CN115803343A

CN115803343A - Multispecific antibodies and uses thereof

Info

Publication number: CN115803343A
Application number: CN202180037470.3A
Authority: CN
Inventors: 黄俊杰; 苏紫琪; 梁世忠; 徐振前; 梁炳辉; 李胜峰
Original assignee: Bio Thera Solutions Ltd
Current assignee: Bio Thera Solutions Ltd
Priority date: 2020-05-26
Filing date: 2021-05-25
Publication date: 2023-03-14
Also published as: WO2021238932A1

Abstract

Multispecific antibodies or antigen-binding fragments thereof are provided that can bind two or more antigens, or two or more epitopes of the same antigen; also provided is the use of the antibody or antigen-binding fragment for the treatment, diagnosis and prognosis of inflammatory diseases, autoimmune diseases or spinal cord injury.

Description

Multispecific antibodies and uses thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a multispecific antibody and application thereof.

Background

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

A bispecific antibody is an engineered antibody that can bind two specific epitopes or proteins of interest simultaneously, and can serve several specific biological functions. Compared with the combined treatment of two monoclonal antibody medicines, the BsAb improves the selectivity and the functionality of the antibody and reduces the treatment cost. BsAb is prepared mainly by chemical coupling, double hybridoma cell method, recombinant gene preparation and other methods.

However, bispecific antibody preparations have difficulties, such as production of a large amount of by-products and inhibition of biological activity.

Disclosure of Invention

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

In some embodiments, the antibody or antigen-binding fragment binds two different antigenic epitopes, a first antigenic epitope x and a second antigenic epitope y, and the antibody or antigen-binding fragment comprises at least 2 polypeptide chains; wherein the first polypeptide chain comprises, in order from the amino terminus, VHa, CLa, VHb and CH1, VHa being the heavy chain variable region associated with a first epitope x, CLa being the first light chain constant region, VHb being the heavy chain variable region associated with a second epitope y, and CH1 being the first constant region of the heavy chain. In some embodiments, the first antigenic epitope x and the second antigenic epitope y are epitopes on different antigens, first antigen a and second antigen b, respectively. In some embodiments, the first antigenic epitope x and the second antigenic epitope y are different epitopes on the same antigen a (or antigen b).

In some embodiments, the antibody or antigen-binding fragment binds to two different antigens, a first antigen a and a second antigen b, and the antibody or antigen-binding fragment comprises at least 2 polypeptide chains; wherein the first polypeptide chain comprises, in order from the amino terminus, VHa, CLa, VHb, and CH1, VHa being the heavy chain variable region associated with a first antigen a, CLa being the first light chain constant region, VHb being the heavy chain variable region associated with a second antigen b, and CH1 being the first constant region of the heavy chain.

In some embodiments, VHa is covalently linked to CLa via linker L1, L1 comprising 2 to 6 amino acids; and/or

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

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

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

In some embodiments, the second polypeptide chain comprises, in order from the amino terminus, VLa, CH1, VLb, and CLb; wherein VLa is a light chain variable region bound to a first antigen a, VLb is a light chain variable region bound to a second antigen b, and CLb is a second light chain constant region.

In some embodiments, VLa is covalently linked to CH1 through linker L3, L3 comprising 2 to 6 amino acids; and/or

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

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

In some embodiments, one of the 2 polypeptide chains further comprises an Fc comprising the hinge region, the second constant region, and the third constant region of the heavy chain. In some embodiments, the Fc is a variant Fc region. In some embodiments, the variant Fc region has one or more amino acid modifications, such as 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 parent Fc region activity. In some embodiments, a variant Fc region may have altered (i.e., 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 modification may alter the affinity of the variant Fc region for an fcyr (fey receptor) relative to a parent Fc region.

In some embodiments, the first polypeptide chain comprises an Fc.

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

In some embodiments, the CLa of the first polypeptide chain is disulfide-linked to the CH1 of the second polypeptide chain, and the CH1 of the first polypeptide chain is disulfide-linked to the CLb of the second polypeptide chain.

In some embodiments, the antigen a, b is a cytokine, cytokine receptor, chemokine receptor, or cell surface protein. In some embodiments, the antibody or antigen binding fragment is capable of specifically binding to a cytokine. In some embodiments, the cytokines include IL-1 α (interleukin IL-1 α), IL-1 β (interleukin IL-1 β), IL-13 (interleukin IL-13), IL-5 (interleukin IL-5), TNF- α (tumor necrosis factor α), TNF- β, and (tumor necrosis factor β), among others. In some embodiments, the antibody or antigen binding fragment is capable of specifically binding to an immune checkpoint protein. In some embodiments, the immune checkpoint proteins include TIM-3 (T cell immunoglobulin domain and mucin domain-3), LAG-3 (lymphocyte activation gene-3 molecule), CTLA-4 (cytotoxic T lymphocyte-associated antigen), TIGIT (T cell Ig and ITIM domain), CD27 (cluster of differentiation 27), OX40 (tumor necrosis factor receptor superfamily member 4), ICOS (lymphocyte suppressor), BTLA (B and T lymphocyte attenuating factor), PD-1 (programmed death receptor 1), and CD137 (cluster of differentiation 137), among others. In some embodiments, the antibody or antigen binding fragment specifically binds to a cell surface protein, such as tumor cell surface protein PD-L1 (programmed death ligand 1), galectin 9, CD48 (cluster of differentiation 48), CD40 (cluster of differentiation 40), CD70 (cluster of differentiation 70), B7H3 (CD 276, cluster of differentiation 276), and HVEM (hermovirus Entry media), among others. In some embodiments, the antibody or antigen binding fragment binds to a chemokine or chemokine receptor, such as CCL1, CCL3, CCL5, CCL7, CCL8, and the like, in the CC chemokine subgroup.

In some embodiments, the 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 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, CD 71) and CD20 (cluster of differentiation 20), PD-L1 and 4-1BB (CD 137, a member of the tumor necrosis factor receptor superfamily), PSMA (prostate specific membrane antigen) and CD28 (co-stimulatory molecule), PD-1 and PD-L1, HER2 (human epidermal growth factor receptor 2) and 4-1BB, PD-1 and TIM-3, PD-1 and CD47 (cluster 47), GITR (glucocorticoid-induced tumor necrosis factor receptor) and CTLA-4, CD40 (cluster 40, tumor necrosis factor receptor superfamily member 5) and 4-1BB, OX40 and 4-1BB, LAG-3 and TIM-3, EGFR (epidermal growth factor receptor 1) and CTLA-4, CD19 (cluster 19) and CD22 (cluster 22), CD16 (cluster 16) and CD30 (cluster 30), CD3 (cluster 3) and CD123 (cluster 123), BCMA (B cell maturation antigen) and CD47, MSLN (mesothelin) and CD47, EGFR and cMET, CD73 and TGF beta (transforming growth factor beta), EGFR and TGF beta, CCR2 (CC chemokine receptor 2) and CSF1R (colony stimulating factor 1 receptor), CD20 and CD3, CD19 and CD47, CDH17 (liver intestine cadherin) and TRAILR2 (TRAIL receptor 2, TRAIL is a tumor necrosis-associated apoptosis-inducing ligand), APLP2 (amyloid-like peptide precursor protein 2) and HER2, IL-1 alpha and IL-1 beta, 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 (kllotho a protein). In some embodiments, the antibody or antigen-binding fragment is capable of specifically binding to both antigen a and antigen b.

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

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

the VHa comprises a heavy chain CDR or heavy chain variable region disclosed in US20190100591A1 or US20180169239 A1;

said VHb comprises the heavy chain CDRs or heavy chain variable regions disclosed in CN1404876A or US 9963508; and/or

The VLa contains a light chain CDR or light chain variable region disclosed in US20190100591A1 or US20180169239 A1; and/or

The VLb comprises light chain CDRs or light chain variable regions as disclosed in CN1404876A or US 9963508.

US20190100591A1, US20180169239A1, CN1404876A, US9963508 are incorporated herein by reference in their entirety.

the VHa comprises amino acids 30-35 (VHaCDR 1, SSYGMS) and/or amino acids 50-66 (VHaCDR 2, TINSNGGSTYPDSVKG) and/or amino acids 99-108 (VHaCDR 3, LGTGTLGFAY) in the sequence shown in SEQ ID NO. 1; and/or

The VHb comprises 31 th to 35 th amino acids (VHbCDR 1, SYTMH) and/or 50 th to 66 th amino acids (VHbCDR 2, FISYDGNYYADSVKG) and/or 99 th to 107 th amino acids (VHbCDR 3, TGWLGPFDY) in a sequence shown in SEQ ID NO. 2; and/or

The VLa contains amino acids 24-34 (VLaCDR 1, KASQDVKTAVS) and/or amino acids 50-56 (VLaCDR 2, WASTRAT) and/or amino acids 89-97 (VLaCDR 3, QQHYSTPWT) in the sequence shown in SEQ ID NO. 3; and/or

The VLb contains amino acids 24 to 35 (VLbCDR 1, RASQSVGSSYLA) and/or amino acids 51 to 57 (VLbCDR 2, GAFSRAT) and/or amino acids 90 to 98 (VLbCDR 3, QQYGSSPWT) in the sequence shown in SEQ ID NO: 4.

the VHa comprises a sequence shown in SEQ ID NO. 1, a sequence with at least 80 percent of identity with the sequence shown in SEQ ID NO. 1, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 1; and/or

The VHb comprises a sequence shown as SEQ ID NO. 2, a sequence with at least 80% of identity with the sequence shown as SEQ ID NO. 2, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown as SEQ ID NO. 2; and/or

The VLa comprises a sequence shown as SEQ ID NO. 3, a sequence with at least 80% identity with the sequence shown as SEQ ID NO. 3, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown as SEQ ID NO. 3; and/or

The VLb comprises a sequence shown in SEQ ID NO. 4, a sequence having at least 80% identity with the sequence shown in SEQ ID NO. 4, or an amino acid sequence having one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 4;

the CLa contains a sequence shown by SEQ ID NO. 5, a sequence with at least 80% of identity with the sequence shown by SEQ ID NO. 5, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown by SEQ ID NO. 5;

the CLb comprises a sequence shown by SEQ ID NO. 6, a sequence with at least 80% of identity with the sequence shown by SEQ ID NO. 6, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown by SEQ ID NO. 6;

the CH1 comprises a sequence shown in SEQ ID NO. 7, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 7, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 7.

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

said VHa comprises the heavy chain CDRs or heavy chain variable regions disclosed in CN101331150A1 or US20150307617 A1;

Said VLa comprises the light chain CDRs or light chain variable regions disclosed in CN101331150A1 or US20150307617 A1; and/or

The VLb comprises the light chain CDRs or light chain variable regions disclosed in CN1404876A or US 99635.

CN101331150A1, US20150307617A1, CN1404876A, US9963508 are incorporated herein by reference in their entirety.

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

the VHa contains 31 th to 35 th amino acids (VHaCDR 1, SYGMH) and/or 50 th to 66 th amino acids (VHaCDR 2, VIAEVGSNQYYADSVKG) and/or 99 th to 111 th amino acids (VHaCDR 3, DNQDTSPDVGIDY) in a sequence shown in SEQ ID NO. 8; and/or

The VHb comprises 31 th to 35 th amino acids (VHbCDR 1, SYTMH) and/or 50 th to 66 th amino acids (VHbCDR 2, FISYDGNYYADSVKG) and/or 99 th to 107 th amino acids (VHbCDR 3, TGWLGPFDY) in a sequence shown as SEQ ID NO. 9; and/or

The VLa comprises amino acids 24-34 (VLaCDR 1, RASQNISPFLN) and/or amino acids 50-56 (VLaCDR 2, AAVGLQS) and/or amino acids 89-97 (VLaCDR 3, QQYTDYPLLT) in the sequence shown in SEQ ID NO. 10; and/or

The VLb contains amino acids 24 to 35 (VLbCDR 1, RASQSVGSSYLA) and/or amino acids 51 to 57 (VLbCDR 2, GAFSRAT) and/or amino acids 90 to 98 (VLbCDR 3, QQYGSSPWT) in the sequence shown in SEQ ID NO: 11.

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

the VHa comprises a sequence shown in SEQ ID NO. 8, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 8, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 8; and/or

The VHb comprises a sequence shown in SEQ ID NO. 9, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 9, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 9; and/or

The VLa comprises a sequence shown as SEQ ID NO. 10, a sequence with at least 80% identity to the sequence shown as SEQ ID NO. 10, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown as SEQ ID NO. 10; and/or

The VLb comprises a sequence shown as SEQ ID NO. 11, a sequence having at least 80% identity with the sequence shown as SEQ ID NO. 11, or an amino acid sequence having one or more conservative amino acid substitutions compared with the sequence shown as SEQ ID NO. 11;

the CLa contains a sequence shown in SEQ ID NO. 12, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 12, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 12;

the CLb comprises a sequence shown in SEQ ID NO. 13, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 13, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 13;

the CH1 comprises a sequence shown in SEQ ID NO. 14, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 14, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 14.

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

the VHb comprises a heavy chain CDR or heavy chain variable region disclosed in US20190100591A1 or US20180169239 A1; and/or

The VLb comprises the light chain CDRs or light chain variable regions disclosed in US20190100591A1 or US20180169239 A1.

the VHa contains 31 th to 35 th amino acids (VHaCDR 1, SYGMH) and/or 50 th to 66 th amino acids (VHaCDR 2, VIAEVGSNQYYADSVKG) and/or 99 th to 111 th amino acids (VHaCDR 3, DNQDTSPDVGIDY) in a sequence shown as SEQ ID NO. 15; and/or

The VHb comprises amino acids 30-35 (VHbCDR 1, SSYGMS), and/or amino acids 50-66 (VHbCDR 2, TINSNGGSTYPDSVKG), and/or amino acids 99-108 (VHbCDR 3, LGTGTLGFAY) in the sequence shown in SEQ ID NO. 16; and/or

The VLa comprises amino acids 24-34 (VLaCDR 1, RASQNISPFLN) and/or amino acids 50-56 (VLaCDR 2, AAVGLQS) and/or amino acids 89-97 (VLaCDR 3, QQYTDYPLLT) in the sequence shown in SEQ ID NO. 17; and/or

The VLb contains amino acids 24-34 (VLbCDR 1, KASQDVKTAVS) and/or amino acids 50-56 (VLbCDR 2, WASTRAT) and/or amino acids 89-97 (VLbCDR 3, QQHYSTPWT) in the sequence shown in SEQ ID NO: 18.

the VHa comprises a sequence shown in SEQ ID NO. 15, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 15, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 15; and/or

The VHb comprises a sequence shown as SEQ ID NO. 16, a sequence with at least 80% of identity with the sequence shown as SEQ ID NO. 16, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown as SEQ ID NO. 16; and/or

The VLa comprises a sequence shown in SEQ ID NO. 17, a sequence having at least 80% identity with the sequence shown in SEQ ID NO. 17, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO. 17; and/or

The VLb comprises a sequence shown in SEQ ID NO. 18, a sequence having at least 80% identity to the sequence shown in SEQ ID NO. 18, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO. 18;

the CLa contains a sequence shown in SEQ ID NO. 19, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 19, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 19;

the CLb comprises a sequence shown in SEQ ID NO. 20, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 20, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 20;

the CH1 comprises a sequence shown in SEQ ID NO. 21, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 21, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 21.

In some embodiments, a sequence that is at least 80% identical is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or a range between any two of these values (including endpoints), or any value therein. In some embodiments, the one or more conservative amino acid substitutions 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 between any two of these values (including endpoints), or any value therein.

