CN116284397A

CN116284397A - Antibodies against B7H6 and uses thereof

Info

Publication number: CN116284397A
Application number: CN202211689018.8A
Authority: CN
Inventors: 成赢; 曹国帅; 李洋洋; 武玉伟
Original assignee: Hefei Tiangang Immune Drugs Co ltd
Current assignee: Hefei Tiangang Immune Drugs Co ltd
Priority date: 2022-12-27
Filing date: 2022-12-27
Publication date: 2023-06-23

Abstract

The invention provides an antibody for resisting B7H6 and application thereof, wherein the antibody comprises heavy chain variable region CDR1, CDR2 and CDR3 sequences shown as SEQ ID NO. 1, 2 and 3 or amino acid sequences with at least 95% identity with SEQ ID NO. 1, 2 and 3; and/or the light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO's 4, 5 and 6, or amino acid sequences having at least 95% identity to 4, 5 and 6, respectively. The antibody provided by the embodiment of the invention can be combined with human and monkey B7H6 protein, and the bispecific antibody prepared by using the antibody has higher human and monkey B7H6 binding activity, and can effectively treat and/or prevent related diseases mediated by B7H 6.

Description

Antibodies against B7H6 and uses thereof

Technical Field

The invention relates to the field of biological medicine, in particular to an antibody for resisting B7H6 and application thereof.

Background

Neoplastic disease is the second most common cause of death worldwide. Currently, about 10 monoclonal antibodies against tumor antigen targets have been marketed globally, such as rituximab (for CD20 target), trastuzumab (for HER 2), bevacizumab (for VEGF) and cetuximab (for EGFR). Currently, no drug is marketed for the B7H6 molecule.

B7H6 is a type I transmembrane protein belonging to the B7 family, which contains 2 immunoglobulin domains. B7H6 is also a ligand for NK cell activating receptor NKp 30. B7H6 is expressed on a variety of tumor cells, but mRNA of B7H6 is not detected in human normal tissue and healthy peripheral blood mononuclear cells. In an inflammatory environment, some pro-inflammatory cytokines such as IL-1 beta and TNF can stimulate cd14+cd16+ monocytes and neutrophils to up-regulate B7H6 expression. The tumor expression profile of B7H6 is very broad, including leukemia, lymphoma, colorectal cancer, non-small cell lung cancer, breast cancer, ovarian cancer, gastric cancer, liver cancer, and the like. The expression of B7H6 has also been linked to metastasis of some cancers, but the role of B7H6 in most tumors is currently unclear.

Currently, although B7H6 is expressed in a variety of tumors, there are few tumor treatments for this molecule. Bispecific antibodies against B7H6 have been developed by researchers in the form of bites, which do not contain the constant regions of the native antibody, and thus have determined that the half-life of such molecules in vivo is very short, greatly limiting their use. Meanwhile, the ability to bind to monkey B7H6 is also a factor in antibody selection for non-clinical studies. Antibodies exist today that differ greatly in their ability to bind to monkey B7H6 and therefore present a number of obstacles in non-clinical studies.

Accordingly, there is still a continuing need to develop a B7H 6-specific antibody with higher affinity and crossing of human monkey species, and a bispecific antibody derived from the B7H 6-specific antibody.

Disclosure of Invention

The present application was made based on the findings of the inventors regarding the following problems and facts:

B7H6 is expressed on a variety of tumor cells, but tumor therapy and research approaches for this molecule are not numerous.

The inventor successfully screens out a murine anti-B7H 6 monoclonal antibody, the monoclonal antibody has higher binding activity with human or monkey B7H6 protein, in addition, the inventor performs humanization on a constant region of the monoclonal antibody, retains a CDR of the murine anti-B7H 6 monoclonal antibody so as to obtain a chimeric antibody, and further, performs humanization on a framework region in a light chain variable region or a heavy chain variable region of the chimeric antibody so as to obtain a fully humanized antibody of the anti-B7H 6, and the humanized antibody not only can specifically target and bind to the human B7H6 protein and the monkey B7H6 protein, but also has the characteristic of low immunogenicity, and can effectively treat and/or prevent related diseases mediated by B7H 6.

Furthermore, the bispecific binding molecule or multispecific binding molecule prepared using the anti-B7H 6 antibody is also capable of specifically targeting binding to human B7H6 protein and monkey B7H6 protein, and in general, the bispecific binding molecule or multispecific binding molecule is capable of targeting binding to a cell highly expressing B7H6 based on the specificity of the bispecific binding molecule or multispecific binding molecule, thereby treating or preventing various diseases such as tumors, and furthermore, the murine anti-B7H 6 monoclonal antibody, bispecific binding molecule or multispecific binding molecule is capable of being used in the study of related diseases highly expressing B7H 6.

Thus, in a first aspect of the invention, the invention provides an antibody or antigen binding fragment. According to an embodiment of the invention, comprising heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO's 1, 2 and 3, respectively, or amino acid sequences having at least 80% identity to SEQ ID NO's 1, 2 and 3; and/or the light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO's 4, 5 and 6, or amino acid sequences having at least 80% identity to 4, 5 and 6, respectively. The antibody or antigen binding fragment according to the embodiment of the invention can be combined with human or monkey B7H6 protein, so that the related diseases mediated by B7H6 can be effectively treated or prevented, and related researches are carried out.

According to embodiments of the present invention, the above-described antibody or antigen binding fragment may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the antibody or antigen binding fragment comprises: a heavy chain variable region CDR1 sequence shown in SEQ ID NO. 1, a heavy chain variable region CDR2 shown in SEQ ID NO. 2, a heavy chain variable region CDR3 shown in SEQ ID NO. 3, a light chain variable region CDR1 shown in SEQ ID NO. 4, a light chain variable region CDR2 shown in SEQ ID NO. 5, and a light chain variable region CDR3 shown in SEQ ID NO. 6.

According to an embodiment of the invention, the antibody or antigen binding fragment comprises: at least one of the heavy chain FR region and the light chain FR region.

According to an embodiment of the invention, at least a portion of at least one of the heavy chain FR region and the light chain FR region is derived from at least one of a human antibody, a primate-derived antibody, and a murine antibody or a mutant thereof.

According to an embodiment of the invention, the antibody or antigen binding fragment comprises: at least one of the sequences of heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 shown in SEQ ID NOS 7-10, respectively; or at least one of the sequences of the heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 shown in SEQ ID NOS 15-18, respectively.

According to an embodiment of the invention, the antibody or antigen binding fragment comprises: at least one of the sequences of the light chain framework regions LFR1, LFR2, LFR3 and LFR4 shown in SEQ ID NO. 11-14 respectively; or at least one of the sequences of the light chain framework regions LFR1, LFR2, LFR3 and LFR4 shown in SEQ ID NOS 19-22, respectively.

According to an embodiment of the invention, the antibody or antigen binding fragment comprises: at least one of the sequences of heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 shown in SEQ ID NOS 7-10, respectively; at least one of the sequences of the light chain framework regions LFR1, LFR2, LFR3 and LFR4 shown in SEQ ID NO. 11-14 respectively; or at least one of the sequences of heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 as shown in SEQ ID NOS 15 to 18, respectively; at least one of the sequences of the light chain framework regions LFR1, LFR2, LFR3 and LFR4 shown in SEQ ID NO. 19-22 respectively.

According to an embodiment of the invention, the antibody or antigen binding fragment comprises: a heavy chain variable region as shown in SEQ ID NO. 23 or SEQ ID NO. 25; and/or a light chain variable region as shown in SEQ ID NO. 24 or SEQ ID NO. 26.

According to an embodiment of the invention, the antibody or antigen binding fragment comprises: 1) A heavy chain variable region as shown in SEQ ID NO. 23, and a light chain variable region as shown in SEQ ID NO. 24; or 2) a heavy chain variable region as set forth in SEQ ID NO. 25, and a light chain variable region as set forth in SEQ ID NO. 26.

According to an embodiment of the invention, the antibody or antigen binding fragment contains at least one of a heavy chain constant region and a light chain constant region, at least a portion of which is derived from at least one of a human antibody, a primate-source antibody, and a murine antibody or a mutant thereof.

According to an embodiment of the invention, the light chain constant region and the heavy chain constant region are both derived from a murine IgG antibody or a mutant thereof or a human IgG antibody or a mutant thereof.

According to embodiments of the invention, the light chain constant region and the heavy chain constant region are both derived from a human IgG1 antibody or a mutant thereof.

According to embodiments of the invention, the light chain constant region comprises a kappa, lambda chain of human or murine origin or variants thereof.

According to embodiments of the invention, the antibody has a heavy chain constant region of the amino acid sequence shown as SEQ ID NO. 27 or 29 and/or a light chain constant region of the amino acid sequence shown as SEQ ID NO. 28 or 30.

According to embodiments of the invention, the antibody or antigen binding fragment has a heavy chain with the amino acid sequence set forth in any one of SEQ ID NO. 31, 33 and 35 and a light chain with the amino acid sequence set forth in any one of SEQ ID NO. 32, 34 and 36.

According to an embodiment of the invention, the antibody or antigen binding fragment has a heavy chain with the amino acid sequence shown in SEQ ID NO. 31 and a light chain with the amino acid sequence shown in SEQ ID NO. 32; the antibody or antigen binding fragment has a heavy chain with an amino acid sequence shown as SEQ ID NO. 33 and a light chain with an amino acid sequence shown as SEQ ID NO. 34; or the antibody or antigen binding fragment has a heavy chain with the amino acid sequence shown in SEQ ID NO. 35 and a light chain with the amino acid sequence shown in SEQ ID NO. 36.

According to embodiments of the invention, the antibody or antigen binding fragment comprises a monoclonal antibody or a polyclonal antibody.

