibody or antigen-binding fragment comprises a first antigen a-binding portion and a second antigen-b binding portion that bind to two different antigens, the first antigen a and the second antigen b, respectively , and the antibody or antigen-binding fragment comprises at least 3 polypeptide chains; wherein, the first polypeptide chain comprises VHa, CH1a, VHb and CLb in sequence from the amino terminus to the carboxyl terminus, and VHa is the heavy chain of the first antigen a binding portion The variable regions, CH1a is the heavy chain first constant region of the first antigen a binding portion, VHb is the heavy chain variable region of the second antigen b binding portion, and CLb is the light chain constant region of the second antigen b binding portion.
The antibody or antigen-binding fragment of claim 1, wherein CH1a and VHb are covalently linked through a linker L1; wherein L1 contains 5 to 33 amino acids, and at least 50% of the amino acids are glycines; and/ or

VHb and CLb are covalently linked through linker L2, which contains 2 to 6 amino acids.
The antibody or antigen-binding fragment of claim 1 or 2, wherein the second polypeptide chain comprises VLa and CLa in sequence from the amino terminus to the carboxyl terminus; wherein, VLa is the light of the first antigen a-binding portion The chain variable region, CLa is the light chain constant region of the first antigen a binding portion.
The antibody or antigen-binding fragment of any one of claims 1-3, wherein the third polypeptide chain comprises VLb and CH1b in sequence from the amino terminus to the carboxyl terminus; wherein, VLb is the second antigen-b binding part of the light chain variable region, CH1b is the heavy chain constant region of the second antigen b binding part.
The antibody or antigen-binding fragment of claim 4, wherein VLb and CH1b are covalently linked through a linker L3, and L3 contains 2 to 6 amino acids.
The antibody or antigen-binding fragment of claim 4 or 5, wherein the first polypeptide chain or the third polypeptide chain comprises Fc, and Fc is a hinge region comprising a heavy chain, the second constant region and the first polypeptide chain. Three constant regions.
The antibody or antigen-binding fragment of any one of claims 1-5, wherein the first polypeptide chain comprises the structure VHa-CH1a-L1-VHb-L2-CLb, and the second polypeptide chain comprises the structure VLa- CLa, the third polypeptide chain comprises the structure VLb-L3-CH1b; or

The first polypeptide chain contains the structure VHa-CH1a-L1-VHb-L2-CLb, the second polypeptide chain contains the structure VLa-CLa, and the third polypeptide chain contains the structure VLb-L3-CH1b-Fc.
The antibody or antigen-binding fragment of any one of claims 1-7, wherein CH1a of the first polypeptide chain and CLa of the second polypeptide chain are connected by a disulfide bond, and the first polypeptide chain The CLb is linked to the CH1b of the third polypeptide chain by a disulfide bond.
The antibody or antigen-binding fragment of any one of claims 1-8, wherein antigens a and b are respectively selected from the group consisting of: 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 TIM-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.
The antibody or antigen-binding fragment of claim 9, wherein the antigen a is OX40 and the antigen b is TIGIT, and the antibody or antigen fragment comprises the following:

The VHa contains the sequence shown in SEQ ID NO: 1 or 2, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 1 or 2, or compared with the sequence shown in SEQ ID NO: 1 or 2 an amino acid sequence with one or more conservative amino acid substitutions; and/or

The VHb contains the sequence shown in SEQ ID NO:3, has at least 80% identity with the sequence shown in SEQ ID NO:3, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO:3 amino acid sequence of amino acid substitutions; and/or

The VLa contains the sequence shown in SEQ ID NO: 4 or 5, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 4 or 5, or compared with the sequence shown in SEQ ID NO: 4 or 5 an amino acid sequence with one or more conservative amino acid substitutions; and/or

The VLb contains the sequence shown in SEQ ID NO:6, a sequence with at least 80% identity to the sequence shown in SEQ ID NO:6, or one or more conserved sequences compared with the sequence shown in SEQ ID NO:6 amino acid sequence of amino acid substitutions; and/or

The CLa contains the sequence shown in SEQ ID NO:7, a sequence with at least 80% identity to the sequence shown in SEQ ID NO:7, or one or more conserved sequences compared with the sequence shown in SEQ ID NO:7 amino acid sequence of amino acid substitutions; and/or

The CLb contains the sequence shown in SEQ ID NO: 8, has at least 80% identity with the sequence shown in SEQ ID NO: 8, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 8 amino acid sequence of amino acid substitutions; and/or

The CH1a contains the sequence shown in SEQ ID NO: 9, has at least 80% identity with the sequence shown in SEQ ID NO: 9, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 9 amino acid sequence of amino acid substitutions; and/or

The CH1b contains the sequence shown in SEQ ID NO: 9, has at least 80% identity with the sequence shown in SEQ ID NO: 9, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 9 Amino acid sequence of amino acid substitutions.
The antibody or antigen-binding fragment of claim 9, wherein said antigen a is TIGIT and antigen b is OX40, said antibody or antigenic fragment comprising the following:

The VHa contains the sequence shown in SEQ ID NO:3, has at least 80% identity with the sequence shown in SEQ ID NO:3, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO:3 amino acid sequence of amino acid substitutions; and/or