In some embodiments, the L1 comprises the sequence shown in SEQ ID NO. 22;

the L2 comprises a sequence selected from the group consisting of the sequence shown in any one of SEQ ID NOs 23-27, a sequence having at least 90% identity to the sequence shown in any one of SEQ ID NOs 23-27, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in any one of SEQ ID NOs 23-27; and/or

The L3 comprises a sequence shown as SEQ ID NO. 28; and/or

The L4 comprises a sequence selected from any one of SEQ ID NOs 29-33, a sequence having at least 90% identity to a sequence set forth in any one of SEQ ID NOs 29-33, or an amino acid sequence having one or more conservative amino acid substitutions as compared to a sequence set forth in any one of SEQ ID NOs 29-33.

In some embodiments, the L2 comprises the sequence set forth in SEQ ID No. 23, a sequence having at least 90% identity to the sequence set forth in SEQ ID No. 23, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID No. 23; the L4 comprises a sequence shown in SEQ ID NO. 29, a sequence with at least 90% of identity with the sequence shown in SEQ ID NO. 29, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 29.

In some embodiments, the L2 comprises the sequence set forth in SEQ ID No. 24, a sequence having at least 90% identity to the sequence set forth in SEQ ID No. 24, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID No. 24; the L4 contains a sequence shown in SEQ ID NO. 30, a sequence with at least 90% of identity with the sequence shown in SEQ ID NO. 30, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 30.

In some embodiments, the L2 comprises the sequence set forth in SEQ ID No. 25, a sequence having at least 90% identity to the sequence set forth in SEQ ID No. 25, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID No. 25; the L4 comprises a sequence shown in SEQ ID NO. 31, a sequence with at least 90% of identity with 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.

In some embodiments, the L2 comprises the sequence set forth in SEQ ID No. 26, a sequence having at least 90% identity to the sequence set forth in SEQ ID No. 26, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID No. 26; the L4 contains a sequence shown in SEQ ID NO. 32, a sequence with at least 90% of identity with the sequence shown in SEQ ID NO. 32, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 32.

In some embodiments, the L2 comprises the sequence set forth in SEQ ID No. 27, a sequence having at least 90% identity to the sequence set forth in SEQ ID No. 27, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID No. 27; the L4 contains a sequence shown in SEQ ID NO. 33, a sequence with at least 90% of identity with the sequence shown in SEQ ID NO. 33, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 33.

In some embodiments, a sequence that is at least 90% identical is about 90% identical, about 91% identical, about 92% identical, about 93% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, or a range between any two of these values (including endpoints) or any value therein. In some embodiments, the one or more conservative amino acid substitutions are about 1, about 2, about 3, about 4, or about 5 conservative amino acid substitutions.

In some embodiments, the L1 comprises the sequence set forth in SEQ ID NO. 22, the L2 comprises the sequence set forth in SEQ ID NO. 23, the L3 comprises a sequence selected from the group consisting of the sequence set forth in SEQ ID NO. 28, and the L4 comprises the sequence set forth in SEQ ID NO. 29.

In some embodiments, the L1 comprises the sequence set forth in SEQ ID NO. 22, the L2 comprises the sequence set forth in SEQ ID NO. 24, the L3 comprises the sequence set forth in SEQ ID NO. 28, and the L4 comprises the sequence set forth in SEQ ID NO. 30.

In some embodiments, L1 comprises the sequence set forth in SEQ ID NO. 22, L2 comprises the sequence set forth in SEQ ID NO. 25, L3 comprises the sequence set forth in SEQ ID NO. 28, and L4 comprises the sequence set forth in SEQ ID NO. 31.

In some embodiments, the L1 comprises the sequence set forth in SEQ ID NO. 22, the L2 comprises the sequence set forth in SEQ ID NO. 26, the L3 comprises the sequence set forth in SEQ ID NO. 28, and the L4 comprises the sequence set forth in SEQ ID NO. 32.

In some embodiments, L1 comprises the sequence set forth in SEQ ID NO. 22, L2 comprises the sequence set forth in SEQ ID NO. 27, L3 comprises the sequence set forth in SEQ ID NO. 28, and L4 comprises the sequence set forth in SEQ ID NO. 33.

In some embodiments, the Fc comprises a sequence set forth in any one of SEQ ID NOs 34-36, a sequence having at least 80% identity to a sequence set forth in any one of SEQ ID NOs 34-36, or an amino acid sequence having one or more conservative amino acid substitutions as compared to a sequence set forth in any one of SEQ ID NOs 34-36. In some embodiments, a sequence of at least 80% identity is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or a range between any two of these values (including endpoints) 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 a range between any two of these values (including endpoints), or any value therein.

In some embodiments, the Fc comprises the sequence set forth in SEQ ID NO 34. In some embodiments, the Fc comprises the sequence set forth in SEQ ID NO 35. In some embodiments, the Fc comprises the sequence set forth in SEQ ID NO 36.

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

1 (VHaCDR 1, SSYGMS), and/or 50-66 (VHaCDR 2, TINSNGGSTYPDSVKG), and/or 99-108 (VHaCDR 3, LGTGTLGFAY); and

amino acids 31 to 35 (VHbCDR 1, SYTMH), and/or amino acids 50 to 66 (VHbCDR 2, FISYDGNKYYADSVKG), and/or amino acids 99 to 107 (VHbCDR 3, TGWLGPFDY) in the sequence shown in SEQ ID NO. 2; and/or

3 (VLaCDR 1, KASQDVKTAVS), and/or 50-56 (VLaCDR 2, WASTRAT), and/or 89-97 (VLaCDR 3, QQHYSTPWT); and

amino acids 24 to 35 (VLbCDR 1, RASQSVGSSYLA), and/or amino acids 51 to 57 (VLbCDR 2, GAFSRAT), and/or amino acids 90 to 98 (VLbCDR 3, QQYGSSPWT) of the sequence shown in SEQ ID NO: 4.

In some embodiments, the antibody or antigen-binding fragment comprises at least a first polypeptide chain and a second polypeptide chain 2 polypeptide chains;

the first polypeptide chain comprises amino acids 30-35 (VHaCDR 1, SSYGMS), and/or amino acids 50-66 (VHaCDR 2, TINSNGGSTYPDSVKG), and/or amino acids 99-108 (VHaCDR 3, LGTGTLGFAY) in the sequence shown in SEQ ID NO. 1; and

The second polypeptide chain comprises amino acids 24-34 (VLaCDR 1, KASQDVKTAVS), and/or amino acids 50-56 (VLaCDR 2, WASTRAT), and/or amino acids 89-97 (VLaCDR 3, QQHYSTPWT) of the sequence shown in SEQ ID NO. 3; and

The invention also provides an antibody or antigen-binding fragment, which binds to two different antigens, a first antigen a and a second antigen b, wherein the first antigen a is TIGIT and the second antigen b is CTLA-4; the antibody or antigen-binding fragment comprises at least a first polypeptide chain and a second polypeptide chain of 2 polypeptide chains; said first polypeptide chain comprises the sequence shown in SEQ ID NO 37, a sequence having at least 80% identity to the sequence shown in SEQ ID NO 37, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO 37; said second polypeptide chain comprises the sequence shown in SEQ ID NO. 38, a sequence having at least 80% identity to the sequence shown in SEQ ID NO. 38, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO. 38; or

Said first polypeptide chain comprises a sequence set forth in SEQ ID NO 39, a sequence having at least 80% identity to a sequence set forth in SEQ ID NO 39, or an amino acid sequence having one or more conservative amino acid substitutions as compared to a sequence set forth in SEQ ID NO 39; said second polypeptide chain comprises the sequence shown in SEQ ID NO. 40, a sequence having at least 80% identity to the sequence shown in SEQ ID NO. 40, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO. 40; or

Said first polypeptide chain comprises a sequence set forth in SEQ ID NO 41, a sequence having at least 80% identity to a sequence set forth in SEQ ID NO 41, or an amino acid sequence having one or more conservative amino acid substitutions as compared to a sequence set forth in SEQ ID NO 41; said second polypeptide chain comprises the sequence set forth in SEQ ID NO 42, a sequence having at least 80% identity to the sequence set forth in SEQ ID NO 42, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence set forth in SEQ ID NO 42; or

Said first polypeptide chain comprises the sequence of SEQ ID NO 43, a sequence having at least 80% identity to the sequence of SEQ ID NO 43, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence of SEQ ID NO 43; the second polypeptide chain comprises the sequence shown as SEQ ID NO. 44, a sequence having at least 80% identity to the sequence shown as SEQ ID NO. 44, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown as SEQ ID NO. 44.

In some embodiments, a sequence of at least 80% identity is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or a range between any two of these values (including endpoints) or any value therein. In some embodiments, the one or more conservative amino acid substitutions 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 a range between any two of these values (including endpoints) or any value therein.

In some embodiments, 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 CTLA-4, the antibody or antigen-binding fragment comprising at least a first polypeptide chain and a second polypeptide chain of 2 polypeptide chains; the first polypeptide chain comprises the sequence shown in SEQ ID NO 37 and the second polypeptide chain comprises the sequence shown in SEQ ID NO 38.

In some embodiments, 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 CTLA-4, the antibody or antigen-binding fragment comprising at least a first polypeptide chain and a second polypeptide chain of 2 polypeptide chains; the first polypeptide chain comprises a sequence shown as SEQ ID NO. 39, and the second polypeptide chain comprises a sequence shown as SEQ ID NO. 40.

In some embodiments, 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 CTLA-4, the antibody or antigen-binding fragment comprising at least a first polypeptide chain and a second polypeptide chain of 2 polypeptide chains; the first polypeptide chain comprises the sequence shown in SEQ ID NO 41 and the second polypeptide chain comprises the sequence shown in SEQ ID NO 42.

In some embodiments, 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 CTLA-4, the antibody or antigen-binding fragment comprising at least a first polypeptide chain and a second polypeptide chain of 2 polypeptide chains; the first polypeptide chain comprises the sequence shown in SEQ ID NO. 43, and the second polypeptide chain comprises the sequence shown in SEQ ID NO. 44.

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

amino acids 31 to 35 (VHaCDR 1, SYGMH), and/or amino acids 50 to 66 (VHaCDR 2, VIAEVGSNQYYADSVKG), and/or amino acids 99 to 111 (VHaCDR 3, DNQDTSPDVGIDY) of the sequence shown in SEQ ID NO. 8; and

amino acids 31 to 35 (VHbCDR 1, SYTMH), and/or amino acids 50 to 66 (VHbCDR 2, FISYDGNKYYADSVKG), and/or amino acids 99 to 107 (VHbCDR 3, TGWLGPFDY) in the sequence shown in SEQ ID NO. 9; and/or

10 (VLaCDR 1, RASQNISPFLN), and/or 50-56 (VLaCDR 2, AAVGLQS), and/or 89-97 (VLaCDR 3, QQYTDYPLT); and

amino acids 24 to 35 (VLbCDR 1, RASQSVGSSYLA), and/or amino acids 51 to 57 (VLbCDR 2, GAFSRAT), and/or amino acids 90 to 98 (VLbCDR 3, QQYGSSPWT) of the sequence shown in SEQ ID NO: 11.

the first polypeptide chain comprises amino acids 31-35 (VHaCDR 1, SYGMH), and/or amino acids 50-66 (VHaCDR 2, VIAEVGSNQYYADSVKG), and/or amino acids 99-111 (VHaCDR 3, DNQDTSPDVGIDY) in the sequence shown in SEQ ID NO. 8; and

The second polypeptide chain comprises amino acids 24 to 34 (VLaCDR 1, RASQNISPFLN) and/or amino acids 50 to 56 (VLaCDR 2, AAVGLQS) and/or amino acids 89 to 97 (VLaCDR 3, QQYTDYPLLT) of the sequence indicated by SEQ ID No. 10; and

The 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 OX40 and the second antigen b is CTLA-4; the antibody or antigen-binding fragment comprises at least a first polypeptide chain and a second polypeptide chain of 2 polypeptide chains; said first polypeptide chain comprises the sequence shown in SEQ ID NO. 45, a sequence having at least 80% identity to the sequence shown in SEQ ID NO. 45, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO. 45; the second polypeptide chain comprises the sequence shown as SEQ ID NO. 46, a sequence having at least 80% identity to the sequence shown as SEQ ID NO. 46, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown as SEQ ID NO. 46. In some embodiments, a sequence that is at least 80% identical is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or a range between any two of these values (including endpoints), or any value therein. In some embodiments, the one or more conservative amino acid substitutions 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 a range between any two of these values (including endpoints) or any value therein.

In some embodiments, 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 CTLA-4, the antibody or antigen-binding fragment comprising at least a first polypeptide chain and a second polypeptide chain of 2 polypeptide chains; the first polypeptide chain comprises the sequence shown in SEQ ID NO. 45, and the second polypeptide chain comprises the sequence shown in SEQ ID NO. 46.

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

amino acids 31 to 35 (VHaCDR 1, SYGMH), and/or amino acids 50 to 66 (VHaCDR 2, VIAEVGSNQYYADSVKG), and/or amino acids 99 to 111 (VHaCDR 3, DNQDTSPDVGIDY) of the sequence shown in SEQ ID NO. 15; and

amino acids 30 to 35 (VHbCDR 1, SSYGMS), and/or amino acids 50 to 66 (VHbCDR 2, TINSNGGSTYPDSVKG), and/or amino acids 99 to 108 (VHbCDR 3, LGTGTLGFAY) in the sequence shown in SEQ ID NO. 16; and/or

Amino acids 24 to 34 (VLaCDR 1, RASQNISPFLN), and/or amino acids 50 to 56 (VLaCDR 2, AAVGLQS), and/or amino acids 89 to 97 (VLaCDR 3, QQYTDYPLLT) of the sequence indicated by SEQ ID NO. 17; and

amino acids 24 to 34 (VLbCDR 1, KASQDVKTAVS), and/or amino acids 50 to 56 (VLbCDR 2, WASTRAT), and/or amino acids 89 to 97 (VLbCDR 3, QQHYSTPWT) in the sequence shown in SEQ ID NO: 18.

the first polypeptide chain comprises 31 th to 35 th amino acids (VHaCDR 1, SYGMH) and/or 50 th to 66 th amino acids (VHaCDR 2, VIAEVGSNQYYADSVKG) and/or 99 th to 111 th amino acids (VHaCDR 3, DNQDTSPDVGIDY) in a sequence shown as SEQ ID NO. 15; and

The second polypeptide chain comprises amino acids 24 to 34 (VLaCDR 1, RASQNISPFLN) and/or amino acids 50 to 56 (VLaCDR 2, AAVGLQS) and/or amino acids 89 to 97 (VLaCDR 3, QQYTDYPLLT) of the sequence shown in SEQ ID NO. 17; and

The 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 OX40 and the second antigen b is TIGIT; the antibody or antigen-binding fragment comprises at least a first polypeptide chain and a second polypeptide chain of 2 polypeptide chains; said first polypeptide chain comprises a sequence set forth in SEQ ID NO 47 or 49, a sequence having at least 80% identity to a sequence set forth in SEQ ID NO 47 or 49, or an amino acid sequence having one or more conservative amino acid substitutions as compared to a sequence set forth in SEQ ID NO 47 or 49; the second polypeptide chain comprises a sequence set forth in SEQ ID NO 48 or 50, a sequence having at least 80% identity to a sequence set forth in SEQ ID NO 48 or 50, or an amino acid sequence having one or more conservative amino acid substitutions as compared to a sequence set forth in SEQ ID NO 48 or 50. In some embodiments, a sequence of at least 80% identity is about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or a range between any two of these values (including endpoints) or any value therein. In some embodiments, the one or more conservative amino acid substitutions 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 a range between any two of these values (including endpoints) or any value therein.

In some embodiments, 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 comprising at least a first polypeptide chain and a second polypeptide chain 2 polypeptide chains; the first polypeptide chain comprises the sequence shown in SEQ ID NO 47 and the second polypeptide chain comprises the sequence shown in SEQ ID NO 48.