According to an embodiment of the invention, the monoclonal antibody comprises at least one of Fv, single chain antibody, fab, single domain antibody, and minimal recognition unit.

According to an embodiment of the invention, the antibody or antigen binding fragment thereof is capable of binding to the amino acid sequences shown in SEQ ID NO. 37 and/or 38.

In a second aspect of the invention, the invention provides a bispecific binding molecule. According to an embodiment of the invention, comprising a first binding region comprising an antibody or antigen binding fragment of the first aspect; and a second binding region, said second binding region having CD3 binding activity. The bispecific binding molecules according to embodiments of the invention can bind to human or monkey B7H6 protein and CD3 protein and can be used in scientific research or to effectively treat or prevent B7H6 and CD3 mediated related diseases.

It will be appreciated by those skilled in the art that the binding activity of the second binding region is not particularly limited and may have other binding activities as long as the diabody has the antibody or antigen-binding fragment of the first aspect and both the antibody or antigen-binding fragment and the second binding region are capable of functioning effectively. In addition, more specific antibodies, such as trispecific, tetraspecific, penta, etc., can also be prepared using the antibodies or antigen-binding fragments described herein, which are capable of targeting to the cell surface of highly expressed antigens, based on their multispecific properties, and eliminating cells that produce such antigens by cell killing.

According to an embodiment of the present invention, the bispecific binding molecule described above may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the bispecific binding molecule comprises a symmetric bispecific binding molecule or an asymmetric bispecific binding molecule.

According to an embodiment of the invention, the bispecific binding molecule is an asymmetric bispecific binding molecule.

According to an embodiment of the invention, the first binding region comprises a peptide chain 1 and a peptide chain 2, wherein the peptide chain 1 comprises the heavy chain variable region as described above and the peptide chain 2 comprises the light chain variable region as described above.

According to an embodiment of the invention, the second binding region comprises at least one of a full length antibody, fv, single chain antibody, fab, single domain antibody, and minimal recognition unit having CD3 binding activity.

According to an embodiment of the invention, the second binding region comprises an anti-CD 3 single chain antibody.

According to an embodiment of the invention, the anti-CD 3 single chain antibody comprises an anti-CD 3 antibody light chain variable region having the amino acid sequence shown in SEQ ID NO. 41 and an anti-CD 3 antibody heavy chain variable region having the amino acid sequence shown in SEQ ID NO. 42.

According to an embodiment of the present invention, the anti-CD 3 single chain antibody further comprises a connecting peptide, wherein the N-terminus of the connecting peptide is linked to the C-terminus of the anti-CD 3 antibody heavy chain variable region, and the C-terminus of the connecting peptide is linked to the N-terminus of the anti-CD 3 antibody light chain variable region; or the N-terminus of the connecting peptide is connected with the C-terminus of the anti-CD 3 antibody light chain variable region, and the C-terminus of the connecting peptide is connected with the N-terminus of the anti-CD 3 antibody heavy chain variable region.

According to an embodiment of the invention, the connecting peptide has the amino acid sequence (GGGGS) n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Those skilled in the art will appreciate that conventional linker peptides in the art, such as conventional flexible amino acid fragments or rigid amino acid fragments, may be used.

According to an embodiment of the invention, the connecting peptide has the amino acid sequence shown in SEQ ID NO. 44.

According to an embodiment of the invention, the anti-CD 3 single chain antibody has an amino acid sequence as shown in SEQ ID NO. 43.

According to an embodiment of the invention, the first binding region further comprises at least one of a first heavy chain constant region and a light chain constant region, at least a portion of which is derived from at least one of a human antibody, a primate-source antibody, and a murine antibody or a mutant thereof.

According to an embodiment of the invention, the first heavy chain constant region and the light chain constant region are both derived from a murine IgG antibody or a mutant thereof or a human IgG antibody or a mutant thereof;

according to an embodiment of the invention, the first heavy chain constant region and the light chain constant region are both derived from a human IgG1 antibody or a mutant thereof.

According to an embodiment of the invention, the first heavy chain constant region has the amino acid sequence shown in SEQ ID NO. 47 and the light chain constant region has the amino acid sequence shown in SEQ ID NO. 48.

According to an embodiment of the invention, the N-terminus of the first heavy chain constant region is linked to the C-terminus of the heavy chain variable region and the N-terminus of the light chain constant region is linked to the C-terminus of the light chain variable region.

According to an embodiment of the invention, the peptide chain 1 has the amino acid sequence shown in SEQ ID NO. 49, and the peptide chain 2 has the amino acid sequence shown in SEQ ID NO. 50.

According to an embodiment of the present invention, the peptide chain 1 and the peptide chain 2 are connected by disulfide bond.

According to an embodiment of the invention, the second binding region further comprises a second heavy chain constant region, at least a portion of which is derived from at least one of a human antibody, a primate-source antibody, and a murine antibody or a mutant thereof.

According to an embodiment of the invention, the second heavy chain constant region is derived from a murine IgG antibody or a mutant thereof or a human IgG antibody or a mutant thereof.

According to an embodiment of the invention, the second heavy chain constant region is derived from a human IgG1 antibody or a mutant thereof. It will be appreciated by those skilled in the art that the amino acid sequences of the first heavy chain constant region and the second heavy chain constant region may be the same or different, for example: when the amino acid sequences of the first heavy chain constant region and the second heavy chain constant region are identical, the two may be linked by disulfide bonds.

According to an embodiment of the invention, the second heavy chain constant region has the amino acid sequence shown as SEQ ID NO. 45.

According to an embodiment of the invention, the N-terminus of the second heavy chain constant region is linked to the C-terminus of the anti-CD 3 single chain antibody.

According to an embodiment of the invention, the second binding region has the amino acid sequence shown as SEQ ID NO. 46.

Nucleic acids encoding the antibodies or antigen binding fragments thereof of the invention or the bispecific binding molecules are within the scope of the invention, from the amino acid sequences thereof, corresponding nucleic acid sequences can be readily obtained by the person skilled in the art.

Thus, in a third aspect of the invention, the invention provides a nucleic acid molecule encoding an antibody or antigen binding fragment of the first aspect or the bispecific binding molecule. An antibody or antigen binding fragment encoded by a nucleic acid molecule according to some embodiments of the invention is capable of binding to a human or monkey B7H6 protein, and is effective in treating or preventing a B7H 6-mediated disease, and a bispecific binding molecule encoded by the nucleic acid molecule is capable of binding to a human or monkey B7H6 protein and a CD3 protein, and is effective in treating or preventing a B7H 6-and CD 3-mediated disease.

According to an embodiment of the present invention, the above-mentioned nucleic acid molecule may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the nucleic acid molecule is DNA.

According to an embodiment of the invention, the nucleic acid molecule comprises SEQ ID NO:39 and 40; or comprises SEQ ID NO: 51. 52 and 53.

It should be noted that, for the nucleic acids mentioned in the present specification and claims, one skilled in the art will understand that either one or both of the complementary double strands are actually included. For convenience, in the present description and claims, although only one strand is shown in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid sequences in the present application include either a DNA form or an RNA form, one of which is disclosed, meaning the other is also disclosed.

In a fourth aspect of the invention, the invention provides an expression vector carrying a nucleic acid molecule as hereinbefore described. The expression vector may include optional control sequences operably linked to the nucleic acid molecule. Wherein the control sequences are one or more control sequences that direct expression of the nucleic acid molecule in a host. The expression vectors presented in the embodiments of the present invention can efficiently express such antibodies or antigen-binding fragments in large amounts in suitable host cells.

"operably linked" herein refers to the linkage of a foreign gene to a vector such that control elements within the vector, such as transcription control sequences and translation control sequences, and the like, are capable of performing their intended functions of regulating transcription and translation of the foreign gene. In the case of attaching the above-mentioned nucleic acid molecule to a vector, the nucleic acid molecule may be directly or indirectly attached to a control element on the vector, as long as the control element is capable of controlling translation, expression, etc. of the nucleic acid molecule. Of course, these control elements may be directly from the carrier itself or may be exogenous, i.e. not from the carrier itself. It will be appreciated by those skilled in the art that the nucleic acid molecules encoding the antibodies or antigen binding fragments may be inserted separately into different vectors, typically into the same vector. The usual vectors may be, for example, plasmids, phages and the like. Such as Plasmid-X.

In a fifth aspect of the invention, the invention provides a method of preparing an antibody or antigen binding fragment or bispecific binding molecule as hereinbefore described comprising: introducing the expression vector described above into a cell; the cells are cultured under conditions suitable for protein expression and secretion to obtain the antibody or antigen binding fragment or bispecific binding molecule. The methods according to some embodiments of the present invention may be effective to obtain large amounts of the antibody or antigen binding fragment or the bispecific binding molecule in vitro.

According to some embodiments of the invention, the above-described method of preparing an antibody or antigen-binding fragment or bispecific binding molecule as described above may further comprise at least one of the following additional technical features:

according to some embodiments of the invention, the cells are not particularly limited, and either prokaryotic or eukaryotic cells may be used.

According to some embodiments of the invention, the cell is a eukaryotic cell.

According to some embodiments of the invention, the eukaryotic cell is a mammalian cell. According to some embodiments of the invention, the recombinant antibody is expressed more efficiently when the cell is a eukaryotic cell, such as a mammalian cell.

In a sixth aspect of the invention, the invention provides a recombinant cell, said recombinant cell being a nucleic acid as described above, or an expression vector, or being capable of expressing an antibody or antigen binding fragment or said bispecific binding molecule as described above. The recombinant cells are obtained by transfecting or transforming the expression vector. According to some embodiments of the invention, the recombinant cells can efficiently and abundantly express the above antibodies or antigen-binding fragments or the bispecific binding molecules under suitable conditions.

