Antibodies against b7h6 and uses thereof

Abstract

The application provides an antibody against B7H6 and application thereof, the antibody comprising heavy chain variable region CDR1, CDR2 and CDR3 sequences shown in the amino acid sequences of SEQ ID NO: 1, 2 and 3 respectively or having at least 95% identity with SEQ ID NO: 1, 2 and 3; and / or light chain variable region CDR1, CDR2 and CDR3 sequences shown in the amino acid sequences of SEQ ID NO: 4, 5 and 6 respectively or having at least 95% identity with 4, 5 and 6. The antibody of the embodiment of the application can be combined with human and monkey B7H6 proteins, and the bispecific antibody prepared by using the antibody has higher human and monkey B7H6 binding activity, and can effectively treat and / or prevent B7H6-mediated related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an antibody against B7H6 and its application. Background Technology

[0002] Cancer is the second leading cause of death worldwide. Currently, approximately 10 monoclonal antibodies targeting tumor antigens are available globally, such as rituximab (targeting CD20), trastuzumab (targeting HER2), bevacizumab (targeting VEGF), and cetuximab (targeting EGFR). There are currently no marketed drugs targeting the B7H6 molecule.

[0003] B7H6 is a type I transmembrane protein belonging to the B7 family, containing two immunoglobulin domains. B7H6 is also a ligand for the NK cell activating receptor NKp30. B7H6 is expressed on various tumor cells, but its mRNA is not detected in normal human tissues or healthy peripheral blood monocytes. In inflammatory environments, pro-inflammatory cytokines such as IL-1β and TNF can stimulate upregulation of B7H6 expression in CD14+CD16+ monocytes and neutrophils. 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, and liver cancer. B7H6 expression is also associated with metastasis in some cancers, but its role in most tumors remains unclear.

[0004] Currently, although B7H6 is expressed in various tumors, there are few tumor treatments targeting this molecule. Researchers have developed a bispecific antibody against B7H6, constructed in BiTE form. This form lacks the constant region of natural antibodies, resulting in a short half-life in vivo, which significantly limits its use. Furthermore, the ability to bind to monkey B7H6 is also a factor in antibody selection for non-clinical research. Existing antibodies show significant differences in their binding ability to human and monkey B7H6, thus presenting many obstacles in non-clinical studies.

[0005] Therefore, further research is needed to develop a B7H6-specific antibody with higher affinity and cross-species interaction between humans and monkeys, as well as a bispecific antibody derived from the aforementioned B7H6-specific antibody. Summary of the Invention

[0006] This application is based on the inventor's discovery of the following problems and facts:

[0007] B7H6 is expressed on various tumor cells, but there are not many tumor treatments and research methods targeting this molecule.

[0008] The inventors of this application have successfully screened a murine anti-B7H6 monoclonal antibody that exhibits high binding activity to human or monkey B7H6 protein. Furthermore, the inventors humanized the constant region of the aforementioned monoclonal antibody while retaining the CDR of the murine anti-B7H6 monoclonal antibody to obtain a chimeric antibody. Further, the inventors humanized the framework region of the light chain variable region or heavy chain variable region of the chimeric antibody to obtain a fully humanized anti-B7H6 antibody. This humanized antibody not only specifically targets and binds to both human and monkey B7H6 proteins but also exhibits low immunogenicity, enabling effective treatment and / or prevention of B7H6-mediated diseases.

[0009] Furthermore, the bispecific or multispecific binding molecules prepared using the anti-B7H6 antibody can also specifically target and bind to human and monkey B7H6 proteins. Typically, based on the specificity of the bispecific or multispecific binding molecules, they can target and bind to cells that highly express B7H6, thereby treating or preventing various diseases, such as tumors. In addition, the murine anti-B7H6 monoclonal antibody, bispecific or multispecific binding molecules can be used for research on diseases related to high expression of B7H6.