In some embodiments, 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 comprising at least a first polypeptide chain and a second polypeptide chain 2 polypeptide chains; the first polypeptide chain comprises a sequence shown as SEQ ID NO. 49, and the second polypeptide chain comprises a sequence shown as SEQ ID NO. 50.

In some embodiments, the antibody or antigen-binding fragment comprises a first polypeptide chain and a third polypeptide chain that are identical in sequence and a second polypeptide chain and a fourth polypeptide chain that are identical in sequence. In some embodiments, the antibody or antigen-binding fragment comprises a first polypeptide chain and a third polypeptide chain of identical sequence whose Fc regions pair to form a disulfide bond, and a second polypeptide chain and a fourth polypeptide chain of identical sequence.

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

The invention also provides nucleic acid molecules encoding the antibodies or antigen-binding fragments. In some embodiments, the nucleic acid molecule is an isolated nucleic acid molecule.

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

The 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 cell is a CHO cell, 293 cell, cos1 cell, cos7 cell, CV1 cell, and murine L cell.

The invention also provides a pharmaceutical composition, which comprises the antibody or antigen-binding fragment and pharmaceutically acceptable auxiliary materials.

The invention also provides methods of treatment and uses. In some embodiments, methods are provided for treating or ameliorating various diseases (such as 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, there is provided a use of the antibody or antigen-binding fragment in a medicament for treating or ameliorating various diseases (e.g., inflammatory diseases, autoimmune diseases, cancer, or spinal cord injury). In some embodiments, there is provided the use of the antibody or antigen-binding fragment in the manufacture of a medicament for treating or ameliorating various diseases (such as inflammatory diseases, autoimmune diseases, cancer, or spinal cord injury).

In some embodiments, the autoimmune or inflammatory disease is selected from the group consisting of: crohn's disease, psoriasis (including plaque psoriasis), arthritis (including rheumatoid arthritis, psoriatic arthritis, osteoarthritis or juvenile idiopathic arthritis), multiple sclerosis, ankylosing spondylitis, ankylosing arthropathy (spondyllothing arthopathiy), systemic lupus erythematosus, uveitis, sepsis, neurodegenerative diseases, neuronal regeneration, spinal cord injury, primary and metastatic cancers, respiratory disorders, asthma, allergic and non-allergic asthma, asthma caused by infection with Respiratory Syncytial Virus (RSV), chronic Obstructive Pulmonary Disease (COPD), conditions involving airway inflammation, hypereosinophilia, fibrosis and excessive mucus production, cystic fibrosis, pulmonary fibrosis, atopic conditions, atopic dermatitis, urticaria, eczema, allergic rhinitis, irritable bowel gastritis, inflammatory and/or autoimmune skin conditions, inflammatory and/or autoimmune organ conditions, inflammatory Bowel Disease (IBD), ulcerative colitis, inflammatory liver disease and/or liver fibrosis, hepatitis b and/or scleroderma caused by hepatic fibrosis and/or liver fibrosis. In some embodiments, the cancer is selected from the group consisting of: hepatocellular carcinoma, glioblastoma, lymphoma, or hodgkin's lymphoma. In some embodiments, the cancer is selected from the group consisting of: melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), pancreatic cancer, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic 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, mature B-cell neoplasm, B-cell chronic lymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, mantle cell lymphoma, follicular lymphoma, cutaneous follicular central lymphoma, marginal zone B-cell lymphoma, hairy cell leukemia, diffuse large B-cell lymphoma, burkitt's lymphoma (Burkitt's lymphoma), plasmacytoma, post-transplant lymphoproliferative disorder, migratory Waldenstrom's macroglobulinemia (Waldenstrom's macroglobulinemia), and anaplastic large cell lymphoma.

The invention also provides diagnostic methods and uses. In some embodiments, a method for detecting the expression of antigen a and/or antigen b in a sample is provided, the sample is contacted with the antibody or antigen-binding fragment, such that the antibody or antigen-binding fragment binds to antigen a and/or antigen b, and the binding thereof, i.e., the amount of antigen a and/or antigen b in the sample, is detected. In some embodiments, the antigen a, b is a cytokine, cytokine receptor, chemokine receptor, or cell surface protein. In some embodiments, the 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, 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, MSMA and CD47, EGFR and cMET, CD73 and CD beta, EGFR and TGF beta, CCR2 and CSF1R, CD20 and CD3, CD19 and CD47, FGMA and TRAH and CD47, FGLN and CD2, FGLN and CD47, EGFR and TIL-1, and TFIL-13, and TFIL-1, and CD13, and TFIL-1, and 13, and TFIL-1, and 13.

In some embodiments, there is provided a use of the antibody or antigen binding fragment in the preparation of a kit for diagnosing an inflammatory disease, an autoimmune disease, a neurodegenerative disease, cancer, or spinal cord injury. In some embodiments, a diagnostic kit comprising the antibody or antigen-binding fragment is provided.

The invention provides multivalent and multispecific antibodies or antigen-binding fragments that can bind to two or more antigens, or two or more epitopes of the same antigen, and uses thereof. The antibodies or antigen-binding fragments of the invention are useful for treating or ameliorating various diseases, such as inflammatory diseases, autoimmune diseases, cancer or spinal cord injury, and also for diagnosis and prognosis of related diseases.

Drawings

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

FIG. 2 is an SDS-PAGE pattern of antibody 1 in an example of the present invention; wherein, lane 1 shows that the antibody 1 is in a non-reducing state, lane M shows a maker, and lane 2 shows that the antibody 1 is in a reducing state.

FIG. 3 is an SDS-PAGE pattern of antibody 2 and antibody 3 in the examples of the present invention; in the figure, CT-20BiAb represents antibody 2, CT-25BiAb represents antibody 3, lane 1 represents antibody 3 in a non-reducing state, lane 2 represents antibody 2 in a non-reducing state, lane 3 represents antibody 3 in a reducing state, lane 4 represents antibody 2 in a reducing state, and M represents maker.

FIG. 4 shows the binding activity of antibody 1, antibody 2, antibody 3 to TIGIT-Fc; wherein CT BiAb means antibody 1, CT-20BiAb means antibody 2, and CT-25BiAb means antibody 3.

FIG. 5 shows the binding activity of antibody 1, antibody 2, antibody 3 to CTLA-4-Fc; wherein CT BiAb means antibody 1, CT-20BiAb means antibody 2, and CT-25BiAb means antibody 3.

FIG. 6 shows the biological activity of antibody detected by Promega's anti-CTLA-4 reporter gene detection system; wherein CTLA-4/TIGIT BiAb represents antibody 1.

FIG. 7 shows the biological activity of antibody detected by Promega's anti-CTLA-4 reporter gene detection system; wherein CT-20BiAb represents antibody 2.

FIG. 8 shows the detection of the biological activity of an antibody using the anti-TIGIT reporter gene detection system; wherein CT BiAb represents antibody 1.

FIG. 9 shows the detection of the biological activity of an antibody using the anti-TIGIT reporter gene detection system; wherein CT-20BiAb represents antibody 2.

FIG. 10 shows the detection of the biological activity of an antibody using the anti-TIGIT reporter assay system; wherein CT-25BiAb represents antibody 3.

FIG. 11 is an SDS-PAGE pattern of antibody 5 (CTLA-4-OX 40 bispecific antibody) in examples of the present invention; here, lane represents a lane, and lane 1, lane 2, and lane 3 are arranged from left to right in this order.

FIG. 12 shows antibodies 5 and OX40 in accordance with examples of the invention ⁺ A binding curve of the cell; OX40 ⁺ The cell is an OX 40-expressing cell; the abscissa represents the log of antibody concentration and the ordinate represents the 2-channel mean fluorescence value read on a flow cytometer; wherein O4 represents antibody 5.

Figure 13 is a graph of a fitted affinity kinetics curve for binding of antibody 5 to OX40 antigen and CTLA-4 antigen in an example of the invention, where O4 represents antibody 5.

FIG. 14 is an SDS-PAGE profile of antibodies 6 and 7 in an example of the invention; wherein lane M represents marker, lane 1 represents antibody 6 in a non-reducing state, lane 2 represents antibody 6 in a reducing state, lane 3 represents antibody 7 in a non-reducing state, and lane 4 represents antibody 7 in a reducing state; the OT-4D-13aa antibody represents antibody 6, and the OT-4D-30aa antibody represents antibody 7.

FIG. 15 is a curve fitted to the affinity kinetics of antibody 7 binding to OX40 antigen and TIGIT antigen in examples of the invention.

FIG. 16 is a graph of the binding of antibody 7 to Jurkat-OX40 cells in an example of the invention; wherein anti-OX40Ab is expressed as OX40 monoclonal antibody (i.e. anti-OX 40), and OT-4D-30a and 4D-30a are both expressed as antibody 7; EC50 in nM is shown.

FIG. 17 is a graph of the binding of antibody 7 to Jurkat TIGIT cells in an example of the invention; wherein anti-Tigit Ab represents TIGIT monoclonal antibody (i.e., anti-TIGIT), and OT-4D-30a represents antibody 7.

Term(s) for

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

Definition of

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

The terms "comprising" or "including" as used herein mean that the compositions and methods and the like include the recited elements, such as components or steps, but not excluding others. "consisting essentially of 8230% \8230means that the compositions and methods exclude other elements that have a fundamental effect on the characteristics of the combination, but do not exclude elements that do not materially affect the composition or method. "consisting of 8230; \8230;" means excluding elements not specifically listed.

The term "polypeptide" is intended to encompass both the singular "polypeptide" and the plural "polypeptide" and refers to a molecule composed of monomers of amino acids linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single chain or multiple chains of two or more amino acids and does not refer to a particular length of the product. Thus, included within the definition of "polypeptide" are peptides, dipeptides, tripeptides, oligopeptides, "proteins," "amino acid chains," or any other term used to refer to two or more amino acid chains, and the term "polypeptide" may be used in place of, or in alternation with, any of the above terms. The term "polypeptide" is also intended to refer to the product of post-expression modification of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modification. The polypeptide may be derived from a natural biological source or produced by recombinant techniques, but it need not be translated from a specified nucleic acid sequence, and it may be produced in any manner, including chemical synthesis.

"amino acid" refers to an organic compound containing both amino and carboxyl groups, such as an alpha-amino acid, which may be encoded by a nucleic acid, either directly or in the form of a precursor. 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 a different codon, which is 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 (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 substitution of one amino acid residue with another amino acid residue having a side chain (R group) of similar chemical nature (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. Examples of classes of amino acids containing chemically similar side chains include: 1) Aliphatic side chain: glycine, alanine, valine, leucine, and isoleucine; 2) Aliphatic hydroxyl side chain: serine and threonine; 3) Amide-containing side chains: asparagine and glutamine; 4) Aromatic side chain: phenylalanine, tyrosine and tryptophan; 5) Basic side chain: lysine, arginine and histidine; 6) Acidic side chain: aspartic acid and glutamic acid.

The term "isolated" as used herein with respect to a cell, nucleic acid, polypeptide, antibody, etc., e.g., "isolated" DNA, RNA, polypeptide, antibody, refers to a molecule that is separated from one or more of the other components, e.g., DNA or RNA, respectively, in the natural environment of the cell. The term "isolated" as used herein also refers to nucleic acids or peptides that are 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. In addition, "isolated nucleic acid" is intended to include nucleic acid fragments that do not occur in nature, and which do not occur in nature. 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 both purified and recombinant polypeptides. Isolated polypeptides, antibodies, and the like are typically prepared by at least one purification step. In some embodiments, an 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 ranges between any two of these values (including endpoints) or any value therein.

The term "recombinant" refers to a polypeptide or polynucleotide, and means a form of a polypeptide or polynucleotide that does not occur in nature, and non-limiting examples may include combinations that produce polynucleotides or polypeptides that do not normally occur.

"homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence that can be aligned. When a position in the compared sequences is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences.

A polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) has a certain percentage (e.g., 90%, 95%, 98%, or 99%) of "identity or sequence identity" with another sequence, meaning that when the sequences are aligned, the percentage of bases (or amino acids) in the two sequences being compared are the same. This alignment and percent identity or sequence identity can be determined using visual inspection or software programs known in the art, such as the software program described in Current Protocols in Molecular Biology, ausubel et al. Preferably, the alignment is performed using default parameters. One alignment program is BLAST using default parameters, such as BLASTN and BLASTP, both using the following default parameters: geneticcode = standard; filter = none; strand = booth; cutoff =60; expect =10; matrix = BLOSUM62; descriptions =50sequences; sortby = HIGHSCORE; databases = non-redundant; genBank + EMBL + DDBJ + PDB + GenBank CDStranslations + SwissProtein + Spupdate + PIR. A biologically equivalent polynucleotide is a polynucleotide having the above specified percentage of identity and encoding a polypeptide having the same or similar biological activity.

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

The terms "polynucleotide" and "oligonucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or analogs thereof. The polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are examples of non-limiting polynucleotides: a gene or gene fragment (e.g., a probe, primer, EST, or SAGE tag), an exon, an intron, a messenger RNA (mRNA), a transfer RNA, ribosomal RNA, ribozyme, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, structural modifications to the nucleotide can be made before or after assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example by conjugation with a labeling component. This term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, embodiments of any polynucleotide of the present disclosure include a double-stranded form and each of two complementary single-stranded forms known or predicted to comprise the double-stranded form.

The term "encoding" as applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide, which polypeptide and/or fragments thereof can be produced by transcription and/or translation, either in its native state or when manipulated by methods well known to those skilled in the art.

"antibody," "antigen-binding fragment," refers to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. The antibody may be a whole antibody and any antigen binding fragment thereof or a single chain thereof. The term "antibody" thus includes any protein or peptide in a molecule that contains at least a portion of an immunoglobulin molecule having biological activity that binds to an antigen. Antibodies and antigen-binding fragments include, but are not limited to, complementarity Determining Regions (CDRs), heavy chain variable regions (VH), light chain variable regions (VL), heavy chain constant regions (CH), light chain constant regions (CL), framework Regions (FR), or any portion thereof of a heavy or light chain or ligand-binding portion thereof, or at least a portion of a binding protein. The CDR regions include the CDR regions of the light chain (VL CDR 1-3) and the CDR regions of the heavy chain (VH CDR 1-3). The antibody or antigen binding fragment of the embodiment of the invention is a bispecific antibody, and the fragment specifically binding to the antigen a and the antigen b is fused: the first polypeptide chain comprises VHa, CLa, VHb, CH1, the first polypeptide chain resembles a heavy chain or a fragment of a heavy chain of an immunoglobulin; the second polypeptide chain comprises the structures VLa, CH1, VLb and CLb, and is analogous to an immunoglobulin light chain.

The term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody, and the constituent form of an antibody fragment of the invention may resemble F (ab') ₂ 、F(ab) ₂ Fab', fab, fv, scFv, etc. Regardless of its structure, an antibody fragment binds to the same antigen that is recognized by an intact antibody. Term(s)"antibody fragments" include 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 particular 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 a short linker peptide of 10 to about 25 amino acids. The linker may be glycine rich to increase flexibility and serine or threonine rich to increase solubility and may link the N-terminus of VH and the C-terminus of VL, or vice versa. Although the protein has the constant region removed and the linker introduced, it retains the specificity of the original immunoglobulin. ScFv molecules are generally known in the art and are described in, for example, U.S. Pat. No. 5,892,019.