It should be noted that the recombinant cells of the present invention are not particularly limited, and may be prokaryotic cells, eukaryotic cells, or phage. The prokaryotic cell can be escherichia coli, bacillus subtilis, streptomycete or proteus mirabilis and the like. The eukaryotic cells comprise fungi such as pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, trichoderma and the like, insect cells such as armyworm and the like, plant cells such as tobacco and the like, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells and the like. In some embodiments, the recombinant cells of the invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

The term "suitable conditions" as used herein refers to conditions suitable for expression of the antibodies or antigen binding fragments or bispecific binding molecules described herein. Those skilled in the art will readily appreciate that conditions suitable for expression of the antibody or antigen binding fragment or the bispecific binding molecule include, but are not limited to, suitable transformation or transfection means, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, suitable cell culture time. The "suitable conditions" are not particularly limited, and one skilled in the art can optimize the conditions for optimal expression of the antibody or antigen binding fragment or the bispecific binding molecule according to the particular circumstances of the laboratory.

In a seventh aspect of the invention, the invention provides an immunoconjugate comprising the antibody or antigen binding fragment or the bispecific binding molecule as described hereinbefore, and a therapeutic agent. As described above, the antibody or antigen binding fragment of the embodiment of the present invention can bind to B7H6 protein effectively, and the bispecific binding molecule can bind to B7H6 protein and CD3 protein of human or monkey, and is effective in treating or preventing B7H6 and CD 3-mediated related diseases, so that the immunoconjugate comprising the antibody or antigen binding fragment can bind to B7H6 protein of human or monkey, and the immunoconjugate comprising the bispecific binding molecule can bind to B7H6 protein and CD3 protein of human or monkey, and has good effect in preventing and/or treating B7H 6-mediated diseases, or B7H6 and CD 3-mediated related diseases.

In an eighth aspect of the invention, the invention provides a composition comprising an antibody or antigen binding fragment, bispecific binding molecule, nucleic acid molecule, expression vector or recombinant cell as described above. As described above, the antibody or antigen binding fragment according to some embodiments of the present invention can effectively bind to human or monkey B7H6 protein, and the bispecific binding molecule can bind to human or monkey B7H6 protein and CD3 protein and effectively inhibit proliferation of tumor cells, and thus, the composition comprising the same can also effectively bind to human or monkey B7H6 protein, or to human or monkey B7H6 protein and CD3 protein, and has good effect of preventing and/or treating B7H 6-mediated diseases, and the kind of the composition is not particularly limited, and may be a food composition or a pharmaceutical composition.

The compositions of the invention may also be administered in combination with each other, or with one or more other therapeutic compounds, for example, with a chemotherapeutic agent. Thus, the composition may also contain a chemotherapeutic agent. The antibodies, or antigen binding fragments thereof, or immunoconjugates of the invention may also be combined with a second therapeutic agent, exemplary agents of which include, but are not limited to, other agents that inhibit B7H6 activity (including other antibodies or antigen binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and/or agents that interfere with B7H6 upstream or downstream signaling.

It is noted that the compositions include combinations that are separated in time and/or space, so long as they are capable of co-acting to achieve the objects of the present invention. For example, the ingredients contained in the composition may be administered to the subject in whole or separately. When the components contained in the composition are separately administered to a subject, the individual components may be administered to the subject simultaneously or sequentially.

In a ninth aspect of the invention, the invention provides a medicament comprising an antibody or antigen binding fragment, bispecific binding molecule, nucleic acid molecule, expression vector, recombinant cell or composition as described hereinbefore. As described above, the antibody or antigen binding fragment of some embodiments of the present invention can effectively bind to human or monkey B7H6 protein, and the bispecific binding molecule can bind to human or monkey B7H6 protein and CD3 protein, and thus, a drug comprising an effective amount of the antibody or antigen binding fragment or the active ingredient of the bispecific binding molecule or a series of substances thereof can also effectively bind to human or monkey B7H6 protein, or to human or monkey B7H6 protein and CD3 protein, with good effect of preventing and/or treating B7H 6-mediated diseases.

According to an embodiment of the present invention, the above-mentioned medicament may further include at least one of the following additional technical features:

according to an embodiment of the invention, the medicament may further comprise a pharmaceutically acceptable carrier.

As used herein, the term "effective amount" or "effective dose" refers to an amount that is functional or active in and acceptable to a human and/or animal.

The effective amount of the antibodies or antigen binding fragments or the bispecific binding molecules of the invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, as dictated by the urgent need for the treatment of the condition.

As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The medicament of the invention contains safe and effective amount of the active ingredients of the invention and pharmaceutically acceptable carriers. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should be matched with the administration mode, wherein the administration mode can be oral administration, nasal administration, intradermal administration, subcutaneous administration, intramuscular administration, intravenous administration or intraperitoneal administration, and the dosage form of the medicine is injection, oral preparation (tablet, capsule, oral liquid), transdermal agent or sustained release agent. For example, by using physiological saline or an aqueous solution containing glucose and other auxiliary agents by conventional methods. The medicament is preferably manufactured under aseptic conditions. The antibody or antigen binding fragment may be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.

Of course, the anti-B7H 6 mab or bispecific binding molecule herein can also be made part of a kit or other diagnostic reagent as desired.

In a tenth aspect of the invention, the invention provides a kit comprising an antibody or antigen binding fragment thereof, a bispecific binding molecule, a nucleic acid molecule, an expression vector or a recombinant cell as described above. As previously described, the antibodies or antigen binding fragments of some embodiments of the invention are effective to bind to human or monkey B7H6 protein, the bispecific binding molecules are effective to bind to human or monkey B7H6 protein and CD3 protein, and therefore, kits comprising the antibodies or antigen binding fragments are effective to qualitatively or quantitatively detect human or monkey B7H6 protein, and kits comprising the bispecific binding molecules are effective to qualitatively or quantitatively detect human or monkey B7H6 protein and CD3 protein. The kit provided by the invention can be used for immunoblotting, immunoprecipitation and the like, and relates to a kit for detection by utilizing the specific binding performance of human or monkey B7H6 and antibodies. These kits may comprise any one or more of the following: an antagonist, an anti-B7H 6 antibody or a drug reference material; a protein purification column; immunoglobulin affinity purification buffers; cell assay diluent; instructions, literature, etc. The anti-B7H 6 antibodies can be used in different types of diagnostic tests, for example, to detect a wide variety of diseases or the presence of drugs, toxins or other proteins in vitro or in vivo, for example, by detecting serum or blood from a subject, for testing for related diseases, or for research studies, using the kit to detect human or monkey B7H6 protein, or human or monkey B7H6 protein and CD3 protein in a test sample. Such related diseases may include B7H6 related diseases, such as cancer. Of course, the antibodies or antigen binding fragments provided herein may also be used in radioimmunoassays, radioimmunotherapy, and the like for the above-described diseases. For the above application scenario, the bispecific binding molecules are equally applicable and will not be described here.

The kit may also include reagents conventionally used for detecting B7H6, or B7H6 and CD3, such as coating solutions and the like.

In an eleventh aspect of the invention, the invention provides the use of an antibody or antigen binding fragment thereof nucleic acid molecule, expression vector, recombinant cell or composition as described hereinbefore for the manufacture of a medicament for the prevention and/or treatment of a B7H6 mediated related disease. As described above, the antibody or antigen-binding fragment of some embodiments of the present invention can effectively bind to human or monkey B7H6 protein, and thus, a drug comprising an effective amount of the antibody or antigen-binding fragment or a series of substances thereof can also effectively bind to human or monkey B7H6 protein, with good prophylactic and/or therapeutic effects on B7H 6-mediated diseases.

According to an embodiment of the present invention, the above-mentioned use for preparing a medicament may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the B7H 6-mediated related disease comprises cancer.

According to an embodiment of the invention, the cancer comprises at least one of the following: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal carcinoma, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, renal cancer, esophageal cancer, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma.

In a twelfth aspect of the invention, the invention provides the use of a bispecific binding molecule, nucleic acid molecule, expression vector, recombinant cell or composition as described hereinbefore for the preparation of a medicament for the prevention and/or treatment of B7H6 and CD3 mediated related diseases. As described above, the bispecific binding molecules of some embodiments of the present invention are capable of binding to human or monkey B7H6 protein and CD3 protein, and thus, a drug comprising an effective amount of the active ingredient of the bispecific binding molecule or a series of substances thereof can also bind to human or monkey B7H6 protein and CD3 protein effectively, with good effect of preventing and/or treating B7H6 and CD3 mediated related diseases.

according to embodiments of the invention, the B7H6 and CD3 mediated related diseases include cancer.

In a thirteenth aspect of the invention, the invention provides the use of an antibody or antigen binding fragment, nucleic acid molecule, expression vector or recombinant cell as described hereinbefore in the preparation of a kit for the detection of B7H6. As previously mentioned, the antibodies or antigen binding fragments of some embodiments of the invention are effective in binding to human or monkey B7H6 protein and blocking binding of the B7H6 protein to its receptor, and thus, the antibodies or antigen binding fragments can be used to prepare kits for detecting B7H6 protein which are effective in the qualitative or quantitative detection of B7H6 protein in humans or monkeys.

In a fourteenth aspect of the invention, the invention provides the use of a bispecific binding molecule, nucleic acid molecule, expression vector or recombinant cell as described hereinbefore for the preparation of a kit for the detection of B7H6 and/or CD3. As previously mentioned, the bispecific binding molecules of some embodiments of the invention are capable of efficiently binding to human or monkey B7H6 protein and CD3 protein, and thus, the bispecific binding molecules can be used to prepare a kit for detecting B7H6 and/or CD3, which is capable of efficiently performing a qualitative or quantitative detection of B7H6 and/or CD3 in a human or monkey.