[0010] Therefore, in a first aspect, the present invention provides an antibody or antigen-binding fragment. According to embodiments of the invention, it comprises heavy chain variable regions CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:1, 2, and 3, or amino acid sequences having at least 80% identity with SEQ ID NO:1, 2, and 3, respectively; and / or light chain variable regions CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO:4, 5, and 6, or amino acid sequences having at least 80% identity with 4, 5, and 6, respectively. The antibody or antigen-binding fragment according to embodiments of the invention can bind to human or monkey B7H6 protein, effectively treating or preventing B7H6-mediated diseases, and enabling related research.

[0011] According to embodiments of the present invention, the above-described antibody or antigen-binding fragment may further include at least one of the following additional technical features:

[0012] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises: the heavy chain variable region CDR1 sequence shown in SEQ ID NO:1, the heavy chain variable region CDR2 shown in SEQ ID NO:2, the heavy chain variable region CDR3 shown in SEQ ID NO:3, the light chain variable region CDR1 shown in SEQ ID NO:4, the light chain variable region CDR2 shown in SEQ ID NO:5, and the light chain variable region CDR3 shown in SEQ ID NO:6.

[0013] According to an embodiment of the present invention, the antibody or antigen-binding fragment includes at least one of a heavy chain FR region and a light chain FR region.

[0014] According to an embodiment of the present 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 human antibodies, primate antibodies, and mouse antibodies or mutants thereof.

[0015] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises at least one of the heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 sequences as shown in SEQ ID NO:7-10; or at least one of the heavy chain framework regions HFR1, HFR2, HFR3 and HFR4 sequences as shown in SEQ ID NO:15-18.

[0016] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises at least one of the light chain framework regions LFR1, LFR2, LFR3 and LFR4 sequences as shown in SEQ ID NO:11-14 respectively; or at least one of the light chain framework regions LFR1, LFR2, LFR3 and LFR4 sequences as shown in SEQ ID NO:19-22 respectively.

[0017] According to embodiments of the present invention, the antibody or antigen-binding fragment comprises: at least one of the heavy chain frame region sequences HFR1, HFR2, HFR3 and HFR4 as shown in SEQ ID NO:7-10; at least one of the light chain frame region sequences LFR1, LFR2, LFR3 and LFR4 as shown in SEQ ID NO:11-14; or at least one of the heavy chain frame region sequences HFR1, HFR2, HFR3 and HFR4 as shown in SEQ ID NO:15-18; or at least one of the light chain frame region sequences LFR1, LFR2, LFR3 and LFR4 as shown in SEQ ID NO:19-22.

[0018] According to embodiments of the present invention, the antibody or antigen-binding fragment includes: 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.

[0019] According to an embodiment of the present invention, the antibody or antigen-binding fragment includes: 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 shown in SEQ ID NO:25 and a light chain variable region as shown in SEQ ID NO:26.

[0020] According to embodiments of the present invention, the antibody or antigen-binding fragment contains at least one of a heavy chain constant region and a light chain constant region, and at least a portion of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a human antibody, a primate antibody, a mouse antibody, or a mutant thereof.

[0021] According to an embodiment of the present invention, both the light chain constant region and the heavy chain constant region are derived from mouse IgG antibodies or their mutants, or human IgG antibodies or their mutants.

[0022] According to an embodiment of the present invention, both the light chain constant region and the heavy chain constant region are derived from human IgG1 antibody or its mutant.

[0023] According to an embodiment of the present invention, the light chain constant region comprises human or mouse κ, λ chains or variants thereof.

[0024] According to embodiments of the present invention, the antibody has a heavy chain constant region of the amino acid sequence shown in SEQ ID NO:27 or 29 and / or a light chain constant region of the amino acid sequence shown in SEQ ID NO:28 or 30.

[0025] According to embodiments of the present invention, the antibody or antigen-binding fragment has a heavy chain having an amino acid sequence shown in any one of SEQ ID NO:31, 33 and 35 and a light chain having an amino acid sequence shown in any one of SEQ ID NO:32, 34 and 36.