The term "antibody" includes a wide variety of polypeptides that can be biochemically distinguished. Those skilled in the art will appreciate that the class of heavy chains includes gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), with some subclasses (e.g., γ 1- γ 4). The nature of this chain determines the "class" of the antibody as IgG, igM, igA, igG or IgE, respectively. The immunoglobulin subclasses (isotypes), e.g., igG1, igG2, igG3, igG4, igG5, etc., have been well characterized and the functional specificity conferred is also known. All immunoglobulin classes are within the scope of the present disclosure. In some embodiments, the immunoglobulin molecule is an IgG class. An IgG typically comprises two identical light chain polypeptides having a molecular weight of about 23,000 daltons and two identical heavy chain polypeptides having a molecular weight of about 53,000-70,000. The four chains are linked by disulfide bonds in a "Y" configuration, in which the light chain begins at the "Y" opening and continues through the variable region surrounding the heavy chain.

The 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 such as Fab-like, and F-like (ab') ₂ Single-chain-like Fvs (scFv).

Light chains can be classified as kappa (. Kappa.) or lambda (. Lamda.). Each heavy chain may be associated with a kappa or lambda light chain. In general, when immunoglobulins are produced by hybridomas, B cells or genetically engineered host cells, the light and heavy chains are joined by covalent bonds and the "tail" portions of the two heavy chains are joined by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the forked end of the Y configuration to the C-terminus of the bottom of each chain. The immunoglobulin kappa light chain variable region is Vkappa; immunoglobulin lambda light chain variable region is V _λ 。

Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used in accordance with function. 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, etc. By convention, the numbering of constant regions increases as they become further away from the antigen binding site or amino terminus of the antibody. The N-terminal part is a variable region and the C-terminal part is a constant region; the CH3 and CL domains actually comprise the carboxy-termini 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 short, non-contiguous amino acid sequences that form the antigen binding domain that specifically bind to an antigen, assuming the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining other amino acids in the antigen binding domain, referred to as the "framework" region, show less intermolecular variability. The framework regions largely adopt a β -sheet conformation with the CDRs forming a loop structure attached to, or in some cases forming part of, the β -sheet structure. Thus, the framework regions allow the CDRs to be positioned in the correct orientation by forming a scaffold via interchain non-covalent interactions. The antigen binding domain with the CDRs at a particular location forms a surface complementary to an epitope on an antigen that facilitates non-covalent binding of an antibody to its antigenic epitope. Amino acids comprising CDRs and framework regions can be identified by known methods by those of ordinary skill in the art for a given heavy or light chain variable region (see Kabat, e., et al, u.s.department of Health and Human Services, sequences of Proteins of Immunological Interest, (1983) and Chothia and leave, j.mol.biol.,196, 901-917 (1987)).

Where two or more definitions are provided for a term used and/or accepted in the art, the definition of the term as used herein includes all such meanings unless explicitly stated to the contrary. One specific example is the use of the term "complementarity determining regions" ("CDRs") to describe 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, U.S. Dept. Of Health and Human Services, sequences of Proteins of Immunological Interest (1983) and Chothia et al, J.mol.biol.196:901-917 (1987), which are incorporated herein by reference in their entirety.

CDRs defined according to Kabat and Chothia include overlaps or subsets of amino acid residues when compared to each other. Nevertheless, 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 number comprising a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can generally determine which specific residues a CDR contains based on the amino acid sequence of the variable region of an antibody.

Kabat et al also defines a numbering system for the variable region sequences applicable to any antibody. One of ordinary skill in the art can apply this "Kabat numbering" system to any variable region sequence without relying on other experimental data beyond the sequence itself. "Kabat numbering" refers to the numbering system proposed by Kabat et al, U.S. Dept. Of Health and Human Services at "Sequence of proteins of Immunological Interest" (1983). Antibodies can also be used with the EU numbering system.

The antibodies disclosed herein may be derived from any animal, including birds and mammals. In some embodiments, the antibody is a human, murine, donkey, rabbit, goat, camel, llama, horse or chicken derived antibody. In another embodiment, the variable region may be of chondrocyclic (condricthoid) origin (e.g. from sharks).

Heavy chainThe constant region includes at least one of a CH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment. The heavy chain constant region of an antibody may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide may comprise a heavy chain constant region derived from an IgG ₁ CH1 Domain of molecules and from IgG ₃ The hinge region of the molecule. In another embodiment, the heavy chain constant region may comprise a portion derived from an IgG ₁ Molecules and moieties derived from IgG ₃ The hinge region of the molecule. In another embodiment, a portion of the heavy chain may comprise a portion derived from IgG ₁ Molecules and moieties derived from IgG ₄ A chimeric hinge region of the molecule.

A "light chain constant region" comprises 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. "light chain-heavy chain pair" refers to a collection of light and heavy chains that can form a dimer 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 (largely amino-terminal) constant region of an immunoglobulin heavy chain. The CH2 domain is 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 well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule, and comprises approximately 108 residues. "hinge region" includes the portion of the heavy chain region that connects the CH1 domain and the CH2 domain. The hinge region comprises about 25 residues and is flexible, thereby enabling independent movement of the two N-terminal antigen-binding regions. The hinge region can be subdivided into three distinct domains: upper, middle and lower hinge domains (rouxetal, j.immunol 161.

"disulfide bond" refers to a covalent bond formed between two sulfur atoms. The thiol group of cysteine may form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by disulfide bonds.

"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 region is from a non-human source (e.g., mouse or primate) and the constant region is from a human source.

"specific binding" or "for" \8230; specific "generally refers to the formation of a relatively stable complex of an antibody or antigen binding fragment and a particular antigen by complementary binding of its antigen binding domain to an epitope. "specificity" can be expressed in terms of the relative affinity of an antibody or antigen-binding fragment for binding to a particular antigen or epitope. For example, an antibody "a" can be considered to have a higher specificity for the same antigen than an antibody "B" if antibody "a" has a greater relative affinity for the antigen than antibody "B". Specific binding can be described by the equilibrium dissociation constant (KD), with a smaller KD implying tighter binding. Methods of determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, biofilm layer optical interferometry, and the like. An antibody that "specifically binds" to antigen a includes an antibody that has an equilibrium dissociation constant KD for antigen a of less than or equal to about 100nM, less than or equal to about 10nM, less than or equal to about 5nM, less than or equal to about 1 nM.

"treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent, slow down, ameliorate, or halt undesirable physiological changes or disorders, such as the progression of a disease, including, but not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration, palliation, alleviation, or abolition (whether partial or total), prolongation of life expectancy in the absence of treatment, and the like, as either detectable or undetectable. Patients in need of treatment include patients already with a condition or disorder, patients susceptible to a condition or disorder, or patients in need of prevention of the condition or disorder, patients who may or are expected to benefit from administration of the antibodies or pharmaceutical compositions disclosed herein for detection, diagnostic procedures, and/or treatment.

"patient" refers to any mammal in need of diagnosis, prognosis or treatment, including humans, dogs, cats, rabbits, mice, horses, cattle, etc.

"about" refers to the conventional error range for corresponding numerical values as would be readily understood by one of ordinary skill in the relevant art. In some embodiments, reference herein to "about" refers to the numerical values recited and ranges of ± 10%, ± 5%, or ± 1% thereof.

“EC ₅₀ "i.e., the half maximum effect concentration (concentration for 50%; of maximum effect ₅₀ ) Refers to a concentration that causes 50% of the maximum effect.

Bispecific antibodies

The present invention provides a novel bispecific antibody or antigen-binding fragment thereof, such as shown in fig. 1, which can modulate the binding activity of the bispecific antibody or antigen-binding fragment thereof to two antigens to which it binds by modulating the positions of and the linkers between the variable region binding to the first antigen and the variable region binding to the second antigen, to enhance the therapeutic effect of the antibody and reduce side effects.

1) Antibodies targeting TIGIT and CTLA-4

The present invention provides bispecific antibodies or antigen-binding fragments thereof having high affinity for TIGIT and CTLA-4 proteins. The antibodies tested exhibit potent binding activity, biological activity, and are useful for therapeutic and diagnostic purposes. For example, these antibodies or antigen-binding fragments effectively block immune checkpoints, activate lymphocytes to release cytokines, and are useful for treating various types of cancer, tumors, or infections, among other related diseases.

Accordingly, one embodiment of the disclosure provides an antibody or antigen-binding fragment that targets TIGIT and CTLA-4 that specifically binds TIGIT and CTLA-4.

In some embodiments, the antibody or antigen-binding fragment comprises at least a first polypeptide chain comprising the sequence set forth in SEQ ID No. 37 and a second polypeptide chain comprising the sequence set forth in SEQ ID No. 38.

In some embodiments, the antibody or antigen-binding fragment comprises at least a first polypeptide chain comprising the sequence of SEQ ID NO 37 except the sequence of the Fc region and a second polypeptide chain comprising the sequence set forth in SEQ ID NO 38.

In some embodiments, the antibody or antigen-binding fragment comprises at least a first polypeptide chain comprising the sequence set forth in SEQ ID No. 39 and a second polypeptide chain comprising the sequence set forth in SEQ ID No. 40, 2 polypeptide chains.

In some embodiments, the antibody or antigen-binding fragment comprises at least a first polypeptide chain comprising the sequence of SEQ ID NO 39 except the sequence of the Fc region and a second polypeptide chain comprising the sequence set forth in SEQ ID NO 40, 2 polypeptide chains.

In some embodiments, the antibody or antigen-binding fragment comprises at least a first polypeptide chain comprising the sequence set forth in SEQ ID No. 41 and a second polypeptide chain comprising the sequence set forth in SEQ ID No. 42, 2 polypeptide chains.

In some embodiments, the antibody or antigen-binding fragment comprises at least a first polypeptide chain comprising the sequence of SEQ ID NO:41 except the sequence of the Fc region and a second polypeptide chain comprising the sequence of SEQ ID NO:42, 2 polypeptide chains.

In some embodiments, the antibody or antigen-binding fragment comprises at least a first polypeptide chain comprising the sequence set forth in SEQ ID No. 43 and a second polypeptide chain comprising the sequence set forth in SEQ ID No. 44, 2 polypeptide chains.

In some embodiments, the antibody or antigen-binding fragment comprises at least a first polypeptide chain comprising a sequence of SEQ ID NO 43 other than the sequence of the Fc region and a second polypeptide chain comprising the sequence set forth in SEQ ID NO 44, 2 polypeptide chains.

2) Antibodies targeting OX40 and CTLA-4

The present invention provides bispecific antibodies or antigen-binding fragments thereof having high affinity for OX40 and CTLA-4 proteins. The antibodies tested exhibit potent binding activity, biological activity, and are useful for therapeutic and diagnostic purposes. For example, these antibodies or antigen-binding fragments are effective in activating T cells and lymphocytes to release cytokines, and are useful in treating various types of cancers, tumors, or infections associated diseases.

Accordingly, one embodiment of the present disclosure provides an antibody or antigen-binding fragment that targets OX40 and CTLA-4, which specifically binds OX40 and CTLA-4.

In some embodiments, the antibody or antigen-binding fragment comprises at least a first polypeptide chain comprising the sequence set forth in SEQ ID No. 45 and a second polypeptide chain comprising the sequence set forth in SEQ ID No. 46, 2 polypeptide chains.

In some embodiments, the antibody or antigen-binding fragment comprises at least a first polypeptide chain comprising the sequence of SEQ ID NO:45 except the sequence of the Fc region and the Fc region of SEQ ID NO:36, and a second polypeptide chain comprising the sequence of SEQ ID NO:46, 2 polypeptide chains.

3) Antibodies targeting OX40 and TIGIT

The present invention provides bispecific antibodies or antigen-binding fragments thereof having high affinity for OX40 and TIGIT proteins. The test antibodies exhibit potent binding activity, biological activity, and are useful for therapeutic and diagnostic purposes. For example, these antibodies or antigen-binding fragments are effective in blocking immune checkpoints, activating T cells, activating lymphocytes to release cytokines, and are useful for treating various types of cancer, tumor, or infection related diseases.

Accordingly, one embodiment of the disclosure provides an antibody or antigen-binding fragment that targets OX40 and TIGIT, which antibody or antigen-binding fragment specifically binds OX40 and TIGIT.

In some embodiments, the antibody or antigen-binding fragment comprises at least a first polypeptide chain comprising the sequence set forth in SEQ ID No. 47 and a second polypeptide chain comprising the sequence set forth in SEQ ID No. 48, 2 polypeptide chains.

In some embodiments, the antibody or antigen-binding fragment comprises at least a first polypeptide chain comprising the sequence of SEQ ID NO 47 except the sequence of the Fc region and a second polypeptide chain comprising the sequence of SEQ ID NO 48, 2 polypeptide chains.

In some embodiments, the antibody or antigen-binding fragment comprises at least a first polypeptide chain comprising the sequence set forth in SEQ ID No. 49 and a second polypeptide chain comprising the sequence set forth in SEQ ID No. 50, 2 polypeptide chains.

In some embodiments, the antibody or antigen-binding fragment comprises at least a first polypeptide chain comprising the sequence of SEQ ID No. 49 except the sequence of the Fc region and a second polypeptide chain comprising the sequence of SEQ ID No. 50, 2 polypeptide chains.

It will also be appreciated by those of ordinary skill in the art that the sequences of the disclosed antibodies or antigen-binding fragments may be substituted with amino acid sequences that differ from the naturally-occurring amino acid sequence of the antibody. For example, the amino acid sequence after substitution can be similar to the starting sequence, such as having a proportion of identity to the starting sequence, such as it can be about 80%, about 85%, about 90%, about 95%, about 98%, or about 99% identity to the starting sequence, or a range between any two of these values (including the end point), or any value therein.

In certain embodiments, an antibody comprises an amino acid sequence having one or more modification groups. For example, bispecific antibodies disclosed herein (bispecific antibodies targeting TIGIT and CTLA-4, bispecific antibodies targeting OX40 and CTLA-4, and bispecific antibodies targeting OX40 and TIGIT) can comprise a flexible linker sequence, or can be modified to add functional groups (e.g., PEG, drugs, toxins, or tags).

The antibodies, antigen binding fragments disclosed herein include modified derivatives, i.e., 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, examples where antibodies can be glycosylated, acetylated, pegylated, phosphorylated, amidated, derivatized by known protecting/blocking groups, proteolytically cleaved, linked to cellular ligands or other proteins, and the like. Any of a number of chemical modifications may be made 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 may be conjugated to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, agent, or PEG.

The antibody can be conjugated or fused to a therapeutic agent, which can include detectable labels (e.g., radioactive labels), immunomodulators, hormones, enzymes, oligonucleotides, photoactive therapeutic agents, diagnostic agents, cytotoxic agents for drugs or toxins, ultrasound enhancing agents, nonradioactive labels, and compositions thereof, and other such agents known in the art.

The antibody may be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antibody is then determined by detecting the luminescence that occurs during the course of the chemical reaction. Examples of chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.

Antibody and method for preparing polynucleotide encoding antibody

Also disclosed are polynucleotides or nucleic acid molecules encoding the antibodies, antigen-binding fragments, and derivatives thereof of the invention. The disclosed polynucleotides may encode a first polypeptide (similar to an immunoglobulin heavy chain or fragment thereof), a second polypeptide (similar to an immunoglobulin light chain), a heavy chain variable region, a light chain variable region, an Fc region, a portion of a heavy chain variable region, or a portion of a light chain variable region. Methods of making antibodies are well known in the art and are described herein. In certain embodiments, the antibodies, antigen-binding fragments, and antibodies disclosed herein 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 to a particular antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce fully human antibodies in response to antigen challenge. Exemplary techniques that can be used to prepare such antibodies are described in U.S. Pat. nos. 6,458,592;6,420,140, the entire contents of which are incorporated herein by reference. The bispecific antibody of the present invention is prepared by fusing the fragments specifically binding to antigen a and antigen b, and some fragments of the bispecific antibody can be obtained by the above-mentioned method for preparing an antibody binding to a single antigen.