In a fifteenth aspect of the invention, the invention provides a method of treating or preventing B7H6, or a B7H6 and/or CD3 mediated related disease. According to an embodiment of the invention, the method comprises administering to the subject at least one of: 1) An antibody or antigen binding fragment as described previously; 2) The bispecific binding molecules described above; 3) A nucleic acid molecule as described above; 4) The expression vector described above; 5) Recombinant cells as described above; 6) A composition as described above; and 7) the aforementioned drugs. As previously described, the bispecific binding molecules are capable of binding to human or monkey B7H6 protein and CD3 protein, the antibodies or antigen binding fragments are capable of binding to human or monkey B7H6 protein, are capable of treating or preventing B7H6, or B7H6 and/or CD3 mediated related diseases, preferably cancer, and thus the methods according to embodiments of the invention are capable of treating or preventing B7H6, or B7H6 and/or CD3 mediated related diseases, such as cancer.

According to an embodiment of the present invention, the above method for treating or preventing a disease may further include at least one of the following additional technical features:

according to embodiments of the invention, the B7H 6-mediated disease or the B7H6 and CD 3-mediated disease comprises cancer.

In a sixteenth aspect of the invention, the invention provides a method of diagnosing B7H6, or a B7H6 and/or CD3 mediated related disease. According to an embodiment of the invention, comprising detecting B7H6, or B7H6 and/or CD3 in a sample to be tested using at least one of the following: 1) An antibody or antigen binding fragment as described previously; 2) The bispecific binding molecules described above; 3) A nucleic acid molecule as described above; 4) The expression vector described above; and 5) determining the content of B7H6 or B7H6 and/or CD3 in the sample to be tested based on the detection result of B7H6 or B7H6 and/or CD3 of the recombinant cells. The antibody or antigen binding fragment, or nucleic acid molecule, expression vector, recombinant cell expressed antibody or antigen binding fragment can be effectively combined with human or monkey B7H6 protein, and the bispecific binding molecule, or nucleic acid molecule, expression vector, recombinant cell expressed bispecific binding molecule can be effectively combined with B7H6, or B7H6 and/or CD3, so that the method can be used for effectively detecting the content of B7H6, or B7H6 and/or CD3 in a sample to be detected from a tested individual, and can be used for effectively diagnosing related diseases caused by B7H6, or B7H6 and/or CD 3.

According to an embodiment of the present invention, the above method for diagnosing a disease may further include at least one of the following additional technical features:

according to an embodiment of the invention, the content of B7H6, or B7H6 and/or CD3 in the test sample is not lower than the minimum criterion for the disease, which is an indication that the test sample is derived from a patient suffering from B7H6, or B7H6 and/or CD 3-induced related disease. The value of the minimum standard can be determined by comparing and verifying the content of B7H6 or B7H6 and/or CD3 in a sample to be tested of a large number of individuals suffering from the B7H6 or B7H6 and/or CD3 related diseases and a large number of healthy individuals.

According to an embodiment of the invention, the sample to be tested comprises at least one of the following: blood, saliva, sweat, tissue, cells, blood, serum, plasma, feces, and urine.

According to embodiments of the invention, the B7H 6-mediated related diseases and the B7H6 and CD 3-mediated related diseases include cancer.

In a sixteenth aspect of the invention, the invention provides a method of staging B7H6, or B7H6 and/or CD3 mediated diseases. According to an embodiment of the invention, comprising detecting B7H6, or B7H6 and/or CD3 in a sample to be tested using at least one of the following: 1) An antibody or antigen binding fragment as described previously; 2) The bispecific binding molecules described above; 3) A nucleic acid molecule as described above; 4) The expression vector described above; and 5) determining the content of B7H6 or B7H6 and/or CD3 in the sample to be tested based on the detection result of B7H6 or B7H6 and/or CD3 of the recombinant cells. The bispecific binding molecules, or nucleic acid molecules, expression vectors and recombinant cell expressed bispecific binding molecules can be effectively bound with B7H6 and/or CD3 of a human or monkey, and the antibodies or antigen binding fragments, or the nucleic acid molecules, expression vectors and recombinant cell expressed antibodies or antigen binding fragments can be effectively bound with B7H6 protein of the human or monkey, so that the method can be used for effectively detecting the content of B7H6, or B7H6 and/or CD3 in a sample to be tested from a tested individual, and evaluating the period of the B7H6, or B7H6 and/or CD3 caused by related diseases based on the content of the B7H6, or B7H6 and/or CD 3.

According to an embodiment of the present invention, the method for staging a disease described above may further include at least one of the following additional technical features:

according to an embodiment of the invention, the content of B7H6, or B7H6 and/or CD3 in the test sample not lower than the standard level of disease in stage IV of the tumor is an indication that the test sample is derived from a patient suffering from stage IV of the tumor, and the content of B7H6, or B7H6 and/or CD3 in the test sample is between the standard levels of disease in stage IV and stage III of the tumor is an indication that the test sample is derived from a patient suffering from stage III of the tumor; the content of B7H6 or B7H6 and/or CD3 in the sample to be tested is between the standard levels of tumor stage III and stage II diseases, which is an indication that the sample to be tested is derived from a patient suffering from tumor stage II; the presence of B7H6, or B7H6 and/or CD3 in the test sample between the standard levels of stage I and stage II disease is an indication that the test sample is derived from a patient with stage I tumor. It will be appreciated by those skilled in the art that the level of B7H6, or B7H6 and/or CD3 in the tumor stage I, II, III, IV will vary depending on the type of tumor, and that the stage of the tumor will be determined by comparing the level of B7H6, or B7H6 and/or CD3 in the sample to be tested with the corresponding standard level of B7H6, or B7H6 and/or CD3 in the tumor stage, or by comparing the level of B7H6, or B7H6 and/or CD3 in the sample to be tested with the level of B7H6, or B7H6 and/or CD3 in the sample from an individual or population of known disease stage. The values of the standard levels of stage I, stage II, stage III and stage IV of the tumor can be determined by comparing and analyzing and verifying differences in the amounts of B7H6 or B7H6 and/or CD3 in a test sample of a plurality of individuals suffering from the angiogenesis-related diseases and a plurality of healthy individuals.

In a seventeenth aspect of the invention, the invention provides a method for assessing the prognosis of B7H6, or B7H6 and/or CD3 mediated related diseases. According to an embodiment of the invention, comprising detecting B7H6, or B7H6 and/or CD3 in a sample to be tested using at least one of the following: 1) The bispecific binding molecules described above; 2) An antibody or antigen binding fragment as described previously; 3) A nucleic acid molecule as described above; 4) The expression vector described above; and 5) determining the content of B7H6 or B7H6 and/or CD3 in the sample to be tested based on the detection result of B7H6 or B7H6 and/or CD3 of the recombinant cells. As previously mentioned, the content of B7H6, or B7H6 and/or CD3 has an important effect on cancer, and after an individual suffering from a related disease is treated, the prognosis of the disease can be effectively assessed by monitoring the content of B7H6, or B7H6 and/or CD3 in the tissue or excreta thereof, such as peripheral blood, urine, etc., for example, the content of B7H6, or B7H6 and/or CD3 in a subject before and after the treatment is compared, or the content of B7H6, or B7H6 and/or CD3 in the subject after the treatment is compared with the content of B7H6, or B7H6 and/or CD3 in a normal individual or a subject suffering from the disease, and the bispecific binding molecules, or nucleic acid molecules, expression vectors or recombinant cell expressed bispecific binding molecules can be effectively bound to B7H6 and/or CD3, or nucleic acid molecules, expression vectors, recombinant cell expressed antibodies or antigen binding fragments can be effectively bound to B7H6, or human antibodies expressed by recombinant cells, and can be effectively detected by the antibodies or CD 6 binding to B7H6, or CD 6 expressed by the antibodies and/or CD3, and the bispecific binding molecules expressed by the nucleic acid molecules, expression vectors and/or CD3 can be effectively detected by the antibodies and CD 7H6 expressed by the antibodies.

According to an embodiment of the present invention, the above method of assessing disease prognosis may further comprise at least one of the following additional technical features:

according to an embodiment of the invention, the sample to be tested is derived from a patient suffering from B7H6, or a B7H6 and/or CD3 mediated related disease, before or after treatment.

According to an embodiment of the invention, the prognostic effect of B7H6, or B7H6 and/or CD3 mediated related diseases is determined based on the content of B7H6, or B7H6 and/or CD3 in the test sample of the patient suffering from B7H6, or B7H6 and/or CD3 mediated related diseases before or after the treatment.

According to an embodiment of the invention, a decrease in B7H6, or B7H6 and/or CD3 content in the test sample of a patient suffering from B7H6, or B7H6 and/or CD3 mediated related disease after treatment is an indication that the patient's prognosis is good.

In an eighteenth aspect of the invention, the invention proposes the use of a bispecific binding molecule, antibody or antigen binding fragment, nucleic acid molecule, expression vector, recombinant cell, composition or medicament as described hereinbefore for the treatment or prophylaxis of B7H6, or B7H6 and/or CD3 mediated related diseases. As described above, the bispecific binding molecules have high binding activity to B7H6 and/or CD3, and the antibodies or antigen binding fragments are capable of binding to B7H6 in humans or monkeys, and are effective in treating or preventing B7H6, or B7H6 and/or CD3 mediated related diseases.