[0026] According to embodiments of the present invention, 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 of the amino acid sequence shown in SEQ ID NO:33 and a light chain of the amino acid sequence shown in SEQ ID NO:34; or the antibody or antigen-binding fragment has a heavy chain of the amino acid sequence shown in SEQ ID NO:35 and a light chain of the amino acid sequence shown in SEQ ID NO:36.

[0027] According to embodiments of the present invention, the antibody or antigen-binding fragment includes monoclonal antibodies or polyclonal antibodies.

[0028] According to embodiments of the present invention, the monoclonal antibody includes at least one of Fv, single-chain antibody, Fab, single-domain antibody, and minimum recognition unit.

[0029] According to embodiments of the present invention, the antibody or its antigen-binding fragment is capable of binding to the amino acid sequence shown in SEQ ID NO:37 and / or 38.

[0030] In a second aspect, the present invention provides a bispecific binding molecule. According to embodiments of the invention, it includes a first binding region comprising the antibody or antigen-binding fragment described in the first aspect; and a second binding region having CD3 binding activity. The bispecific binding molecule according to embodiments of the invention can bind to human or monkey B7H6 and CD3 proteins, and can be applied to scientific research, or effectively treat or prevent B7H6 and CD3-mediated diseases.

[0031] Those skilled in the art will understand that the binding activity of the second binding region is not particularly limited and may also have other binding activities, as long as the bispecific antibody has the antibody or antigen-binding fragment described in the first aspect, and both the antibody or antigen-binding fragment and the second binding region can effectively function. Furthermore, the antibody or antigen-binding fragment described in this application can be used to prepare more specific antibodies, such as trispecific, tetraspecific, or pentaspecific antibodies. Based on the multispecificity of these antibodies, the antibody or antigen-binding fragment of this invention can target the surface of cells that highly express multiple antigens, eliminating cells producing these antigens through cell-killing action.

[0032] According to embodiments of the present invention, the above-mentioned bispecific binding molecule may further include at least one of the following additional technical features:

[0033] According to embodiments of the present invention, the bispecific binding molecule includes a symmetrical bispecific binding molecule or an asymmetrical bispecific binding molecule.

[0034] According to an embodiment of the present invention, the bispecific binding molecule is an asymmetric bispecific binding molecule.

[0035] According to an embodiment of the present invention, the first binding region comprises peptide chain 1 and peptide chain 2, wherein peptide chain 1 includes the aforementioned heavy chain variable region, and peptide chain 2 includes the aforementioned light chain variable region.

[0036] According to an embodiment of the present invention, the second binding region includes at least one of a full-length antibody, Fv, single-chain antibody, Fab, single-domain antibody, and minimum recognition unit having CD3 binding activity.

[0037] According to an embodiment of the present invention, the second binding region includes an anti-CD3 single-chain antibody.

[0038] According to an embodiment of the present invention, the anti-CD3 single-chain antibody includes an anti-CD3 antibody light chain variable region and an anti-CD3 antibody heavy chain variable region, wherein the anti-CD3 antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO:41, and the anti-CD3 antibody light chain variable region has the amino acid sequence shown in SEQ ID NO:42.

[0039] According to an embodiment of the present invention, the anti-CD3 single-chain antibody further includes a linker peptide, wherein the N-terminus of the linker peptide is connected to the C-terminus of the variable region of the heavy chain of the anti-CD3 antibody, and the C-terminus of the linker peptide is connected to the N-terminus of the variable region of the light chain of the anti-CD3 antibody; or the N-terminus of the linker peptide is connected to the C-terminus of the variable region of the light chain of the anti-CD3 antibody, and the C-terminus of the linker peptide is connected to the N-terminus of the variable region of the heavy chain of the anti-CD3 antibody.