In certain embodiments, the antibodies are prepared so as not to elicit an adverse immune response in the animal (e.g., 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, the antibody may be humanized, primatized, deimmunized or a chimeric antibody may be prepared. These types of antibodies are derived from non-human antibodies, typically murine or primate antibodies, which retain or substantially retain the antigen binding properties of the parent antibody but are less immunogenic in humans. This can be achieved by a variety of methods, including (a) grafting the entire variable region of non-human origin to a constant region of human origin to produce a chimeric antibody; (b) Grafting at least a portion of one or more non-human Complementarity Determining Regions (CDRs) into a framework and constant region of human origin, with or without retention of critical framework residues; or (c) transplanting the entire variable regions of non-human origin, but "hiding" them by replacing surface residues with portions of human-like origin. Typically, framework residues in the human framework region will be substituted with corresponding residues from the CDR donor antibody, such as residues capable of improving antigen binding. These framework substitutions can be identified by methods well known in the art, for example, by modeling the interaction of the CDRs with framework residues to identify framework residues that play a significant role in antigen binding and by sequence alignment to identify framework residues that are aberrant at particular positions. (see U.S. Pat. Nos. 5,585,089, riechmann et al, nature 332 (1988); incorporated herein by reference in its entirety). Antibodies can be humanized using a variety of techniques well known in the art, such as CDR grafting (EP 239,400, WO 91/09967; U.S. Pat. Nos. 5,225,539,5,530,101 and 5,585,089), repair or resurfacing (EP 592,106; EP519,596; padlan, et al, molecular Immunology 28 (4/5): 489-498 (1991); studnicka et al, protein Engineering 7 (6): 805-814 (1994); roguska, et al, proc. Natl. Sci. USA 91 969-973 (1994)), and chain rearrangement (U.S. Pat. No. 5,565,332), the entire contents of which are incorporated herein by reference.

Deimmunization may also be used to reduce the immunogenicity of antibodies. In the present invention, the term "deimmunization" includes the alteration of antibodies to modify T cell epitopes (see, for example, WO/9852976A1 and WO/0034317A 2). For example, the heavy and light chain variable region sequences from the starting antibody are analyzed and a human T cell epitope "map" is generated from each variable region, showing the position of the epitope relative to the Complementarity Determining Regions (CDRs) and other key residues within the sequence. Individual T cell epitopes from the T cell epitope map are analyzed to identify alternative amino acid substitutions with 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 subsequently incorporated into a series of binding polypeptides. The genes comprising the modified variable regions and the complete heavy and light chains of the human constant regions are then cloned into expression vectors, and the plasmids are subsequently transferred into cell lines to produce complete antibodies. The antibodies are then compared in appropriate biochemical and biological experiments to identify the best antibody.

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, the scFv in bispecific antibodies of the invention may be found in techniques for the production of single chain units (U.S. Pat. Nos. 4,694,778, bird, science 242 (1988), huston et al, proc. Natl. Acad. Sci. USA55:5879-5883 (1988) and Ward et al, nature 334-554 (1989) and Nie et al, antibody Therapeutics 3 (1): 18-62 (2020)). Single chain fusion peptides are produced by amino acid bridging of the heavy and light chain fragments of the Fv region to form a single chain unit. Techniques for assembling functional Fv fragments in E.coli can also be used (Skerra et al, science 242.

Examples of techniques that can be used to produce single chain Fv (scFv) s and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498, and Huston et al, methods in Enzymology 203-46-88 (1991), shu et al, proc. Natl.Sci.USA 90. For certain uses, including the use of antibodies in humans and in vitro detection assays, chimeric, humanized or fully human antibodies may be used. Chimeric antibodies are molecules in which different portions of the antibody are derived from different animal species, such as antibodies having the variable regions of a murine monoclonal antibody and the constant regions of a human immunoglobulin. Methods for producing chimeric antibodies are known in the art, see Morrison, science 229 (1985); oi et al, bioTechniques 4 (1986); gillies et al, j.immunol.methods 125 (1989); neuberger et al, nature372:604-608 (1984); takeda et al, nature 314; and U.S. Pat. nos. 5,807,715, 4,816,567, and 4,816,397, which are incorporated herein by reference in their entirety.

Furthermore, another efficient method for producing recombinant antibodies is disclosed in Newman, biotechnology 10, 1455-1460 (1992), which in particular is capable of producing primate antibodies comprising monkey variable region and human constant region sequences, the entire contents of which are incorporated herein by reference. In addition, this technique is also mentioned in commonly assigned U.S. Pat. nos. 5,658,570, 5,693,780, and 5,756,096, each of which is incorporated herein by reference in its entirety.

Antibodies can be prepared by a variety of methods known in the art, including phage display methods using antibody libraries from immunoglobulin sequences. Reference may also be made to U.S. Pat. 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, the entire contents of each of which are incorporated herein by reference.

Fully human antibodies that recognize selective epitopes can be produced using a technique known as "guided selection". In this method, a selected non-human monoclonal antibody (e.g., a mouse antibody) is used to direct the screening of fully human antibodies that recognize the same epitope (see U.S. Pat. No. 5,565,332, the entire contents of which are incorporated herein by reference).

In another embodiment, DNA encoding the desired monoclonal antibody can be isolated and sequenced using conventional methods (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Isolated and subcloned hybridoma cells can serve as a source of such DNA. Once isolated, the DNA may be placed into an expression vector and then transfected into prokaryotic or eukaryotic host cells such as E.coli cells, simian COS cells, chinese Hamster Ovary (CHO) cells, or myeloma cells that do not produce other immunoglobulins. Isolated DNA (which may be synthetic as described herein) may also be used to prepare the sequences of the constant and variable regions of antibodies, as described in U.S. Pat. No. 5,658,570, which is incorporated herein by reference in its entirety. This method extracts RNA from selected cells and converts it to cDNA, which is then amplified by PCR techniques using Ig-specific primers. Suitable probes for this purpose are also mentioned in U.S. Pat. No. 5,658,570.

In addition, using conventional recombinant DNA techniques, one or more CDRs of an antibody of the invention can be inserted into a framework region, e.g., into a human framework region, to construct a humanized non-fully human antibody. The framework regions may 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 series of human framework regions). Some polynucleotides may encode antibodies produced by the framework region and CDR combination that specifically bind to at least one epitope of an antigen of interest. One or more amino acid substitutions may be made within the framework regions, and amino acid substitutions may be selected which improve the binding of the antibody to its antigen. Alternatively, substitution or deletion of cysteine residues in one or more of the variable regions involved in interchain disulfide bond formation can be performed in this manner, thereby producing an antibody molecule lacking one or more interchain disulfide bonds. Other variations of polynucleotides within the skill of the art are also encompassed by the present invention.

Antibody-producing cell lines can be selected, constructed and cultured using techniques well known to those skilled in the art. These techniques are described in various laboratory manuals and major publications. In this regard, the techniques described below as being suitable for use with the present invention are referenced to Current Protocols in Immunology, coligan et al, eds., green Publishing Associates and Wiley-Interscience, john Wiley and Sons, new York (1991), the entire contents of which, including supplements, are incorporated by reference in their entirety.

In some embodiments, the expression antibody vector comprises 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 insertion. 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 promoters of cytomegalovirus, and other cellular promoters such as actin can also be used. 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, among others. Commonly used mammalian cells include 293 cells, cos1 cells, cos7 cells, CV1 cells, murine L cells, CHO cells, and the like.

In some embodiments, the inserted gene fragment contains a selection marker, and common selection markers include dihydrofolate reductase, glutamine synthetase, neomycin resistance, hygromycin resistance, and the like, to facilitate selection and isolation of cells that have been successfully transfected. The constructed plasmid is transfected to host cells without the genes, and the cells successfully transfected grow in large quantities through selective culture medium culture to produce the target protein to be obtained.

In addition, mutations can be introduced in the nucleotide sequence encoding the antibody of the present invention using standard techniques known to those skilled in the art, including but not limited to site-directed mutations resulting in amino acid substitutions and PCR-mediated mutations. Variants (including derivatives) encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions or less than 2 amino acid substitutions relative to the original heavy chain variable region VH CDR1, VH CDR2, VH CDR3 and light chain variable region VL CDR1, VL CDR2 or VL CDR 3. 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.

Method of treatment

The invention also provides methods of treatment and uses. In some embodiments, there is provided a method for treating or ameliorating various types of cancer, tumor, or infection, among other related diseases, comprising administering to a patient an effective dose of the bispecific antibody: antibodies targeting TIGIT and CTLA-4, antibodies targeting OX40 and CTLA-4, or antibodies targeting OX40 and TIGIT. In some embodiments, the bispecific antibodies are provided for use in treating or ameliorating a cancer, tumor, or infection associated disorder. In some embodiments, the use of said bispecific antibody in the preparation of a medicament for treating or ameliorating a disease associated with cancer, a tumor, or an infection is provided.

The specific dosage and treatment regimen for any particular patient will depend upon a variety of factors including the specific antibody or derivative used, the age and weight of the patient, general health, sex and diet, and the time of administration, frequency of excretion, drug combination, and the severity of the particular disease being treated. These factors are judged by a medical caregiver who is within the purview of one of ordinary skill in the art. The dosage will also depend upon the individual patient to be treated, the route of administration, the type of formulation, the nature of the compound employed, the severity of the disease and the effect desired. The dosage employed can be determined by pharmacological and pharmacokinetic principles well known in the art.

Methods of administration of the antibodies or derivatives include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural, and oral injection. The pharmaceutical compositions may be administered by any convenient route, for example by infusion or bolus injection, absorbed through epithelial or cutaneous mucosa (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be co-administered with other biologically active agents. Thus, a pharmaceutical composition comprising an antibody of the invention may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (e.g. by powder, ointment, drops or transdermal patch), bucally or by oral or nasal spray.

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

The mode of administration may be systemic or local. Furthermore, it may be desirable to introduce the antibodies of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be assisted by an intraventricular catheter connected to, for example, a reservoir (which may be an Ommaya reservoir). Pulmonary administration is also possible, for example by use of an inhaler or nebulizer, and also by use of nebulized formulations.

The antibodies of the invention may be administered locally to the area in need of treatment; this may be done by, but is not limited to, the following: local infusion during surgery, for example in combination with topical application of a post-operative wound dressing, is achieved by injection, through a catheter, by means of a suppository or by means of an implant which is a porous, non-porous or gelatinous material, including membranes (e.g. silicone rubber membranes) or fibres. Preferably, when administering the proteins of the invention (including antibodies), 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 by it by constructing it as part of a suitable nucleic acid expression vector, which can then be made intracellular by administering such parts of the vector, for example, by using a retroviral vector (see U.S. Pat. No. 4,980,286), or by direct injection, or by using microprojectile bombardment (e.g., gene gun; biolistic, dupont), or coated with lipids or cell surface receptors or transfection reagents, or by ligation with a homologous heterologous cassette peptide known to enter the nucleus (see, e.g., joliot al, 1991, proc, natl. Acad. Sci. Usa 88-1864-1868), and the like. Alternatively, the nucleic acid may be introduced into the cell by homologous recombination and integrated into the host cell DNA for expression.

In some embodiments, an antibody of the invention is administered to a patient at a dose of 0.01mg/kg to 100mg/kg of patient body weight, or 0.1mg/kg to 20mg/kg of patient body weight. A second or more doses of the antibody or antigen-binding fragment may be administered subsequently to the initial dose, at about the same or less dose as the initial dose, wherein the subsequent doses may be separated by at least 1 to 3 days; or at least one week. The dosage and frequency of administration of the antibodies of the invention can be reduced by enhancing the uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as lipidation.

Various known delivery systems may be used to administer the antibodies or derivatives of the invention or polynucleotides encoding same, e.g., encapsulated in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, receptor-mediated endocytosis (see, e.g., wu and Wu,1987, j.biol. Chem.262 4429-4432), construction of nucleic acids as part of a retrovirus or other vector, and the like.

Combination therapy

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

In some embodiments, the bispecific antibodies of the invention are administered in combination with a chemotherapeutic agent. In some embodiments, a bispecific antibody of the invention is an antibody that targets TIGIT and CTLA-4, an antibody that targets OX40 and CTLA-4, or an antibody that targets OX40 and TIGIT. In some embodiments, chemotherapeutic agents that can be administered with the antibodies of the invention include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and actinomycin D), antiestrogens (e.g., tamoxifen), antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2 b, glutamic acid, mithramycin, mercaptopurine, and 6-thioguanine), cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytarabine, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, and vincristine sulfate), hormones (e.g., medroxyprogesterone, estramustine sodium phosphate, ethinylestradiol, estradiol, megestrol acetate, mesterone, diethylstilbestrol diphosphate, clenolide, and testolactone), nitrogen mustard derivatives (e.g., melphalan, chlorambucil, diethyldithiocarbamate, and tirothiapine), steroids and combinations thereof (e.g., vinpocetine, and other compounds such as, vincamine, vinca, and epothilones), and other compounds (e.g., etoposide, vinca) and vinca sulfate).

In some embodiments, an antibody of the invention is administered in combination with a cytokine. 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, and the like.

In some embodiments, a bispecific antibody of the invention is administered in combination with a chemotherapeutic agent. Examples of chemotherapeutic agents include immunotherapeutic agents, including but not limited to therapeutic antibodies suitable for treating a patient. Some examples of therapeutic antibodies include octatuzumab (simtuzumab), abamectin (abagovacab), adalimumab (adecarmumab), afurtuzumab (aftuzumab), alemtuzumab (alemtuzumab), altumumab (alemtuzumab), amatuximab (amatuximab), anandazumab (anatuximab), anandazumab (anatuzumab), aximomab (arcitumomab) bavituximab, betuzumab (bectuximab), bevacizumab (bectumomab), bevacizumab (bevacizumab), bivacizumab (bivatuzumab), brettanimab (blinatumomab), brentuximab (brentuximab), trastuzumab (cantuzumab), catatumumab (cataxomab), cetuximab (cetuximab), cetuximab (citazumab) cetuximab (cixutuzumab), clevatuzumab (clivatuzumab), cleavazumab (contuzumab), dalmatizumab (daratumumab), choletuzumab (drozitumab), durigotuzumab (duligomab), duriguzumab (dumb), delumumab (detumab), daceutuzumab (dacetuzumab), dalotuzumab (dalotuzumab), ecklomuzumab (ecomexib), elotuzumab (eltuzumab), ennitumoxitumumab (enstuximab), ertumxozumab (ertaxolomab), iralizumab (etaxeb), fartuzumab (fartuzumab), certitumumab (fituzumab), certitumumab (certitumumab), certitumumab (gimuzumab), certitumumab (jituzumab), rituximab (filituzumab), rituximab (rituxab), etaxeb (etaxeb), fartuzumab (fartuzumab), and fituzumab (giu), and gemtuzumab (jiuttuzumab (jiugueb), voratuzumab (flantuzumab), futuximab (futuximab), ganituzumab (ganitumab), gimtuzumab (gemtuzumab), giltuzumab (girtuzumab), girentuzumab (giretuzumab), gram-Bartuzumab (glemba), ibritumomab (ibritumomab), iggozumab (ovomab), immatuzumab (imgatuzumab), edutaximab (indatuximab), imrituzumab (inotuzumab), infatuzumab (intetumumab), epipilimumab (ipilimumab), iritumomab (iramtuzumab), labetuzumab (Latuzumab), lexatuzumab (lefluzumab), rituzumab (lintuzumab), wobuzumab (Lortumab), rabetuzumab (Latuzumab), lexatuzumab (lexatuzumab), rituzumab (lexuzumab (lintuzumab), rituximab (lintuzumab), wobutab (Lortuzumab (Lortumab), lurtuzumab (Lortuzumab), and Cartuzumab (Lurtumab (Lurtuzumab) mapatumumab (mapatumumab), matuzumab (matuzumab), milatumuzumab (matuzumab), murumumab (milatumumab), minrimumab (minretumumab), mitumumab (mitumumab), molital (moxetumumab), nannatazumab (narnatatumumab), nanitumumab (natumumab), neimtumumab (necitumumab), nimotumumab (nimotumumab), nofititumumab (nofemtomomumab), oxkatuzumab (ocatuzumab), ofatumumab (ofatumumab), olanzumab (olamtuzumab), onartuzumab (nonartuzumab), opotuzumab (oportuzumab), oguzumab (orituzumab), panitumumab (paratuzumab), satuzumab (satuzumab), omab (sauttuzumab), omab (omab), milatumumab (matuzumab), and nimotuzumab (matuzumab), pertuzumab (patritumab), pemtuzumab (pemtumumab), pertuzumab (pertuzumab), rituximab (pintumomab), pertuzumab (pertuzumab), latanotuzumab (racotumumab), largetuzumab (radretumab), rilotumumab (rilotumumab), rituximab (rituximab), lobitumumab (robatumumab), sartuzumab (satumoma), sibutruzumab (sibutroumab), sitotumumab (situmumab), solituximab (solituzumab), and pertuzumab (pemphigob) tacatuzumab, tacrolituzumab (tapilizumab), tranitumumab (tenatumumab), ternetuzumab (teprotuzumab), terputuzumab (teprotuzumab), texatuzumab (tigitumumab), trastuzumab (trastuzumab), tukuzumab (tucotuzumab), zetuzumab (tucotuzumab), ulizumab (ublituximab), veltuzumab (veltuzumab), vortuzumab (voretuzumab), voltuzumab (voltuzumab), and zalutumumab (zalutumuzumab), and the like.