According to an embodiment of the present invention, the above-mentioned use may further comprise at least one of the following additional technical features:

In a nineteenth aspect of the invention, the invention provides the use of a bispecific binding molecule, antibody or antigen binding fragment, nucleic acid molecule, expression vector or recombinant cell as described hereinbefore for diagnosis of B7H6, or B7H6 and/or CD3, or for staging of B7H6, or B7H6 and/or CD3 mediated related diseases or for assessment of B7H6, or B7H6 and/or CD3 mediated related disease prognosis. As described above, the bispecific binding molecules, or nucleic acid molecules, expression vectors or recombinant cell-expressed bispecific binding molecules, provided herein can be effectively bound to B7H6 and/or CD3, and the antibodies or antigen-binding fragments, or nucleic acid molecules, expression vectors, recombinant cell-expressed antibodies or antigen-binding fragments, can be effectively bound to B7H6 in humans or monkeys, so that the methods described herein can be used to effectively detect B7H6, or B7H6 and/or CD3 content in a test sample derived from a subject, and can be used to effectively diagnose, stage and prognosticate B7H6, or B7H6 and/or CD 3-mediated related diseases.

The term "subject" or "individual" as used herein refers generally to mammals, such as primates and/or rodents, and in particular humans, monkeys or mice.

The invention has the beneficial effects that:

1) Compared with the existing B7H6 monoclonal antibody, the murine monoclonal antibody against B7H6 has higher B7H6 protein binding activity, and the B7H6 protein comprises human B7H6 protein and monkey B7H6 protein.

2) The humanized antibody obtained by humanizing the murine anti-B7H 6 monoclonal antibody has higher B7H6 protein binding activity than the existing B7H6 monoclonal antibody, the B7H6 protein comprises human B7H6 protein and monkey B7H6 protein, and the humanized antibody has lower immunogenicity, higher safety and longer efficacy.

Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Drawings

The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:

FIG. 1A is a graph of ELISA results for B7H6 antibodies binding to human B7H6 according to an embodiment of the invention;

FIG. 1B is a graph of ELISA results for B7H6 antibodies binding to monkey B7H6 according to an embodiment of the invention;

FIG. 2A is a flow chart of results of binding of B7H6 antibodies to CHO cells overexpressing human B7H6 according to an embodiment of the invention;

FIG. 2B is a flow chart of results of binding of B7H6 antibodies to CHO cells overexpressing monkey B7H6 according to an embodiment of the invention;

FIG. 3 is a graph showing the results of an in vitro cytotoxicity of B7H6/CD3 bispecific binding protein and PBMC against HCT-15 cells according to an embodiment of the invention.

Detailed Description

Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.

It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless clearly defined otherwise herein in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

Antibodies or antigen binding fragments of the invention are typically prepared by biosynthetic methods. The coding nucleic acids according to the invention can be prepared by various known methods, conveniently by the person skilled in the art, based on the nucleotide sequences according to the invention. Such as, but not limited to: PCR, DNA synthesis, etc., and specific methods can be found in J.Sam Brookfield, guidelines for molecular cloning experiments. As one embodiment of the present invention, the coding nucleic acid sequence of the present invention can be constructed by a method of synthesizing nucleotide sequences in segments followed by overlap extension PCR. Wherein the antibody or antigen fragment is numbered and defined using the Kabat numbering system.

Antibodies of the invention include murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.

The term "murine antibody" is herein a monoclonal antibody to human B7H6 prepared according to the knowledge and skill in the art. The preparation is performed by injecting the test subjects with an antigen, and then isolating hybridomas expressing antibodies having the desired sequence or functional properties. In a preferred embodiment of the invention, the murine B7H6 antibody or antigen binding fragment thereof may further comprise a light chain constant region of murine kappa, lambda chain or variants thereof, or further comprise a heavy chain constant region of murine IgG1, igG2, igG3 or variants thereof.

The term "chimeric antibody (chimeric antibody)" refers to an antibody in which a variable region of a murine antibody is fused to a constant region of a human antibody, and which can reduce an immune response induced by the murine antibody. Cloning variable region genes from mouse hybridoma cells, cloning constant region genes of human antibodies as required, connecting the mouse variable region genes and the human constant region genes into chimeric genes, inserting the chimeric genes into human vectors, and finally expressing chimeric antibody molecules in eukaryotic expression systems or prokaryotic expression systems. In a preferred embodiment of the invention, the antibody light chain of the B7H6 chimeric antibody further comprises a light chain Fc region of a human kappa, lambda chain or variant thereof. The antibody heavy chain of the B7H6 chimeric antibody further comprises a heavy chain constant region of human IgG1, igG2, igG3, igG4, or variants thereof.

The term "humanized antibody (humanized antibody)", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting the CDR sequences of a mouse into the framework of the variable region of a human antibody, i.e., into the framework sequences of a different type of human germline antibody. Such framework sequences may be obtained from public DNA databases including germline antibody gene sequences or published references. Germline DNA sequences for human heavy and light chain variable region genes, for example, can be found in the "VBase" human germline sequence database. To avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region framework sequences may be subjected to minimal reverse or back-mutations to maintain activity.

Herein, the "monoclonal antibody" refers to an antibody having a single antigen binding site.

Herein, the "diabody" refers to an antibody having two different antigen binding sites.

As used herein, the term "mutant" or "variant" may refer to a molecule comprising a mutation of one or more nucleotides or amino acids of any naturally occurring or engineered molecule.

The term "complementarity determining region" or "CDR sequence" refers to an amino acid sequence in an antibody responsible for antigen binding, e.g., generally comprising: amino acid residues in the light chain variable region in the vicinity of 23-34 (L1), 50-56 (L2) and 89-97 (L3), and in the vicinity of 31-35B (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable region (Kabat et al, sequences ofProteins ofImmunological Interest,5th Ed.Public Health Service,National Institutes ofHealth,Bethesda,MD. (1991)); and/or from "hypervariable loops" (e.g., 26-32 (LI), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and amino acid residues near 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and LeskJ. Mol. Biol.196:901-917 (1987)).

In this context, the term "identity" is used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences when compared to the amino acid sequence or nucleic acid sequence of a reference sequence, using conventional methods, e.g., see, ausubel et al, eds. (1995), current Protocols in Molecular Biology, chapter 19 (Greene Publishing andWiley-Interscience, new York); and the ALIGN program (Dayhoff (1978), atlas ofProtein Sequence and Structure 5: support.3 (National Biomedical Research Foundation, washington, D.C.), there are many algorithms for alignment and determination of sequence identity, including homology alignment algorithms of needle et al (1970) J.mol.biol.48:443, computer programs using these algorithms are also available and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or the programs available in Peason et al (1988) Proc.Natl.Acad.Sci.85:2444, smith-Waterman algorithm (Meth.mol.70:173-187 (1997), and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al (1990) J.mol.biol.215:403-410), and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or the programs available in BLAST-5:35, and the programs available in the company of Witsin the open area, inc. 35, and the methods of the company, inc. 35, and the methods provided by the methods of the company, inc. Proc.Natl.Acad.Sci.85:2444, and the methods of the invention.

One skilled in the art may replace, add and/or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids to the sequences of the invention to obtain variants of the sequences of the antibodies or functional fragments thereof without substantially affecting the activity of the antibodies (retaining at least 95% of the activity). They are all considered to be included within the scope of the present invention. Such as substitution of amino acids with similar properties in the variable region. The variant sequences of the invention may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity (or homology) to a reference sequence. Sequence identity as described herein can be measured using sequence analysis software. Such as computer programs BLAST, in particular BLASTP or TBLASTN, using default parameters. The amino acid sequences mentioned in the present invention are all shown in N-terminal to C-terminal fashion.

As previously mentioned, a mab of the invention may be a full length antibody or may comprise only a functional fragment thereof (e.g., fab, F (ab') 2, or scFv fragment), or may be modified to affect function. The invention includes anti-B7H 6 antibodies with modified glycosylation patterns. In some applications, it may be useful to modify to remove undesired glycosylation sites, or antibodies in which no fucose moiety is present on the oligosaccharide chain, for example to enhance Antibody Dependent Cellular Cytotoxicity (ADCC) function. In other applications, galactosylation modifications may be made to alter Complement Dependent Cytotoxicity (CDC).

Herein, the "full length antibody" is a tetrapeptide chain structure formed by connecting two identical light chains and two identical heavy chains through inter-chain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin a (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE). The same class of immunoglobulins can also be of different subclasses, e.g., igG1, igG2, igG3, igG4, depending on the amino acid composition. Immunoglobulin light chains are classified as either kappa chains or lambda chains depending on the constant region.

The term "functional fragment" as used herein refers in particular to an antibody fragment such as a CDR-grafted antibody, fab ', F (ab') 2, fv or scFv, nanobody, or any fragment which should be able to increase half-life by chemical modification, e.g. addition of a poly (alkylene) glycol such as polyethylene glycol ("pegylation, pegylation") (a pegylated fragment known as Fv-PEG, scFv-PEG, fab-PEG, F (ab ') 2-PEG or Fab' -PEG) ("PEG" is polyethylene glycol) which has B7H6 binding activity, or by incorporation into liposomes. Preferably, the functional fragment will consist of or comprise a partial sequence of the heavy chain variable region or the light chain variable region of its source antibody, which partial sequence is sufficient to retain the same binding specificity and sufficient affinity as its source antibody, preferably at least equal to 1/100, more preferably at least equal to 1/10, for B7H 6. Such functional fragments will comprise a minimum of 3 amino acids, preferably 5, 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which they are derived.

In the present invention, the term "antigen-binding fragment" as used without being otherwise specified generally refers to an antigen-binding antibody fragment, which may comprise a portion of an intact antibody, typically an antigen-binding or variable region, as exemplified by CDR-grafted antibodies, fab ', F (ab') 2, fv or scFv, nanobodies, and the like.

Herein, the term "CDR-grafted antibody" refers to the grafting of CDRs of a species mab to antibody variable regions of another species. For example, the CDRs of a murine mab may be grafted to the variable regions of a human antibody in order to replace the CDRs of a human antibody, allowing the human antibody to acquire the antigen binding specificity of the murine mab while reducing its heterology.