[0040] According to embodiments of the present invention, the linker peptide has an amino acid sequence (GGGGS)n, where 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 understand that conventional linkers, such as conventional flexible or rigid amino acid fragments, can be used.

[0041] According to an embodiment of the present invention, the linker peptide has the amino acid sequence shown in SEQ ID NO:44.

[0042] According to an embodiment of the present invention, the anti-CD3 single-chain antibody has an amino acid sequence as shown in SEQ ID NO:43.

[0043] According to an embodiment of the present invention, the first binding region further includes at least one of a first heavy chain constant region and a light chain constant region, wherein at least a portion of at least one of the first heavy chain constant region and the light chain constant region is derived from at least one of a human antibody, a primate antibody, a mouse antibody, or a mutant thereof.

[0044] According to an embodiment of the present invention, both the first heavy chain constant region and the light chain constant region are derived from mouse IgG antibody or its mutant or human IgG antibody or its mutant;

[0045] According to an embodiment of the present invention, both the first heavy chain constant region and the light chain constant region are derived from human IgG1 antibody or its mutant.

[0046] According to an embodiment of the present 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.

[0047] According to an embodiment of the present invention, the N end of the first heavy chain constant region is connected to the C end of the heavy chain variable region, and the N end of the light chain constant region is connected to the C end of the light chain variable region.

[0048] According to an embodiment of the present invention, peptide chain 1 has the amino acid sequence shown in SEQ ID NO:49, and peptide chain 2 has the amino acid sequence shown in SEQ ID NO:50.

[0049] According to an embodiment of the present invention, peptide chain 1 and peptide chain 2 are linked by disulfide bonds.

[0050] According to an embodiment of the present invention, the second binding region further includes a second heavy chain constant region, at least a portion of which is derived from at least one of a human antibody, a primate antibody, and a mouse antibody or a mutant thereof.

[0051] According to an embodiment of the present invention, the second heavy chain constant region is derived from a mouse IgG antibody or a mutant thereof, or a human IgG antibody or a mutant thereof.

[0052] According to an embodiment of the present invention, the second heavy chain constant region is derived from a human IgG1 antibody or a mutant thereof. Those skilled in the art will understand 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 the same, they can be linked by disulfide bonds.

[0053] According to an embodiment of the present invention, the second heavy chain constant region has the amino acid sequence shown in SEQ ID NO:45.

[0054] According to an embodiment of the present invention, the N-terminus of the second heavy chain constant region is connected to the C-terminus of the anti-CD3 single-chain antibody.

[0055] According to an embodiment of the present invention, the second binding region has the amino acid sequence shown in SEQ ID NO:46.

[0056] The nucleic acid encoding the antibody or its antigen-binding fragment or the bispecific binding molecule of the present invention is within the scope of the present invention, and based on its amino acid sequence, those skilled in the art can easily obtain the corresponding nucleic acid sequence.

[0057] Therefore, in a third aspect, the present invention provides a nucleic acid molecule encoding the antibody or antigen-binding fragment or the bispecific binding molecule described in the first aspect. According to some specific embodiments of the present invention, the antibody or antigen-binding fragment encoded by the nucleic acid molecule can bind to human or monkey B7H6 protein, effectively treating or preventing B7H6-mediated diseases; the bispecific binding molecule encoded by the nucleic acid molecule can bind to both human or monkey B7H6 protein and CD3 protein, effectively treating or preventing B7H6 and CD3-mediated diseases.

[0058] According to embodiments of the present invention, the above-mentioned nucleic acid molecule may further include at least one of the following additional technical features:

[0059] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0060] According to embodiments of the present invention, the nucleic acid molecule comprises the nucleotide sequences shown in SEQ ID NO: 39 and 40; or comprises the nucleotide sequences shown in SEQ ID NO: 51, 52 and 53.

[0061] It should be noted that those skilled in the art should understand that the nucleic acids mentioned in this specification and claims actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases in this specification and claims, the other complementary strand is actually disclosed as well. In addition, the nucleic acid sequences in this application include DNA or RNA forms; disclosure of one means that the other is also disclosed.