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

Pharmaceutical composition

The invention also provides a pharmaceutical composition. Such compositions comprise an effective amount of the antibody or antigen-binding fragment and an acceptable carrier. In some embodiments, the pharmaceutical composition further comprises an anti-cancer agent (e.g., an immune checkpoint inhibitor).

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

The term "adjuvant" refers to a diluent, adjuvant, excipient, or carrier that is administered to a patient along with an active ingredient. Such pharmaceutical excipients may be sterile liquids, such as water and oils, including those of petroleum, animal or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a 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, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. If desired, the compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antimicrobial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as 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 may be formulated as a suppository, using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable Pharmaceutical carriers are described in Remington's Pharmaceutical Sciences of e.w. martin, which is hereby incorporated by reference. Such compositions will contain a clinically effective dose of the antibody or antigen-binding fragment, preferably in purified form, together with an appropriate amount of carrier to provide a form of administration suitable for the patient. The formulation should be suitable for the mode of administration. The parent formulation may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

In some embodiments, the composition is formulated according to conventional procedures as a pharmaceutical composition suitable for intravenous injection into a human. Compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer. The composition may also include a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the site of injection. Generally, the active ingredients are provided in unit dosage forms, either separately or in admixture, e.g., as a dry lyophilized powder or as an anhydrous concentrate, in a sealed container (e.g., ampoule or sachet) which is indicative of the serving size of the active agent. In the case of administration of the composition by infusion, the composition may be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. In the case of administering the composition by injection, an ampoule of sterile water or saline for injection may be used so that the active ingredients may be mixed before administration.

The compounds of the present invention may be formulated in neutral or salt form. Pharmaceutically acceptable salts include salts derived from anions such as hydrochloric, phosphoric, acetic, oxalic, tartaric acids, and the like, and salts derived from cations such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others based on the teachings of the present invention are within the scope of the invention.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified; for example, TIGIT-Fc antigen, CTLA-4-Fc antigen, TIGIT-his antigen can be purchased from Shanghai near-shore science and technology Co., ltd or Acrobiosystems, and the anti-TIGIT reporter gene detection system can be obtained from Promega (Cat. No. JA 1400), or can be prepared according to a known method.

Reference antibody

anti-TIGIT antibody (reference antibody) was expressed in CHO cells and purified. The heavy chain of the anti-TIGIT antibody is:

the light chain of the anti-TIGIT antibody is:

anti-CTLA-4 antibody (reference antibody) in CHO cells expression and purification of the obtained. The heavy chain of the anti-CTLA-4 antibody is as follows:

the light chain of the anti-CTLA-4 antibody is:

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

or

The light chain of anti-OX40 is:

bispecific antibodies targeting TIGIT and CTLA-4

EXAMPLE 1 method for producing antibody

The structure of the bispecific antibody is shown in figure 1, which is similar to an immunoglobulin and comprises a first polypeptide chain and a third polypeptide chain having the same sequence, and a second polypeptide chain and a fourth polypeptide chain having the same sequence; wherein the first polypeptide chain comprises the structure VHa-L1-CLa-L2-VHb-CH1-Fc and the second polypeptide chain comprises the structure VLa-L3-CH1-L4-VLb-CLb; antigen a is TIGIT, and antigen b is CTLA-4.

And (3) performing sequence optimization on the amino acid sequences of the first polypeptide and the second polypeptide of the antibody according to the codon preference characteristic of a host cell CHO (Chinese hamster ovary cell) to obtain DNA sequences of the first polypeptide and the second polypeptide. To facilitate expression in CHO cells, a signal peptide and a Kozak sequence (Kozak sequence is a nucleic acid sequence located after the end cap structure of the eukaryotic mRNA 5') are added to the sequences of the first and second polypeptides, respectively, and a stop codon is added to the end of the sequences. At the same time, the cloning to a mammalian expression vector pCDNA3.1 is facilitated ^TM (+) (Invitrogen, cat # V79020), with restriction sites added to both ends of the sequence, hind III at the 5 'end and EcoR I at the 3' end.

Cloning of the optimized and synthesized sequence clones to pCDNA3.1 separately ^TM And (c) in the (+) vector, respectively extracting a plurality of plasmids, wherein the first polypeptide and the second polypeptide adopt ExpicCHO according to a plasmid molar ratio of 1 ^TM The expression system (Gibco, cat # A29133) was transiently expressed. The protein was harvested and purified with protein A as per the instructions.

The related amino acid sequences of the bispecific antibody targeting TIGIT and CTLA-4 are shown in a table 1, and the related nucleic acid sequences of the bispecific antibody targeting TIGIT and CTLA-4 are shown in a table 2. The linkers are in table 1 in bold italics and underlined; the first polypeptide of the antibody 1 is shown as SEQ ID NO. 37, and the second polypeptide of the antibody 1 is shown as SEQ ID NO. 38; the first polypeptide of the antibody 2 is shown as SEQ ID NO. 39, and the second polypeptide of the antibody 2 is shown as SEQ ID NO. 40; the first polypeptide of the antibody 3 is shown as SEQ ID NO. 41, and the second polypeptide of the antibody 3 is shown as SEQ ID NO. 42; the first polypeptide of antibody 4 is shown in SEQ ID NO. 43, and the second polypeptide of antibody 4 is shown in SEQ ID NO. 44. In Table 2, the small letter underlined in italic at the 5 'end (5' end to 3 'end) is a Hind III restriction site, a Kozak sequence and a leader peptide sequence in this order, and the small letter underlined in italic at the 3' end (5 'end to 3' end) is a stop codon and an EcoR I restriction site in this order; since

antibodies

1, 2, 3, and 4 differ only in linker sequence, only the nucleic acid sequences of the first and second polypeptides of antibody 1, and the nucleic acid sequence of the linker, are listed in table 2.

TABLE 1 amino acid sequence of bispecific antibody targeting TIGIT and CTLA-4

TABLE 2 nucleic acid sequences encoding bispecific antibodies targeting TIGIT and CTLA-4

Example 2 antibody purity assay

And (3) carrying out gel electrophoresis detection on the purified antibody: and detecting the purity of the purified antibody. As shown in fig. 2, the purity of antibody 1 was high; as shown in fig. 3, the purity of antibody 2 and antibody 3 was high.

Example 3 detection of antibody binding Activity

1) And (3) detecting the binding activity of the purified bispecific antibody, and detecting whether the antibody can normally bind to TIGIT or CTLA-4. The procedure for the antibody binding to TIGIT assay is as follows: coating 1 mu g/ml of TIGIT-Fc antigen by PBS (phosphate buffer solution), wherein each hole is 100 mu l, and then placing the coated membrane in a refrigerator at the temperature of 2-8 ℃ overnight for coating; the next day, 250. Mu.l of PBS containing 3% BSA was added and blocked in a 37 ℃ incubator for 2 hours; then washing with PBST for 2 times, and then respectively adding anti-TIGIT, the antibody 1, the antibody 2 and the antibody 3 which are diluted in a gradient manner, wherein the initial concentration is 3 mu g/ml, and the dilution is 1/3 times, and the total number is 10 dilution gradients; add 100. Mu.l of diluted antibody to each well and incubate 1 hr at 37 ℃ in an incubatorWhen the current is over; washed 3 times with PBST, 100 μ l of enzyme-labeled secondary antibody (Sigma goat anti-human IgG kappa, igG light chain coupled HRP, cat # a7164-1 ML) diluted 1 fold was added per well and incubated for 1 hour at 37 ℃ in an incubator; washing with PBST for 5 times, adding TMB developing solution for developing color for about 20min, and adding 1M H ₂ SO ₄ The termination is carried out and the OD450 value is read within 30 min.

As shown in figure 4, the binding capacity of the

antibodies

1 and 2 to human TIGIT-Fc is close to that of anti-TIGIT results, and the binding capacity of the antibody 3 to human TIGIT-Fc is lower.

2) The binding assay of the antibodies of the invention to CTLA-4-Fc is performed in a similar manner to that described above. As shown in FIG. 5, all of antibody 1, antibody 2 and antibody 3 were able to bind to CTLA-4-Fc, and antibody 1 and antibody 2 were able to bind strongly. Example 4 detection of antibody affinity

1) In order to determine the influence of linker lengths (L2 and L4) on the ability of bispecific antibodies to bind to hTIGIT-his protein (Shanghai near technologies, inc., CJ 58), the affinity constants of anti-TIGIT, antibody 1, antibody 2, and antibody 3 and hTIGIT-his protein were determined using a Biacore T200 instrument. The specific test process is as follows: diluting the hTIGIT-his antigen by HBS-EP + buffer in 5 gradients with initial concentration of 50nM, and then diluting in half; respectively diluting the protein anti-TIGIT to be detected, the antibody 1, the antibody 2 and the antibody 3 to be detected to be 2.5 mu g/ml by using HBS-EP + buffer; the apparatus was used for detection with Biacore T200, protein A chip (GE healthcare, cat # 29127556) and the apparatus set up as follows: antibody capture binding time (contact time) 25s, flow rate 5. Mu.l/min; sample binding time (contact time) 120s, dissociation time (dissociation time) 300s, and flow rate 30. Mu.l/min; the regeneration (regeneration) conditions were: glycine-HCl (pH 1.5), regeneration time 60s, flow rate 30 μ l/min; the detection channels (flow path) are 2-1 and 4-3.

As shown in Table 3, the affinity of antibody 1 to hTIGIT-his is not reduced or even increased to some extent compared with anti-TIGIT; compared with anti-TIGIT, the affinity of the

antibodies

2 and 3 to hTIGIT-his is basically similar.

2) And (3) detecting the affinity constants of anti-CTLA-4, antibody 1, antibody 2 and antibody 3 and h CTLA-4-his protein (Shanghai near-shore science and technology, inc., CP 33) by using a Biacore T200 instrument. The specific test process is as follows: diluting hCTLA-4-his antigen with HBS-EP + buffer gradient for 6 gradients with initial concentration of 100nM, and then diluting in half; respectively diluting the protein anti-CTLA-4 to be detected, the antibody 1, the antibody 2 and the antibody 3 to be detected to 5 mu g/ml by using HBS-EP + buffer; the detection was carried out using an instrument, biacore T200, protein A chip (GE healthcare, cat # 29127556), set up as follows: antibody capture binding time (contact time) 15s, flow rate 10. Mu.l/min; sample binding time (contact time) 180s, dissociation time (dissociation time) 300s, flow rate 30. Mu.l/min; the regeneration (regeneration) conditions were: glycine-HCl (pH 1.5), regeneration time 60s, flow rate 30 μ l/min; the detection channels (flow path) are 2-1 and 4-3.

As shown in Table 4, the affinity of antibody 1 and antibody 2 was substantially close to that of hCTLA-4-his as compared with that of anti-CTLA-4; antibody 3 has a lower affinity for hCTLA-4-his than anti-CTLA-4.

TABLE 3 affinity of antibodies to hTIGIT-his

TABLE 4 affinity of the antibody for hCTLA-4-his

Example 5 detection of biological Activity of antibodies

1) In order to further identify the biological activity of the antibody, the biological activity of the antibody was tested by using the anti-CTLA-4 reporter gene assay system (Promega, cat. No. JA 1400), which was referred to the product manual.

As shown in FIG. 6, the control antibody anti-CTLA-4 has good biological activity, and can activate the IL-2 promoter in the reporter system so as to activate the expression of downstream luciferase; the activation ability of antibody 1 is relatively weak and the biological activity is relatively low. As shown in FIG. 7, the activation ability of antibody 2 was relatively weak and the biological activity was relatively low compared to anti-CTLA-4.

2) The biological activity of the antibody is determined by using an anti-TIGIT reporter gene detection system, and the detection process is similar to that of the anti-CTLA-4 reporter gene detection system of Promega.

As shown in fig. 8-10, antibody 1, antibody 2 and antibody 3 did not exhibit much poorer biological activity, even though the activation abilities of antibody 1 and antibody 2 were relatively better, compared to the control antibody anti-TIGIT-4.

In combination with the detection of the biological activity of the antibody, the increase of the lengths of the linkers L2 and L4 has no obvious influence on the function of the TIGIT target, but has an influence on the function of the CTLA-4 target: the longer the linker length, the lower the biological activity against the CTLA-4 target; the antibody 1 of the present invention has excellent biological activity.

From the current TIGIT target clinical trial (Genentech MTIG-7129 clinical trial (NCT 0356716) or OncoMed company OMP-313M32 clinical trial (NCT 03119428)), it can be seen that the target drug generally requires higher doses, and the 2018 abstract of the cancer immunotherapy society (SITC 2018) discloses that TIGIT target antibody OMP-313M32 has no serious adverse events; however, for anti-CTLA-4 antibodies, it has been clinically found that up to 60% of patients treated with Yervoy, an anti-CTLA-4 antibody, develop immune-related adverse events: 10% -30% are serious (grade 3-4) immune-related adverse events, and the risk of developing immune-related adverse events in patients receiving treatment with Yervoy is dose-dependent (Martins F, sofiya L, sykiotis G P, et al. Additive effects of immune-checkpoint inhibitors: epidemic, management and Clinical [ J ]. Nature Reviews Clinical Oncology,2019,16 (9): 563-580); the biological activity of the antibody 1 aiming at CTLA-4 is reduced to a certain extent compared with that of anti-CTLA-4, so that the blocking of TIGIT and CTLA-4 targets can be ensured, and the side effect brought by the anti-CTLA-4 antibody can be reduced or avoided.

Bispecific antibodies targeting OX40 and CTLA-4

EXAMPLE 6 method for producing antibody

The structure of the bispecific antibody is shown in figure 1, which is similar to an immunoglobulin and comprises a first polypeptide chain and a third polypeptide chain having the same sequence, and a second polypeptide chain and a fourth polypeptide chain having the same sequence; wherein the first polypeptide chain comprises the structure VHa-L1-CLa-L2-VHb-CH1-Fc and the second polypeptide chain comprises the structure VLa-L3-CH1-L4-VLb-CLb; antigen a is OX40, and antigen b is CTLA-4.