In this context, the term "Fab antibody" or "Fab" generally refers to an antibody comprising only Fab molecules, consisting of VH and CH1 of the heavy chain and the complete light chain, linked by a disulfide bond between the light and heavy chains.

In this context, the term "nanobody" (single domain antibody or VHH antibody), which was originally described as an antigen binding immunoglobulin (variable) domain of a "heavy chain antibody" (i.e. "antibody lacking a light chain") (Hamers-Casterman C, ataroucht, muydermans S, robinson G, hamers C, songa EB, bendahman N, hamers R.: "Naturallyoccurring antibodies devoid oflight chains"; nature 363,446-448 (1993)), comprises only a heavy chain variable region (VH) and conventional CH2 and CH3 regions, which specifically bind antigen via the heavy chain variable region.

As used herein, the term "Fv antibody" generally refers to an antibody consisting of only the light chain variable region (VL) and the heavy chain variable region (VH) joined by a non-covalent bond, which is the smallest functional fragment of an antibody that retains the intact antigen-binding site.

As used herein, the term "single chain antibody" or "scFv" is a fragment of an antibody in which the heavy and light chain variable regions are linked by a short peptide.

As used herein, a "Knob intonation hole structure" is a button (Knob) forming mutation in the CH3 region of the heavy chain constant region of an antibody that facilitates heavy chain occlusion to form a heterodimer, e.g., by mutating amino acids in the CH3 domain of the heavy chain constant region of a human IgG1 (in this application, the T366S, L368A, Y407V, Y349C mutation, i.e., "hole", in one chain and the T366W, S354C mutation, i.e., "Knob") in the other chain. Wherein the amino acid numbering herein is according to the Kabat numbering system, e.g. "T366S" means that the T amino acid at position 366 according to the Kabat numbering system is replaced by an S amino acid.

The amino acid or nucleic acid sequences involved in the present invention are shown in Table 1.

Table 1:

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the present invention will be described in detail by examples. In the examples and test examples, the experimental methods were carried out under conventional conditions without specifying the specific conditions.

The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

EXAMPLE 1 preparation of anti-human B7H6 hybridoma monoclonal antibodies

This example was used to obtain murine monoclonal antibodies against human B7H6, wherein the immunizing antigens were human B7H6 (ACRO, B76-H52H8, SEQ ID NO: 37) and monkey B7H6 protein (ACRO, B76-C52Ha, SEQ ID NO: 38), and C57bl/6 mice (9 weeks old, purchased from Shanghai rice, body weight 20g or so).

Immunized mice were immunized 3 times intraperitoneally with purified antigen and complete Freund's adjuvant, and the immune response was detected after bleeding through the tail vein. Serum was screened by ELISA and flow cytometry by conventional procedures to obtain mice bearing anti-human B7H6 immunoglobulins. And spleen cells from mice with the highest anti-B7H 6 immunoglobulin were taken out and fused with murine myeloma cell SP2/0 (ATCC No. CRL-1581). The fused hybridoma cells were resuspended in HAT complete medium (RPMI-1640 medium containing 20% FBS, 1 XHAT and 1 XOPI) and plated in 96-well cell culture plates and incubated at 37℃with 5% CO 2. HAT complete medium was added at day 5 post-fusion, 50 μl/well. From day 7 to day 8 after fusion, the medium was replaced with HT complete medium (RPMI-1640 medium containing 20% FBS, 1 XHT and 1 XOPI) at 200. Mu.L/well, depending on the cell growth density.

Flow cytometry binding assays were performed on day 10-11 post-fusion, based on cell growth density. The positive wells were changed and expanded into 24 well plates in time according to cell density. The cell lines engrafted into 24-well plates were retested for seed protection and first subcloning. Seed protection is carried out when the first subclone screening is positive, and the second subclone is carried out. And performing seed conservation and protein expression on the positive secondary subcloning. Antibodies were further prepared by serum-free cell culture and purified by protein G affinity chromatography for subsequent functional activity detection. .

EXAMPLE 2 hybridoma cell sequencing

The total number of candidate hybridoma cells selected in example 1 above was cultured to 10 ⁶ Cells were collected by centrifugation at 800rpm for 10 minutes and total RNA was extracted with Trizol kit (Invitrogen); the cDNA library (Invitrogen) was synthesized by reverse transcription using total RNA as a template, and the corresponding B7H6 antibody variable region nucleic acid sequence of the hybridoma cells was PCR amplified using cDNA as a template. The primer sequences used in the PCR amplification reaction are complementary to the antibody variable region first framework region or the signal peptide region and the constant region (specific sequencesReference is made to Larrick, j.w., et al, (1990) scand.j.immunol.,32, 121-128 and Coloma, j.j.et al, (1991) BioTechniques,11, 152-156). PCR amplification was performed in a 50. Mu.L reaction system, with the addition of 2. Mu.L of cDNA, 5. Mu.L of 10 XPCR buffer, 2. Mu.L of upstream and downstream primers (5. Mu.M), 2. Mu.L of dNTP, 1. Mu.L of Taq enzyme (Takara, ex Taq), H ₂ O38. Mu.L; pre-denaturation at 95 ℃ for 5min, and entering into temperature circulation for PCR amplification. The reaction conditions are as follows: denaturation at 94℃for 30S, annealing at 58℃for 45S, extension at 72℃for 50S for 32 cycles, and then extension at 72℃for 7min. After sequencing the amplified product, the heavy chain variable region (SEQ ID NO: 23) and light chain variable region (SEQ ID NO: 24) sequences of anti-B7H 6 murine monoclonal antibody 4-99 were obtained.

EXAMPLE 3 humanization of anti-human B7H6 monoclonal antibodies

Based on example 2, by aligning the IMGT human antibody heavy and light chain variable region germline gene database and the MOE software, heavy chain or light chain variable region germline genes with high homology with the murine monoclonal antibody are respectively selected as templates, and CDRs of the murine monoclonal antibody are respectively transplanted into corresponding human templates to form variable region sequences in sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Wherein the amino acid residues are determined and annotated by the Kabat numbering system.

In order to maintain the conformation of the CDR regions, for residues at the VL and VH binding interface, residues near the CDR and embedded within the protein, residues with direct interactions with the CDR are back mutated to ensure that the activity of the variable region is not affected.

Example 4 transient expression of B7H6 humanized antibodies

After the variable region framework of the B7H6 monoclonal antibody is selected and subjected to back mutation, the variable region sequence of the heavy chain of the obtained humanized B7H6 antibody is shown as SEQ ID NO. 25, and the variable region sequence of the light chain is shown as SEQ ID NO. 26. The nucleotide sequence (SEQ ID NO: 39) encoding the heavy chain of the humanized antibody and the nucleotide sequence (SEQ ID NO: 40) encoding the light chain of the humanized antibody were recombined onto the pTT5 plasmid, respectively, by molecular biological techniques. B7H6 antibodies were prepared by transient transfection of ExpiCHO-S cells (Gibco, cat. A29127) with the above pTT5 vector carrying the light and heavy chains of the humanized anti-human B7H6 antibody, as follows:

the day before transfection, the cell density of the ExpiCHO-S cells was adjusted to (3.about.4). Times.10 ⁶ /mL，37℃，8％CO ₂ The culture was continued overnight with shaking at 120 rpm. On the day of transfection, cells grew to 7X 10 ⁶ ～1×10 ⁷ Per mL, viability was greater than 95% and cells were diluted to 6X 10 using freshly pre-warmed ExpiCHO medium (Gibco, cat. A2910002) ⁶ Per mL, the pTT5 plasmid carrying the light and heavy chains of the humanized anti-human B7H6 antibody described above and ExpiFectamine CHO transfection reagent (Gibco, cat No. a 29129) were used at 2:1 molar ratio into ExpiCHO-S cells at 37℃with 8% CO ₂ Shaking culture at 120 rpm. Mixing ExpiFectamine CHO Enhancer and ExpiCHO Feed for 18-22 hr, immediately adding transfected cells, mixing, and mixing at 32deg.C with 5% CO ₂ Shaking culture at 120 rpm. On day 5 after transfection, 8mLExpiCHO Feed was added to the cells again and culture was continued after mixing. The change of the cell number and the cell activity rate is observed every day, and the cells are harvested after the cell activity rate is reduced to below 80 percent or after the cells are cultured for 10 to 14 days by centrifugation. The expressed supernatant was filtered with a 0.22 μm filter membrane, an antibody having an Fc domain was captured from the expressed supernatant by using a MabSelectprism A affinity column (GE company, cat. No. 17549854), the column was equilibrated with a phosphate buffer of pH7.2, the supernatant was eluted with an elution buffer (100 mM citric acid, pH 2.7), and finally the purified antibody was concentrated and displaced with PBS buffer, and the purity of the purified antibody was 95% or higher by SDS-PAGE, to thereby obtain a humanized B7H6 antibody.

EXAMPLE 5 ELISA binding assay for murine B7H6 antibody

ELISA experiments were used to detect the binding properties of murine B7H6 antibody 4-99 obtained in example 1. Human B7H6 protein (ACRO biosystems, B76-H52H 8) and monkey B7H6 protein (ACRO biosystems, B76-C52 Ha) were each coated with a coating buffer (35 mM NaHCO) ₃ ，15mM Na ₂ CO ₃ pH 9.6) was diluted to 2. Mu.g/mL and 100. Mu.L per well was added to the ELISA plate overnight at 4 ℃. Followed by 3 washes with PBST (0.05% Tween20-PBS, pH 7.2). mu.L of blocking buffer (1% BSA,0.05% Tween20-PBS, pH 7.2) was added to the plate, and the plate was allowed to stand at room temperature for 2 hours and washed 3 times with PBST . Murine antibodies 4-99 were added to each well and incubated for 1 hour at room temperature. The mixture was washed 3 times with PBST. mu.L of HRP-goat anti-mouse IgG secondary antibody (boster, cat. BA 1051) diluted with blocking buffer was added to each well and incubated for 1 hour at room temperature. Washing with PBST for 3 times, adding TMB into each hole, reacting at room temperature in a dark place for 2-5 minutes, stopping the reaction with 2M sulfuric acid in each hole, and finally reading the OD450 value with an ELISA reader. Wherein FIG. 1A shows that the murine anti-B7H 6 antibody (4-99) of the present invention can bind to human B7H6 protein, and FIG. 1B shows that the murine anti-B7H 6 antibody (4-99) of the present invention can also bind to monkey B7H6 protein.