The VHb contains the sequence shown in SEQ ID NO: 2, has at least 80% identity with the sequence shown in SEQ ID NO: 2, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 2 amino acid sequence of amino acid substitutions; and/or

The VLa contains the sequence shown in SEQ ID NO: 6, has at least 80% identity with the sequence shown in SEQ ID NO: 6, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 6 amino acid sequence of amino acid substitutions; and/or

The VLb contains the sequence shown in SEQ ID NO:5, a sequence with at least 80% identity to the sequence shown in SEQ ID NO:5, or one or more conserved sequences compared with the sequence shown in SEQ ID NO:5 amino acid sequence of amino acid substitutions; and/or

The CLa contains the sequence shown in SEQ ID NO:7, a sequence with at least 80% identity to the sequence shown in SEQ ID NO:7, or one or more conserved sequences compared with the sequence shown in SEQ ID NO:7 amino acid sequence of amino acid substitutions; and/or

The CLb contains the sequence shown in SEQ ID NO: 8, has at least 80% identity with the sequence shown in SEQ ID NO: 8, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 8 amino acid sequence of amino acid substitutions; and/or

The CH1a contains the sequence shown in SEQ ID NO: 9, has at least 80% identity with the sequence shown in SEQ ID NO: 9, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 9 amino acid sequence of amino acid substitutions; and/or

The CH1b contains the sequence shown in SEQ ID NO: 9, has at least 80% identity with the sequence shown in SEQ ID NO: 9, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 9 Amino acid sequence of amino acid substitutions.
The antibody or antigen-binding fragment of claim 9, wherein the antigen a is TIGIT and the antigen b is PD-1, and the antibody or antigen fragment comprises the following:

The VHa contains the sequence shown in SEQ ID NO: 10, has at least 80% identity with the sequence shown in SEQ ID NO: 10, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 10 amino acid sequence of amino acid substitutions; and/or

The VHb contains the sequence shown in SEQ ID NO: 11, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 110, or one or more conserved sequences compared with the sequence shown in SEQ ID NO: 11 amino acid sequence of amino acid substitutions; and/or

The VLa contains the sequence shown in SEQ ID NO: 12, has at least 80% identity with the sequence shown in SEQ ID NO: 12, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 12 amino acid sequence of amino acid substitutions; and/or

The VLb contains the sequence shown in SEQ ID NO: 13, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 13, or one or more conserved sequences compared with the sequence shown in SEQ ID NO: 13 amino acid sequence of amino acid substitutions; and/or

The CLa contains the sequence shown in SEQ ID NO:7, a sequence with at least 80% identity to the sequence shown in SEQ ID NO:7, or one or more conserved sequences compared with the sequence shown in SEQ ID NO:7 amino acid sequence of amino acid substitutions; and/or

The CLb contains the sequence shown in SEQ ID NO: 8, has at least 80% identity with the sequence shown in SEQ ID NO: 8, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 8 amino acid sequence of amino acid substitutions; and/or

The CH1a contains the sequence shown in SEQ ID NO: 9, has at least 80% identity with the sequence shown in SEQ ID NO: 9, or has one or more conserved sequences compared with the sequence shown in SEQ ID NO: 9 amino acid sequence of amino acid substitutions; and/or

The CLb contains the sequence shown in SEQ ID NO: 14, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 14, or a sequence compared with the sequence shown in SEQ ID NO: 14 or amino acid sequence of multiple conservative amino acid substitutions; and/or.
The antibody or antigen-binding fragment of any one of claims 5-12, wherein the L1 contains a sequence selected from any of SEQ ID NO: 15-18, which is the same as SEQ ID NO: 15 - a sequence that is at least 90% identical to the sequence shown in any one of 18, or an amino acid sequence that has one or more conservative amino acid substitutions compared to the sequence shown in any one of SEQ ID NOs: 15-18; and/ or

The L2 contains the sequence shown in SEQ ID NO: 19; and/or

The L3 contains the sequence shown in SEQ ID NO: 20.
The antibody or antigen-binding fragment of any one of claims 5-13, wherein the Fc comprises the sequence shown in SEQ ID NO: 21 or 22, and has the same sequence as the sequence shown in SEQ ID NO: 21 or 22 A sequence that is at least 80% identical, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 21 or 22.
An antibody or antigen-binding fragment, characterized in that the antibody or antigen-binding fragment binds 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 three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO: 23, which is the same as SEQ ID NO: 23. The sequence shown in ID NO: 23 has at least 80% identity, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 23; the second polypeptide chain contains SEQ ID The sequence shown in NO: 24, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 24, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 24; The third polypeptide chain contains the sequence shown in SEQ ID NO: 25, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 25, or a sequence compared with the sequence shown in SEQ ID NO: 25. or amino acid sequence of multiple conservative amino acid substitutions; or