Sequence of a second polypeptide of the synthetic antibody: the 5 'end contains HindIII enzyme cutting site and leader peptide, and the 3' end contains stop codon and EcoRI enzyme cutting site; sequence of the first polypeptide of the synthetic antibody: the 5 'end contains HindIII restriction site and leader peptide, and the 3' end contains stop codon and EcoRI restriction site.

Plasmids carrying the nucleic acid sequences of the first and second polypeptides, respectively, are simultaneously transiently transfected into 293F cells. 293F cells are cultured by CD 293 TGE Medium (BPM Cell Culture, cat # CM-1156), when the Cell activity is more than 95%, the cells are passaged to 80-100 ten thousand/mL by using a fresh Culture Medium, and when the Cell density is about 150-200 ten thousand/mL after 24h, transfection can be carried out. The plasmid was first washed with a water bath at 65 ℃ for 30 minutes using 0.5. Mu.g of plasmid per 100 million cells and 3. Mu.L of PEI (Polysciences, cat. No. 24765-2) per 1. Mu.g of plasmid. The plasmid and PEI were each diluted with medium (the volume of the two solutions added together is 5% of the total volume) and allowed to stand at room temperature for 5 minutes. The PEI solution was then added to the plasmid solution, mixed well and allowed to stand at room temperature for 20 minutes. The complex solution was added to the cells, and after shaking Culture at 37 ℃ for 24 hours, CD Feed X Supplement (BPM Cell Culture, cat. No. CF-1116) was added to the cells in a total volume ratio of 10%. After 5 days, cell supernatants were harvested and purified using Protein a to obtain bispecific antibody 5.

The amino acid sequences of bispecific antibody 5 targeting OX40 and CTLA-4 are shown in Table 5, and the nucleic acid sequences of bispecific antibody 5 targeting TIGIT and CTLA-4 are shown in Table 6. The linkers are shown in Table 5 in bold italics and underlined.

TABLE 5 amino acid sequences of bispecific antibodies targeting OX40 and CTLA-4

TABLE 6 nucleic acid sequences encoding bispecific antibodies targeting OX40 and CTLA-4

Example 7 antibody purity assay

Antibody 5 was subjected to reducing and non-reducing treatments, respectively, and then subjected to SDS-PAGE electrophoresis. As shown in fig. 11, the reduced and non-reduced bands are clear and free of miscellaneous bands, indicating that the sample purity is high: non-reducing antibody 5 is greater than 250kDa in size on an electrophoretogram; the first polypeptide of reducing antibody 5 is approximately 90kDa in size on an electrophoretogram, and the second polypeptide of reducing antibody 5 is approximately 50kDa in size on an electrophoretogram; the size is as expected. It can be seen that bispecific antibody 5 is normally expressed in cells; antibody 5 expressed in 293F cells was purified and used for further testing.

Example 8 detection of antibody binding Activity

The ELISA plates were coated with CTLA-4-Fc antigen and OX40-Fc antigen, respectively, overnight at 4 ℃. Blocking was performed the next day with 5% BSA blocking solution at 37 ℃ for 2 hours. After washing the plate, add respectivelyThe antibody 5, CTLA-4 monoclonal antibody (i.e., anti-CTLA-4), and OX40 monoclonal antibody (i.e., anti-OX 40) were diluted in 2-fold gradient starting from 2. Mu.g/mL, and 12 concentrations (the last one was blank 0 concentration) were diluted in a total of 12 wells, and each concentration was incubated at 37 ℃ for 2 hours. After washing the plates, anti-human Kappa light chain secondary antibody diluted 1. After washing the plate, adding TMB single-component color developing solution to develop for 10-15 minutes in a dark place, terminating the reaction with 0.1M sulfuric acid, and reading at the wavelength of 450 nm. EC calculation from ELISA binding curves ₅₀ The values, results are shown in Table 7.

Wherein, the preparation method of the OX40-Fc antigen comprises the following steps: from the protein database Uniprot, the amino acid sequence of human OX40 (P43489) was found, in which the amino acid sequence of the extracellular region of human OX40 is residues 1 to 216; from the protein database Uniprot, the amino acid sequence of IgG1-Fc (P01857) residues 104 to 330 are found; then, obtaining nucleotide sequences corresponding to OX40 extracellular region and Fc through artificial synthesis (general purpose company), and inserting the nucleotide sequences into pCDNA3.0 vector (Invitrogen) through enzyme digestion connection to obtain recombinant plasmid; the plasmid was then transiently transfected into HEK293 cells by PEI, and after 7 days of culture, the supernatant was collected and finally purified to obtain OX40-Fc (i.e., hOX 40-Fc).

As shown in Table 7, antibody 5 retains the ability to bind to both OX40 and CTLA-4 antigens; antibody 5 binds to OX40 well and antibody 5 binds to CTLA-4 poorly compared to monoclonal antibodies.

TABLE 7 EC for binding of antibody to antigen ₅₀ Value of

Example 9 detection of antibody affinity

Coupling the OX40 monoclonal antibody to be detected, the CTLA-4 monoclonal antibody and the antibody 5 with biotin respectively. The SA sensor was pre-wetted in PBS for 10 minutes, the biotin-labeled antibody was diluted to 10. Mu.g/mL with PBST (hereinafter referred to as dilution buffer) containing 0.5% BSA at pH 6.8, and the SA sensors were each biotinylated at the above 3 positionsThe antibody was immobilized to a signal of about 4nm. CTLA-4-Fc antigen and hOX40-Fc antigen to be tested were diluted to 400nM in dilution buffer, and diluted in 2-fold gradient with this as the starting concentration, for a total of 6 concentration points (400, 200, 100, 50, 25, 0 nM). Diluting buffer solution, gradient drug diluent solution, and regeneration buffer solution (1M MgCl with pH of 8.4) ₂ ) And the neutralization buffer (PBST) was added sequentially to the corresponding columns of the 96-well plate, and the following steps were performed: (1) baseline: baseline detection in dilution buffer for 60 seconds; (2) association: binding for 300 seconds in antibody gradient dilutions and sample blanks (dilution buffer); (3) association: dissociation in dilution buffer for 360 seconds; (4) regeneration: regeneration in regeneration buffer for 5 seconds; (5) neutrallization: neutralizing in a neutralization buffer for 5 seconds; (6) regeneration and neutratization cycles were performed 3 times.

The Data is processed and analyzed by ForteBio Data Analysis 8.2, and the sample Data is fitted after deduction of reference (sample blank) signals to obtain an affinity constant K _D . The affinity results for antibody 5 and CTLA-4-Fc antigen are shown in Table 8, and the affinity results for antibody 5 and OX40-Fc antigen are shown in Table 9.

TABLE 8 kinetic parameters for the affinity binding of antibodies to CTLA-4 antigen

TABLE 9 kinetic parameters for affinity binding of antibodies to OX40 antigen

As shown in table 8, antibody 5 has higher affinity for CTLA-4 antigen than monoclonal antibody; as shown in Table 9, the affinity of antibody 5 for the OX40 antigen was substantially similar compared to that of the monoclonal antibody.

Example 10 antibodies to OX40 ⁺ Binding Activity of cells

Jurkat cells overexpressing human OX40 (OX 40) were generated by transfecting pCMV vector (Invitrogen) with human OX40cDNA ⁺ Cells), OX40 was taken separately ⁺ Cells were centrifuged to remove the medium and washed once with PBS, leaving the cell pellet. OX40 monoclonal antibody and antibody 5 were diluted 3-fold from the antibody concentration of 100nM, respectively, for a total of 10 concentrations. Resuspending the antibody solutions at different concentrations in the corresponding cell pellet (OX 40) ⁺ Cells), about 200 million cells are required for each concentration. After incubation at 4 ℃ for 2 hours, the supernatant was centrifuged and washed once with PBS. To each sample was added 1. The supernatant was centrifuged off and washed once with PBS. Each sample was resuspended in PBS and then tested in a C6 flow cytometer with channel 2 selected by the light source. The binding curve is shown in FIG. 12, and the statistics of the positive rate and fluorescence value are shown in tables 10-11.

As shown in FIG. 12 and tables 10-11, antibody 5 compares to OX40 as compared to monoclonal antibodies ⁺ The binding activity of the cells was comparable.

TABLE 10 OX40 monoclonal antibodies with OX40 ⁺ Binding positivity and fluorescence value of cells

TABLE 11 antibodies 5 and OX40 ⁺ Binding positivity and fluorescence value of cells

Example 11 antibodies bind to OX40 antigen and CTLA-4 antigen simultaneously

OX40-Fc antigen was coupled to biotin, the SA sensor was pre-wetted in PBS for 10 minutes for use, the biotin-labeled OX40-Fc antigen was diluted to 5. Mu.g/Ml with PBST (hereinafter referred to as dilution buffer) containing 0.5% BSA at pH 6.8, and the SA sensor was immobilized in the above-described labeled biotinylated OX40-Fc antigen solution to a signal of about 0.8nM. Diluting the antibody 5 to be detected to 50nM by using a dilution buffer; CTLA-4-Fc antigen was diluted to 200nM with dilution buffer. Diluting buffer solution, antibody 5 diluent, CTLA-4-Fc antigen diluent, and regeneration buffer solution (1M MgCl with pH of 8.4) ₂ ) And adding the neutralization buffer solution (PBST) into corresponding columns of the 96-well plate in sequence, and operating the following steps: (1) baseline: baseline detection in dilution buffer for 60 seconds; (2) association: combine in antibody gradient dilutions and sample blanks (dilution buffer) for 300 seconds; (3) baseline: baseline detection in dilution buffer for 60 seconds; (4) association: combine in antibody gradient dilutions and sample blanks (dilution buffer) for 300 seconds; (5) association: dissociation in dilution buffer for 300 seconds; (6) regeneration: regeneration in regeneration buffer for 5 seconds; (7) neutrallization: neutralizing in a neutralization buffer for 5 seconds; (8) regeneration and neutrallization were cycled 3 times.

As shown in fig. 13, the sensor bound antibody 5 after immobilization of OX40-Fc antigen, with significant binding; at the moment, CTLA-4-Fc antigen is combined, and a certain combination signal value still exists; and no significant signal drop was seen at the dissociation step; indicating that antibody 5 is capable of binding both OX40 antigen and CTLA-4 antigen.

Bispecific antibodies targeting OX40 and TIGIT

EXAMPLE 12 preparation of antibodies

The structure of the bispecific antibody is shown in figure 1, which is similar to an immunoglobulin, and comprises a first polypeptide chain and a third polypeptide chain having the same sequence, and a second polypeptide chain and a fourth polypeptide chain having the same sequence; wherein the first polypeptide chain comprises the structure VHa-L1-CLa-L2-VHb-CH1-Fc and the second polypeptide chain comprises the structure VLa-L3-CH1-L4-VLb-CLb; antigen a is OX40 and antigen b is TIGIT.

Nucleic acid sequences corresponding to the first polypeptide and the second polypeptide of the bispecific antibody are obtained by artificial synthesis, and then the nucleic acid sequences are respectively connected to a pCDNA3.0 vector (purchased from Invitrogen company) through enzyme digestion connection; 2 recombinant plasmids for expressing the full antibody were obtained. The plasmid was transiently transfected into HEK293 cells by PEI according to the manufacturer' S instructions using the Freedom CHO-S kit (purchased from Invitrogen), and the supernatant was collected after 7 days of culture to obtain a bispecific antibody protein sample by purification.

The amino acid sequences related to bispecific antibodies 6 and 7 targeting OX40 and TIGIT are shown in table 12, and the nucleic acid sequences related to bispecific antibodies 6 and 7 targeting TIGIT and TIGIT are shown in table 13. In Table 12 the linkers are in bold italics and underlined.

TABLE 12 amino acid sequences of OX40 and TIGIT-targeted bispecific antibodies

TABLE 13 nucleic acid sequences encoding bi-specific antibodies targeting OX40 and TIGIT

EXAMPLE 13 antibody purity testing

The composition and purity of the bispecific antibody was analyzed by SDS-PAGE under reducing and non-reducing conditions. As shown in fig. 14, under non-reducing conditions, antibodies 6 and 7 migrated in a single band; under reducing conditions, antibody 6 and antibody 7 each produced two bands; antibody 6 and antibody 7 are a single species and these 2 polypeptide chains are efficiently paired to form an IgG-like protein.

Example 14 detection of antibody affinity

ForteBio affinity assay can be performed by reference to conventional methods (Estep Patricia, et al. High throughput solution-based measurement of affinity-affinity and affinity binding. Mabs,2013,5 (2): 270-8), and the specific procedure is as follows: the sensor is balanced under the line of an analysis buffer solution (such as PBS) for 20 minutes, then a machine is used for detecting for 60 seconds to establish a signal baseline, the purified antibodies 6 and 7 are loaded on the machine to a corresponding sensor (ForteBio), and finally the ForteBio affinity measurement is carried out; adsorbing antibodies 6 and 7 with a protein sensor, and separately detecting binding to and dissociation from OX40-his (Acrobiosystems) and Tigit-his (Acrobiosystems), respectively, for about 5min each; finally, kinetic analysis was performed using the 1.

TABLE 14 affinity of the antibodies to OX40-his and TIGIT-his

As shown in table 14, antibody 6 was able to bind significantly to OX40 antigen, but with weaker affinity than anti-OX40; the antibody 7 can obviously bind to OX40 antigen and has the affinity equivalent to that of anti-OX40; antibody 7 also binds significantly to TIGIT antigen with an affinity substantially similar to that of anti-TIGIT.

Example 15 antibodies bind to both OX40 antigen and TIGIT antigen

Similarly to the Fortebio affinity assay method described above, bispecific antibodies were tested for simultaneous binding to OX40-his antigen (Acrobiosystems) and TIGIT-his antigen (Acrobiosystems). The antibody 7 in the above example was adsorbed by a protein sensor, and binding to and dissociation from OX40 antigen was detected, and then binding to and dissociation from TIGIT antigen was detected.

As shown in fig. 15, antibody 7 was clearly able to bind both OX40 and TIGIT antigens without steric hindrance. EXAMPLE 16 binding Activity of antibodies to OX40 or TIGIT overexpressing Jurkat cells

1) Jurkat-OX40 cells overexpressing human OX40 (i.e., OX40 as described above) were generated by transfecting pCMV vector (Invirogen) with human OX40cDNA ⁺ Cells), jurkat-hOX40 cells (0.5X 10) ⁶ Individual cells) were incubated with different concentrations of antibody for 40 minutes on ice in PBS. The cells were then washed twice and incubated with secondary antibodies in PBS (containing 0.1% BSA) on ice for 25 minutes. Cells were washed twice and analyzed by flow cytometry on an Accuri C6 system (BD Biosciences).

As shown in FIG. 16, antibody 7 (OT-4D-30 a) binds significantly to Jurkat-OX40 cells, with a binding capacity close to that of anti-OX40.

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

As shown in FIG. 17, antibody 7 was clearly able to bind to Jurkat-Tigit cells with slightly weaker binding than anti-TIGIT.