EXAMPLE 6 construction of CHO-K1 cells overexpressing human and cynomolgus monkey B7H6

HEK293T cells were plated in T150 flasks and cultured in DMEM complete medium. After overnight incubation, either the endotoxin-free psPAX2, pMD2.G and pCDH-CMV-FCGR3A-IRES-puro (pCDH-CMV-MCS-IRES-puro vector with the coding sequence of the human B7H6 protein (SEQ ID NO: 54)) or the cynomolgus monkey protein coding sequence (SEQ ID NO: 55) vector was inserted between the multiple cloning sites according to a 7:3:10 to 1.5mL of Opti-MEM (Gibco, cat. No. 31985070) medium, and 100. Mu.LP 3000 transfection reagent. mu.L of Lipofectamine 3000 (thermo, cat. L3000008) transfection reagent was added to 1.5mL of Opti-MEM medium and mixed well. DNA diluent and liposome diluent are mixed according to the volume ratio of 1:1, incubating for 5-10min at room temperature, adding into 293T cells, culturing for 48 hours, and harvesting virus supernatant. The virus supernatant was centrifuged at 4 ℃,2000g for 10min, the supernatant was filtered with a 0.45 μm filter and added to PEG8000 solution (Shanghai, inc.), thoroughly mixed, left overnight at 4 ℃, and centrifuged at 2200g for 90min with a white precipitate at the bottom of the centrifuge tube, and the virus was resuspended with sterile PBS buffer.

Polybrene (sigma, TR-1003) (final concentration 8. Mu.g/mL) was added to DME/F12 medium, and after mixing, an appropriate amount of virus solution was added. 2E5 CHO-K1 cells were placed in 24-well plates, medium with virus was added, and fresh medium was replaced after 8 hours in an incubator. Detecting the expression level of B7H6 on the surface of CHO-K1 cells in 48 hours by a flow cytometry, carrying out limited dilution after positive group cells appear, namely, after the cells are digested, diluting the density to 4 cells per milliliter, inoculating the cells into a 96-hole flat bottom plate, adding 200 mu L into each hole, culturing for 2 weeks, detecting the expression level of B7H6 on the surface of each cloned cell by a flow cytometry after the cells become obvious single cell mass, and obtaining the full positive cells which are CHO-K1 cells over expressing human B7H6 or cynomolgus monkey CD16 meeting the requirement.

EXAMPLE 7 murine B7H6 antibody flow cytometry binding experiments

The human B7H 6-overexpressing CHO-K1 cells obtained in example 6 or cynomolgus monkey CD 16-overexpressing CHO-K1 cells were diluted to 2X 10 with PBS ⁶ Per mL, 100. Mu.L/tube was added to a 1.5mL EP tube, 10. Mu.L/tube goat serum was added thereto, and the mixture was blocked at 4℃for 30min. B7H6 antibody (4-99) and control antibody 12G4 (CN 114395045A) and control antibody B7H2#14/CD3#1 (US 20210107983A 1) were added individually at gradient concentrations (5-fold dilution, final concentration up to 10. Mu.g/mL) and incubated at 4℃for 30min. To the EP tube, 1mL of PBS was added, centrifuged at 3500 rpm.times.5 min at 4℃and the supernatant was discarded and washed once with PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in 100. Mu.L/tube PBS, to which 0.1. Mu.L/tube Alexa-647-labeled goat anti-mouse antibody secondary antibody (Invitrogen) was added, and incubated at 4℃for 30min in the absence of light. Washed twice with PBS and the supernatant was discarded after centrifugation. Cells were resuspended in 200 μl/tube PBS and examined by flow cytometry. The results in FIGS. 2A and 2B show that the B7H6 antibodies 4-99 of the invention can bind to human B7H6 and monkey B7H6 with higher affinity than the control.

Example 8 preparation of bispecific antibodies to 7B 6 H6XCD 3 and detection of binding Activity and cell killing Capacity

Based on the experimental results of examples 1 to 7, the inventors designed a bispecific antibody B7H6/CD3, and examined the binding activity and cell killing ability of the bispecific antibody.

Design and preparation of bispecific antibody of 8.1B7H6/CD3

The antibody contains two monovalent units, one of which is in the form of an anti-CD 3 scFv-Fc, the variable region amino acids are from the CD3 clone UCHT-1 (SEQ ID NO:41, 42) and the other monovalent unit is in the form of an IgG-like version (one heavy chain + one light chain, sequence from SEQ ID NO:49, SEQ ID NO: 50) of humanized anti-B7H 6, and this bispecific antibody is designated B7H6/CD3. The bispecific antibody contains three polypeptide chains, respectively: the heavy chain of the IgG-like antibody against B7H6, the light chain of the IgG-like antibody against B7H6, the heavy chain of the scFv of the CD3 single chain antibody (SEQ ID NO: 46), wherein the heavy chain constant regions of both heavy chains are derived from human antibody IgG. Because of the special asymmetric structure of the molecule, different amino acid mutations are introduced in the constant regions of the two chains in order to reduce homodimer production. Also, in order to prevent activation of crosslinking by fcγ receptor, a (L234A/L235A) mutation was introduced in the heavy chain constant region.

Preparation of bispecific antibodies the bispecific antibodies were prepared by combining plasmid ratios (1:1 or other ratios) and performing one-step affinity purification as described in example 6.2, wherein the relevant sequences of the bispecific antibodies B7H6/CD3 are shown in table 3.

TABLE 3 Table 3

	B7H6 heavy chain	B7H6 light chain	CD3 heavy chain
				Amino acid sequence	SEQ ID NO：49	SEQ ID NO：50	SEQ ID NO：46
Nucleotide sequence	SEQ ID NO：51	SEQ ID NO：52	SEQ ID NO：53

8.2 cell killing experiments

Colorectal cancer HCT-15 cells were counted after digestion and cell density was adjusted to 2X 10 ⁵ /mL. The RTCA instrument (agilent) was turned on, instrument type DP was selected, experimental mode was selected, and cell information and drug information were filled in. Entering a schedule setting experiment step, adding 50 mu L of fresh culture medium (89% RPMI 1640 culture medium+10% fetal bovine serum+1% green streptomycin) into the plates, putting the plates into an instrument, closing the plates, and clicking the plates to start in the first step. Taking out the plate after Done, adding 100 mu L of cell suspension, standing for 15-30 min at room temperature to prevent edge effect, putting into an instrument, and starting clicking. After growth to log phase, suspension was continued, 50. Mu.L of PBMC (4X 10) ⁶ /mL) and add a gradient of bispecific antibody, click on, analyze after a period of time. FIG. 3 shows that the killing efficiency of PBMC against colorectal cancer HCT-15 cells gradually increases with increasing B7H6/CD3 bispecific antibody concentration, demonstrating that B7H6/CD3 can promote PBMC to kill B7H6 specifically ⁺ Is a tumor cell of (a).

In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.

While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims

1. An antibody or antigen-binding fragment comprising:

Heavy chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO 1, 2 and 3, respectively, or amino acid sequences having at least 80% identity to SEQ ID NO 1, 2 and 3; and/or

Light chain variable region CDR1, CDR2, CDR3 sequences as shown in SEQ ID NO 4, 5 and 6 or amino acid sequences having at least 80% identity to 4, 5 and 6, respectively.

2. The antibody or antigen-binding fragment of claim 1, comprising:

a heavy chain variable region CDR1 sequence shown in SEQ ID NO. 1, a heavy chain variable region CDR2 shown in SEQ ID NO. 2, a heavy chain variable region CDR3 shown in SEQ ID NO. 3, a light chain variable region CDR1 shown in SEQ ID NO. 4, a light chain variable region CDR2 shown in SEQ ID NO. 5, and a light chain variable region CDR3 shown in SEQ ID NO. 6.

3. The antibody or antigen-binding fragment of claim 1, comprising: at least one of a heavy chain FR region and a light chain FR region;

optionally, at least a portion of at least one of the heavy chain FR region and the light chain FR region is derived from at least one of a human antibody, a primate-derived antibody, and a murine antibody or a mutant thereof.

4. An antibody or antigen-binding fragment according to claim 3, comprising:

At least one of the sequences of heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 shown in SEQ ID NOS 7-10, respectively; or (b)

At least one of the sequences of the heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 shown in SEQ ID NOS 15-18, respectively.

5. An antibody or antigen-binding fragment according to claim 3, comprising:

at least one of the sequences of the light chain framework regions LFR1, LFR2, LFR3 and LFR4 shown in SEQ ID NO. 11-14 respectively; or (b)

At least one of the sequences of the light chain framework regions LFR1, LFR2, LFR3 and LFR4 shown in SEQ ID NO. 19-22 respectively.

6. The antibody or antigen-binding fragment thereof of any one of claims 4-5, comprising: at least one of the sequences of heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 shown in SEQ ID NOS 7-10, respectively; at least one of the sequences of the light chain framework regions LFR1, LFR2, LFR3 and LFR4 shown in SEQ ID NO. 11-14 respectively; or alternatively

At least one of the sequences of heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 shown in SEQ ID NOS 15-18, respectively; at least one of the sequences of the light chain framework regions LFR1, LFR2, LFR3 and LFR4 shown in SEQ ID NO. 19-22 respectively.