The first polypeptide chain contains the sequence shown in SEQ ID NO: 26, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 26, or a sequence compared with the sequence shown in SEQ ID NO: 26. or amino acid sequence of multiple conservative amino acid substitutions; the second polypeptide chain contains the sequence shown in SEQ ID NO: 24, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 24, or a sequence with SEQ ID NO: 24 Compared with the sequence shown in NO: 24, the amino acid sequence has one or more conservative amino acid substitutions; the third polypeptide chain contains the sequence shown in SEQ ID NO: 25, and has at least 80% of the sequence shown in SEQ ID NO: 25. A sequence of % identity, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 25; or

The first polypeptide chain contains the sequence shown in SEQ ID NO: 27, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 27, or a sequence compared with the sequence shown in SEQ ID NO: 27. or amino acid sequence of multiple conservative amino acid substitutions; the second polypeptide chain contains the sequence shown in SEQ ID NO: 28, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 28, or a sequence with SEQ ID NO: 28 Compared with the sequence shown in NO: 28, the amino acid sequence has one or more conservative amino acid substitutions; the third polypeptide chain contains the sequence shown in SEQ ID NO: 25, and has at least 80% of the sequence shown in SEQ ID NO: 25. A sequence of % identity, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 25; or

The first polypeptide chain contains the sequence shown in SEQ ID NO: 29, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 29, or a sequence compared with the sequence shown in SEQ ID NO: 29. or amino acid sequence of multiple conservative amino acid substitutions; the second polypeptide chain contains the sequence shown in SEQ ID NO: 28, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 28, or a sequence with SEQ ID NO: 28 Compared with the sequence shown in NO: 28, the amino acid sequence has one or more conservative amino acid substitutions; the third polypeptide chain contains the sequence shown in SEQ ID NO: 25, and has at least 80% of the sequence shown in SEQ ID NO: 25. A sequence of % identity, or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 25.
An antibody or antigen-binding fragment, characterized in that the antibody or antigen-binding fragment binds 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 OX40; The antibody or antigen-binding fragment comprises at least three polypeptide chains: a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO: 30, which is the same as SEQ ID NO: 30. The sequence shown in ID NO: 30 has at least 80% identity, or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 30; the second polypeptide chain contains SEQ ID The sequence shown in NO: 31, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 31, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO: 31; The third polypeptide chain contains the sequence shown in SEQ ID NO:32, a sequence with at least 80% identity to the sequence shown in SEQ ID NO:32, or a sequence compared with the sequence shown in SEQ ID NO:32. or amino acid sequence of multiple conservative amino acid substitutions; or

The first polypeptide chain contains the sequence shown in SEQ ID NO:33, a sequence with at least 80% identity to the sequence shown in SEQ ID NO:33, or a sequence compared with the sequence shown in SEQ ID NO:33. or amino acid sequence of multiple conservative amino acid substitutions; the second polypeptide chain contains the sequence shown in SEQ ID NO: 31, a sequence with at least 80% identity to the sequence shown in SEQ ID NO: 31, or a sequence with SEQ ID NO: 31 Compared with the sequence shown in NO: 31, the amino acid sequence has one or more conservative amino acid substitutions; the third polypeptide chain contains the sequence shown in SEQ ID NO: 32, and has at least 80% of the sequence shown in SEQ ID NO: 32. A sequence of % identity, or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 32.
An antibody or antigen-binding fragment, characterized in that the antibody or antigen-binding fragment binds 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 PD-1 The antibody or antigen-binding fragment comprises at least three polypeptide chains: the first polypeptide chain, the second polypeptide chain and the third polypeptide chain; the first polypeptide chain contains the sequence shown in SEQ ID NO: 34, A sequence with at least 80% identity to the sequence shown in SEQ ID NO: 34, or an amino acid sequence with one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 34; the second polypeptide chain contains The sequence shown in SEQ ID NO:35, a sequence with at least 80% identity to the sequence shown in SEQ ID NO:35, or an amino acid with one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:35 sequence; the third polypeptide chain contains the sequence shown in SEQ ID NO:36, a sequence with at least 80% identity to the sequence shown in SEQ ID NO:36, or compared with the sequence shown in SEQ ID NO:36 An amino acid sequence with one or more conservative amino acid substitutions.
The antibody or antigen-binding fragment of any one of claims 15-17, wherein the antibody comprises a first polypeptide chain and a fourth polypeptide chain with the same sequence, a second polypeptide chain with the same sequence and The fifth polypeptide chain, as well as the third polypeptide and the sixth polypeptide chain having the same sequence, the Fc regions of the third polypeptide chain and the sixth polypeptide chain are paired to form a disulfide bond.
A nucleic acid molecule encoding the antibody or antigen-binding fragment of any one of claims 1-18.
A vector or host cell comprising the nucleic acid molecule of claim 19.
A pharmaceutical composition, characterized in that, the pharmaceutical composition comprises the antibody or antigen-binding fragment according to any one of claims 1-18 and a pharmaceutically acceptable carrier.
Use of the antibody or antigen-binding fragment of any one of claims 1-18 in the preparation of a medicament for the treatment or improvement of inflammatory diseases, autoimmune diseases, cancer or spinal cord injury, or in the preparation of a medicament for diagnosing inflammation Use in kits for sexually transmitted diseases, autoimmune diseases, cancer or spinal cord injury.