Claims

An antibody or antigen-binding fragment, wherein the antibody or antigen-binding fragment binds to two different antigens, a first antigen a and a second antigen b, and wherein the antibody or antigen-binding fragment comprises at least 2 polypeptide chains; wherein the first polypeptide chain comprises, in order from the amino terminus, VHa, CLa, VHb, and CH1, VHa being the heavy chain variable region associated with a first antigen a, CLa being the first light chain constant region, VHb being the heavy chain variable region associated with a second antigen b, and CH1 being the first constant region of the heavy chain.
The antibody or antigen-binding fragment of claim 1 wherein VHa is covalently linked to CLa via a linker L1, L1 comprising 2 to 6 amino acids; and/or

The CLa and the VHb are covalently linked through a linker L2; wherein L2 contains 10 to 30 amino acids and at least 50% of the amino acids are glycine.
The antibody or antigen-binding fragment of claim 1 or 2, wherein the second polypeptide chain comprises, in order from the amino terminus, VLa, CH1, VLb, and CLb; wherein VLa is a light chain variable region associated with a first antigen a, VLb is a light chain variable region associated with a second antigen b, and CLb is a second light chain constant region.
The antibody or antigen-binding fragment of claim 3, wherein VLa is covalently linked to CH1 through a linker L3, wherein L3 comprises from 2 to 6 amino acids; and/or

CH1 is covalently linked to VLb via linker L4; wherein L4 contains 10 to 30 amino acids and at least 50% of the amino acids are glycine.
The antibody or antigen-binding fragment of claim 4 wherein L1 comprises the sequence set forth in SEQ ID NO. 22; and/or

The L2 comprises a sequence selected from the group consisting of the sequence shown in any one of SEQ ID NOs 23-27, a sequence having at least 90% identity to the sequence shown in any one of SEQ ID NOs 23-27, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in any one of SEQ ID NOs 23-27; and/or

The L3 comprises a sequence shown as SEQ ID NO. 28; and/or

The L4 comprises a sequence selected from any one of SEQ ID NO 29-33, a sequence having at least 90% identity with a sequence selected from any one of SEQ ID NO 29-33, or an amino acid sequence having one or more conservative amino acid substitutions as compared to a sequence selected from any one of SEQ ID NO 29-33.
The antibody or antigen-binding fragment of any one of claims 1-5, wherein one of the 2 polypeptide chains further comprises an Fc comprising a hinge region, a second constant region, and a third constant region of a heavy chain.
The antibody or antigen-binding fragment of claim 6, wherein the first polypeptide chain comprises the structure VHa-L1-CLa-L2-VHb-CH1 and the second polypeptide chain comprises the structure VLa-L3-CH1-L4-VLb-CLb; or

The first polypeptide chain comprises the structure VHa-L1-CLa-L2-VHb-CH1-Fc and the second polypeptide chain comprises the structure VLa-L3-CH1-L4-VLb-CLb.
The antibody or antigen-binding fragment of any one of claims 1 to 7, wherein antigens a and 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, 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, MSMA and CD47, EGFR and cMET, CD73 and CD beta, EGFR and TGF beta, CCR2 and CSF1R, CD20 and CD3, CD19 and CD47, FGMA and TRAH and CD47, FGLN and CD2, FGLN and CD47, EGFR and TIL-1, and TFIL-13, and TFIL-1, and CD13, and TFIL-1, and 13, and TFIL-1, and 13.
The antibody or antigen-binding fragment of claim 8, wherein antigen a is TIGIT and antigen b is CTLA-4, the antibody or antigen-binding fragment comprising the following:

the VHa comprises amino acids 30-35 (VHaCDR 1, SSYGMS) and/or amino acids 50-66 (VHaCDR 2, TINSNGGSTYPDSVKG) and/or amino acids 99-108 (VHaCDR 3, LGTGTLGFAY) in the sequence shown in SEQ ID NO. 1; and/or

The VHb comprises 31 th to 35 th amino acids (VHbCDR 1, SYTMH) and/or 50 th to 66 th amino acids (VHbCDR 2, FISYDGNYYADSVKG) and/or 99 th to 107 th amino acids (VHbCDR 3, TGWLGPFDY) in a sequence shown in SEQ ID NO. 2; and/or

The VLa contains amino acids 24-34 (VLaCDR 1, KASQDVKTAVS) and/or amino acids 50-56 (VLaCDR 2, WASTRAT) and/or amino acids 89-97 (VLaCDR 3, QQHYSTPWT) in the sequence shown in SEQ ID NO. 3; and/or

The VLb contains amino acids 24 to 35 (VLbCDR 1, RASQSVGSSYLA) and/or amino acids 51 to 57 (VLbCDR 2, GAFSRAT) and/or amino acids 90 to 98 (VLbCDR 3, QQYGSSPWT) in the sequence shown in SEQ ID NO: 4.
The antibody or antigen-binding fragment of claim 9, wherein the antibody or antigen-binding fragment comprises the following:

the VHa comprises a sequence shown in SEQ ID NO. 1, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 1, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 1; and/or

The VHb comprises a sequence shown as SEQ ID NO. 2, a sequence with at least 80% of identity with the sequence shown as SEQ ID NO. 2, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown as SEQ ID NO. 2; and/or

The VLa comprises a sequence shown as SEQ ID NO. 3, a sequence with at least 80% identity with the sequence shown as SEQ ID NO. 3, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown as SEQ ID NO. 3; and/or

The VLb comprises a sequence shown in SEQ ID NO. 4, a sequence having at least 80% identity with the sequence shown in SEQ ID NO. 4, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO. 4;

the CLa contains a sequence shown by SEQ ID NO. 5, a sequence with at least 80% of identity with the sequence shown by SEQ ID NO. 5, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown by SEQ ID NO. 5;

the CLb comprises a sequence shown by SEQ ID NO. 6, a sequence with at least 80% of identity with the sequence shown by SEQ ID NO. 6, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown by SEQ ID NO. 6;

the CH1 comprises a sequence shown in SEQ ID NO. 7, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 7, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 7.
The antibody or antigen-binding fragment of claim 8, wherein antigen a is OX40 and antigen b is CTLA-4, the antibody or antigen-binding fragment comprising the following:

the VHa contains 31 th to 35 th amino acids (VHaCDR 1, SYGMH) and/or 50 th to 66 th amino acids (VHaCDR 2, VIAEVGSNQYYADSVKG) and/or 99 th to 111 th amino acids (VHaCDR 3, DNQDTSPDVGIDY) in a sequence shown in SEQ ID NO. 8; and/or

The VHb comprises 31 th to 35 th amino acids (VHbCDR 1, SYTMH) and/or 50 th to 66 th amino acids (VHbCDR 2, FISYDGNYYADSVKG) and/or 99 th to 107 th amino acids (VHbCDR 3, TGWLGPFDY) in a sequence shown as SEQ ID NO. 9; and/or

The VLa comprises amino acids 24-34 (VLaCDR 1, RASQNISPFLN) and/or amino acids 50-56 (VLaCDR 2, AAVGLQS) and/or amino acids 89-97 (VLaCDR 3, QQYTDYPLLT) in the sequence shown in SEQ ID NO. 10; and/or

The VLb contains amino acids 24 to 35 (VLbCDR 1, RASQSVGSSYLA) and/or amino acids 51 to 57 (VLbCDR 2, GAFSRAT) and/or amino acids 90 to 98 (VLbCDR 3, QQYGSSPWT) in the sequence shown in SEQ ID NO: 11.
The antibody or antigen-binding fragment of claim 11, wherein the antibody or antigen-binding fragment comprises the following:

the VHa comprises a sequence shown in SEQ ID NO. 8, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 8, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 8; and/or

The VHb comprises a sequence shown in SEQ ID NO. 9, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 9, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 9; and/or

The VLa comprises a sequence shown as SEQ ID NO. 10, a sequence with at least 80% identity to the sequence shown as SEQ ID NO. 10, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown as SEQ ID NO. 10; and/or

The VLb comprises a sequence shown as SEQ ID NO. 11, a sequence having at least 80% identity with the sequence shown as SEQ ID NO. 11, or an amino acid sequence having one or more conservative amino acid substitutions compared with the sequence shown as SEQ ID NO. 11;

the CLa contains a sequence shown in SEQ ID NO. 12, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 12, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 12;

the CLb comprises a sequence shown in SEQ ID NO. 13, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 13, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 13;

the CH1 comprises a sequence shown in SEQ ID NO. 14, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 14, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 14.
The antibody or antigen-binding fragment of claim 8, wherein the antibody or antigen-binding fragment comprises the following:

the VHa contains 31 th to 35 th amino acids (VHaCDR 1, SYGMH) and/or 50 th to 66 th amino acids (VHaCDR 2, VIAEVGSNQYYADSVKG) and/or 99 th to 111 th amino acids (VHaCDR 3, DNQDTSPDVGIDY) in a sequence shown as SEQ ID NO. 15; and/or

The VHb comprises amino acids 30-35 (VHbCDR 1, SSYGMS), and/or amino acids 50-66 (VHbCDR 2, TINSNGGSTYPDSVKG), and/or amino acids 99-108 (VHbCDR 3, LGTGTLGFAY) in the sequence shown in SEQ ID NO. 16; and/or

The VLa comprises amino acids 24-34 (VLaCDR 1, RASQNISPFLN) and/or amino acids 50-56 (VLaCDR 2, AAVGLQS) and/or amino acids 89-97 (VLaCDR 3, QQYTDYPLLT) in the sequence shown in SEQ ID NO. 17; and/or

The VLb contains amino acids 24-34 (VLbCDR 1, KASQDVKTAVS) and/or amino acids 50-56 (VLbCDR 2, WASTRAT) and/or amino acids 89-97 (VLbCDR 3, QQHYSTPWT) in the sequence shown in SEQ ID NO: 18.
The antibody or antigen-binding fragment of claim 13, wherein the antibody or antigen-binding fragment comprises the following:

the VHa comprises a sequence shown in SEQ ID NO. 15, a sequence with at least 80 percent of identity with the sequence shown in SEQ ID NO. 15, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 15; and/or

The VHb comprises a sequence shown in SEQ ID NO. 16, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 16, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 16; and/or

The VLa comprises a sequence shown in SEQ ID NO. 17, a sequence having at least 80% identity to the sequence shown in SEQ ID NO. 17, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO. 17; and/or

The VLb comprises a sequence shown as SEQ ID NO. 18, a sequence with at least 80% identity to the sequence shown as SEQ ID NO. 18, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown as SEQ ID NO. 18;

the CLa contains a sequence shown in SEQ ID NO. 19, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 19, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 19;

the CLb comprises a sequence shown as SEQ ID NO. 20, a sequence with at least 80% of identity with the sequence shown as SEQ ID NO. 20, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown as SEQ ID NO. 20;

the CH1 comprises a sequence shown in SEQ ID NO. 21, a sequence with at least 80% of identity with the sequence shown in SEQ ID NO. 21, or an amino acid sequence with one or more conservative amino acid substitutions compared with the sequence shown in SEQ ID NO. 21.
An antibody or antigen-binding fragment that binds two distinct antigens, a first antigen a and a second antigen b, wherein the first antigen a is TIGIT and the second antigen b is CTLA-4; the antibody or antigen-binding fragment comprises at least a first polypeptide chain and a second polypeptide chain 2 polypeptide chains; said first polypeptide chain comprises the sequence shown in SEQ ID NO 37, a sequence having at least 80% identity to the sequence shown in SEQ ID NO 37, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO 37; said second polypeptide chain comprises the sequence shown in SEQ ID NO. 38, a sequence having at least 80% identity to the sequence shown in SEQ ID NO. 38, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO. 38; or

Said first polypeptide chain comprises the sequence shown in SEQ ID NO:39, a sequence having at least 80% identity to the sequence shown in SEQ ID NO:39, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO: 39; said second polypeptide chain comprises the sequence of SEQ ID NO. 40, a sequence having at least 80% identity to the sequence of SEQ ID NO. 40, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence of SEQ ID NO. 40; or

Said first polypeptide chain comprises the sequence shown in SEQ ID NO. 41, a sequence having at least 80% identity to the sequence shown in SEQ ID NO. 41, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO. 41; said second polypeptide chain comprises the sequence shown in SEQ ID NO. 42, a sequence having at least 80% identity to the sequence shown in SEQ ID NO. 42, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO. 42; or

Said first polypeptide chain comprises the sequence shown in SEQ ID NO 43, a sequence having at least 80% identity to the sequence shown in SEQ ID NO 43, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO 43; the second polypeptide chain comprises the sequence shown as SEQ ID NO. 44, a sequence having at least 80% identity to the sequence shown as SEQ ID NO. 44, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown as SEQ ID NO. 44.
An antibody or antigen-binding fragment comprising at least a first polypeptide chain and a second polypeptide chain 2 polypeptide chains;

the first polypeptide chain comprises a sequence shown as SEQ ID NO. 37, and the second polypeptide chain comprises a sequence shown as SEQ ID NO. 38; or

Said first polypeptide chain comprises the sequence shown in SEQ ID NO:39 and said second polypeptide chain comprises the sequence shown in SEQ ID NO: 40; or

The first polypeptide chain comprises a sequence shown as SEQ ID NO. 41, and the second polypeptide chain comprises a sequence shown as SEQ ID NO. 42; or

The first polypeptide chain comprises the sequence shown in SEQ ID NO. 43, and the second polypeptide chain comprises the sequence shown in SEQ ID NO. 44.
An antibody or antigen-binding fragment that binds to two different antigens a first antigen a and a second antigen b, wherein the first antigen a is OX40 and the second antigen b is CTLA-4; the antibody or antigen-binding fragment comprises at least a first polypeptide chain and a second polypeptide chain of 2 polypeptide chains; said first polypeptide chain comprises the sequence shown in SEQ ID NO:45, a sequence having at least 80% identity to the sequence shown in SEQ ID NO:45, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO: 45; the second polypeptide chain comprises the sequence shown as SEQ ID NO. 46, a sequence having at least 80% identity to the sequence shown as SEQ ID NO. 46, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown as SEQ ID NO. 46.
An antibody or antigen-binding fragment comprising at least a first polypeptide chain and a second polypeptide chain 2 polypeptide chains; the first polypeptide chain comprises the sequence shown in SEQ ID NO. 45, and the second polypeptide chain comprises the sequence shown in SEQ ID NO. 46.
An antibody or antigen-binding fragment that binds to two distinct 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 a first polypeptide chain and a second polypeptide chain 2 polypeptide chains; said first polypeptide chain comprises the sequence shown in SEQ ID NO. 47, a sequence having at least 80% identity to the sequence shown in SEQ ID NO. 47, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO. 47; said second polypeptide chain comprises the sequence shown in SEQ ID NO. 48, a sequence having at least 80% identity to the sequence shown in SEQ ID NO. 48, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO. 48; or

Said first polypeptide chain comprises the sequence shown in SEQ ID NO. 49, a sequence having at least 80% identity to the sequence shown in SEQ ID NO. 49, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO. 49; the second polypeptide chain comprises the sequence shown in SEQ ID NO. 50, a sequence having at least 80% identity to the sequence shown in SEQ ID NO. 50, or an amino acid sequence having one or more conservative amino acid substitutions as compared to the sequence shown in SEQ ID NO. 50.
An antibody or antigen-binding fragment comprising at least a first polypeptide chain and a second polypeptide chain 2 polypeptide chains;

said first polypeptide chain comprises the sequence shown in SEQ ID NO 47 and said second polypeptide chain comprises the sequence shown in SEQ ID NO 48; or

The first polypeptide chain comprises a sequence shown as SEQ ID NO. 49, and the second polypeptide chain comprises a sequence shown as SEQ ID NO. 50.
A nucleic acid molecule encoding the antibody or antigen-binding fragment of any one of claims 1-20.
A vector comprising the nucleic acid molecule of claim 21.
A host cell comprising the nucleic acid molecule of claim 21 or the vector of claim 22.
A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of claims 1-20.
A method of treating or ameliorating various diseases comprising administering to a patient an effective amount of the antibody or antigen-binding fragment of any one of claims 1-20.
Use of the antibody or antigen-binding fragment of any one of claims 1-20 in the manufacture of a medicament for treating or ameliorating various diseases.
The use of claim 26, wherein the disease is selected from the group consisting of: inflammatory diseases, autoimmune diseases, cancer and spinal cord injury.
A diagnostic kit comprising the antibody or antigen-binding fragment of any one of claims 1-20.