7. The antibody or antigen-binding fragment thereof of claim 6, comprising:

A heavy chain variable region as shown in SEQ ID NO. 23 or SEQ ID NO. 25; and/or

The light chain variable region as shown in SEQ ID NO. 24 or SEQ ID NO. 26.

8. The antibody or antigen-binding fragment thereof of claim 7, comprising:

1) A heavy chain variable region as shown in SEQ ID NO. 23, and a light chain variable region as shown in SEQ ID NO. 24; or (b)

2) A heavy chain variable region as shown in SEQ ID NO. 25, and a light chain variable region as shown in SEQ ID NO. 26.

9. The antibody or antigen-binding fragment of any one of claims 1 to 8, wherein the antibody or antigen-binding fragment comprises at least one of a heavy chain constant region and a light chain constant region, at least a portion of which is derived from at least one of a human antibody, a primate-derived antibody, and a murine antibody or a mutant thereof;

optionally, the light chain constant region and the heavy chain constant region are both derived from a murine IgG antibody or mutant thereof or a human IgG antibody or mutant thereof;

optionally, the light chain constant region and the heavy chain constant region are both derived from a murine IgG1 antibody or a mutant thereof or a human IgG1 antibody or a mutant thereof;

optionally, the antibody has a heavy chain constant region of the amino acid sequence shown as SEQ ID NO. 27 or 29 and/or a light chain constant region of the amino acid sequence shown as SEQ ID NO. 28 or 30.

10. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment has a heavy chain with an amino acid sequence set forth in any one of SEQ ID NOs 31, 33 and 35 and a light chain with an amino acid sequence set forth in any one of SEQ ID NOs 32, 34 and 36.

11. The antibody or antigen-binding fragment of claim 10, wherein the antibody or antigen-binding fragment has a heavy chain of the amino acid sequence shown in SEQ ID No. 31 and a light chain of the amino acid sequence shown in SEQ ID No. 32;

the antibody or antigen binding fragment has a heavy chain with an amino acid sequence shown as SEQ ID NO. 33 and a light chain with an amino acid sequence shown as SEQ ID NO. 34; or (b)

The antibody or antigen binding fragment has a heavy chain with the amino acid sequence shown in SEQ ID NO. 35 and a light chain with the amino acid sequence shown in SEQ ID NO. 36.

12. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment comprises a monoclonal antibody or a polyclonal antibody;

optionally, the monoclonal antibody comprises at least one of a full length antibody, fv, single chain antibody, fab, single domain antibody, and minimal recognition unit;

optionally, the antibody or antigen binding fragment thereof is capable of binding to the amino acid sequences shown in SEQ ID NO. 37 and/or 38.

13. A bispecific binding molecule comprising:

a first binding region comprising the antibody or antigen binding fragment of any one of claims 1-12; and

a second binding region, said second binding region having CD3 binding activity.

14. The bispecific binding molecule of claim 13, wherein the bispecific binding molecule comprises a symmetric bispecific binding molecule or an asymmetric bispecific binding molecule;

optionally, the bispecific binding molecule is an asymmetric bispecific binding molecule.

15. The bispecific binding molecule according to claim 13, characterized in that the first binding region comprises a peptide chain 1 and a peptide chain 2, wherein the peptide chain 1 comprises the heavy chain variable region according to any one of claims 7 to 8, and the peptide chain 2 comprises the light chain variable region according to any one of claims 7 to 8;

optionally, the second binding region comprises at least one of a full length antibody, fv, single chain antibody, fab, single domain antibody, and minimal recognition unit having CD3 binding activity;

optionally, the second binding region comprises an anti-CD 3 single chain antibody;

optionally, the anti-CD 3 single chain antibody comprises an anti-CD 3 antibody light chain variable region having the amino acid sequence shown in SEQ ID No. 41 and an anti-CD 3 antibody heavy chain variable region having the amino acid sequence shown in SEQ ID No. 42;

Optionally, the anti-CD 3 single chain antibody further comprises a linker peptide, wherein the N-terminus of the linker peptide is linked to the C-terminus of the anti-CD 3 antibody heavy chain variable region, and the C-terminus of the linker peptide is linked to the N-terminus of the anti-CD 3 antibody light chain variable region; or the N-terminal of the connecting peptide is connected with the C-terminal of the anti-CD 3 antibody light chain variable region, and the C-terminal of the connecting peptide is connected with the N-terminal of the anti-CD 3 antibody heavy chain variable region;

optionally, the connecting peptide has an amino acid sequence (GGGGS) n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

optionally, the linker peptide has the amino acid sequence shown as SEQ ID NO. 44;

optionally, the anti-CD 3 single chain antibody has an amino acid sequence as shown in SEQ ID NO. 43.

16. The bispecific binding molecule of claim 13, wherein the first binding region further comprises at least one of a first heavy chain constant region and a light chain constant region, at least a portion of the at least one of which is derived from at least one of a human antibody, a primate-source antibody, and a murine antibody or a mutant thereof;

optionally, the first heavy chain constant region and the light chain constant region are both from a human IgG antibody or mutant thereof;

Optionally, the first heavy chain constant region and the light chain constant region are both from a human IgG1 antibody or a mutant thereof;

optionally, the first heavy chain constant region has the amino acid sequence shown in SEQ ID NO. 47 and the light chain constant region has the amino acid sequence shown in SEQ ID NO. 48;

optionally, the N-terminus of the first heavy chain constant region is linked to the C-terminus of the heavy chain variable region and the N-terminus of the light chain constant region is linked to the C-terminus of the light chain variable region;

optionally, the peptide chain 1 has an amino acid sequence shown in SEQ ID NO. 49, and the peptide chain 2 has an amino acid sequence shown in SEQ ID NO. 50;

optionally, the peptide chain 1 and the peptide chain 2 are linked by a disulfide bond.

17. The bispecific binding molecule of claim 13, wherein the second binding region further comprises a second heavy chain constant region, at least a portion of which is derived from at least one of a human antibody, a primate-source antibody, and a murine antibody or a mutant thereof;

optionally, the second heavy chain constant region is derived from a human IgG antibody or mutant thereof;

optionally, the second heavy chain constant region is derived from a human IgG1 antibody or a mutant thereof;

Optionally, the second heavy chain constant region has the amino acid sequence shown as SEQ ID NO. 45;

optionally, the N-terminus of the second heavy chain constant region is linked to the C-terminus of the anti-CD 3 single chain antibody;

optionally, the second binding region has the amino acid sequence shown as SEQ ID NO. 46;

optionally, the first heavy chain constant region and the second heavy chain constant region are linked by a knob-intoo-hole structure.

18. A nucleic acid molecule encoding the antibody or antigen-binding fragment of any one of claims 1 to 12 or the bispecific binding molecule of any one of claims 13 to 17.

19. An expression vector carrying the nucleic acid molecule of claim 18.

20. A recombinant cell carrying the nucleic acid molecule of claim 18, the expression vector of claim 19 or being capable of expressing the antibody or antigen-binding fragment of any one of claims 1 to 12 or the bispecific binding molecule of any one of claims 13 to 17.

21. The recombinant cell according to claim 20, wherein the recombinant cell is obtained by introducing the expression vector of claim 19 into a host cell;

Optionally, the recombinant cell is a eukaryotic cell;

optionally, the recombinant cell is a mammalian cell.

22. A composition comprising the antibody or antigen-binding fragment of any one of claims 1 to 12, the bispecific binding molecule of any one of claims 13 to 17, the nucleic acid molecule of claim 18, the expression vector of claim 19, or the recombinant cell of any one of claims 20 to 21.

23. A medicament comprising an antibody or antigen-binding fragment according to any one of claims 1 to 12, a bispecific binding molecule according to any one of claims 13 to 17, a nucleic acid molecule according to claim 18, an expression vector according to claim 19, a recombinant cell according to any one of claims 20 to 21 or a composition according to claim 22.

24. A kit comprising the antibody or antigen-binding fragment of any one of claims 1 to 12, the bispecific binding molecule of any one of claims 13 to 17, the nucleic acid molecule of claim 18, the expression vector of claim 19, or the recombinant cell of any one of claims 20 to 21.

25. Use of the antibody or antigen binding fragment of any one of claims 1 to 12, the nucleic acid molecule of claim 18, the expression vector of claim 19, the recombinant cell of any one of claims 20 to 21 or the composition of claim 22 in the manufacture of a medicament for the prevention and/or treatment of a B7H6 mediated related disease;

optionally, the B7H 6-mediated related disease comprises cancer;

optionally, the cancer comprises at least one of: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal carcinoma, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, renal cancer, esophageal cancer, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma.

26. Use of a bispecific binding molecule according to any one of claims 13 to 17, a nucleic acid molecule according to claim 18, an expression vector according to claim 19, a recombinant cell according to any one of claims 20 to 21 or a composition according to claim 22 for the preparation of a medicament for the prevention and/or treatment of B7H6 and CD3 mediated related diseases;

Optionally, the B7H6 and CD3 mediated related diseases include cancer;

optionally, the cancer comprises at least one of: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, myeloid leukemia, skin cancer, thyroid cancer, renal cancer, esophageal cancer, melanoma, and pancreatic cancer.

27. Use of the antibody or antigen binding fragment of any one of claims 1 to 12, the nucleic acid molecule of claim 18, the expression vector of claim 19 or the recombinant cell of any one of claims 20 to 21 in the preparation of a kit for detecting B7H6.

28. Use of a bispecific binding molecule according to any one of claims 13 to 17, a nucleic acid molecule according to claim 18, an expression vector according to claim 19 or a recombinant cell according to any one of claims 20 to 21 for the preparation of a kit for the detection of B7H6 and/or CD3.