[0062] In a fourth aspect, the present invention provides an expression vector carrying the aforementioned nucleic acid molecule. The expression vector may include an optional control sequence operatively linked to the nucleic acid molecule. The control sequence may be one or more control sequences that direct the expression of the nucleic acid molecule in a host. The expression vector proposed in the embodiments of the present invention can efficiently and massively express the antibody or antigen-binding fragment in suitable host cells.

[0063] In this article, "operably ligated" refers to ligating a foreign gene to a vector so that the control elements within the vector, such as transcriptional and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. When ligating the aforementioned nucleic acid molecules to a vector, the nucleic acid molecules can be directly or indirectly linked to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecules. These control elements can be directly derived from the vector itself or be exogenous, i.e., not derived from the vector itself. Those skilled in the art will understand that nucleic acid molecules used to encode antibody or antigen-binding fragments can be independently inserted into different vectors, but commonly they are inserted into the same vector. Commonly used vectors include plasmids, bacteriophages, etc., such as the Plasmid-X plasmid.

[0064] In a fifth aspect, the present invention provides a method for preparing the aforementioned antibody or antigen-binding fragment or bispecific binding molecule, comprising: introducing the aforementioned expression vector into cells; and culturing the cells under conditions suitable for protein expression and secretion to obtain the antibody or antigen-binding fragment or bispecific binding molecule. The method proposed according to some specific embodiments of the present invention can effectively obtain large quantities of the antibody or antigen-binding fragment or the bispecific binding molecule in vitro.

[0065] According to some specific embodiments of the present invention, the above-described methods for preparing the aforementioned antibodies or antigen-binding fragments or bispecific binding molecules may further include at least one of the following additional technical features:

[0066] According to some specific embodiments of the present invention, the cells are not particularly limited, and either prokaryotic cells or eukaryotic cells can be used.

[0067] According to some specific embodiments of the present invention, the cells are eukaryotic cells.

[0068] According to some specific embodiments of the present invention, the eukaryotic cells are mammalian cells. According to some specific embodiments of the present invention, when the cells are eukaryotic cells, such as mammalian cells, the expression efficiency of the recombinant antibody is higher.

[0069] In a sixth aspect, the present invention provides a recombinant cell, wherein the recombinant cell expresses the aforementioned nucleic acid, or expression vector, or is capable of expressing the aforementioned antibody or antigen-binding fragment or the aforementioned bispecific binding molecule. The recombinant cell is obtained by transfection or transformation of the expression vector. According to some specific embodiments of the present invention, the recombinant cell can efficiently and extensively express the aforementioned antibody or antigen-binding fragment or the aforementioned bispecific binding molecule under suitable conditions.

[0070] It should be noted that the recombinant cells described in this invention are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, and Trichoderma; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some embodiments, the recombinant cells described in this 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.

[0071] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the antibody or antigen-binding fragment or the bispecific binding molecule described in this application. Those skilled in the art will readily understand that suitable conditions for the expression of the antibody or antigen-binding fragment or the bispecific binding molecule include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the antibody or antigen-binding fragment or the bispecific binding molecule based on the specific environment of their laboratory.

[0072] In a seventh aspect, the present invention provides an immunoconjugate comprising the aforementioned antibody or antigen-binding fragment or the bispecific binding molecule, and a therapeutic agent. As previously described, the antibody or antigen-binding fragment of the embodiments of the present invention can effectively bind to the B7H6 protein, and the bispecific binding molecule can bind to human or monkey B7H6 protein and CD3 protein, effectively treating or preventing B7H6 and CD3-mediated diseases. Therefore, the immunoconjugate containing the antibody or antigen-binding fragment can also bind to human or monkey B7H6 protein, and the immunoconjugate containing the bispecific binding molecule can also bind to human or monkey B7H6 protein and CD3 protein. The immunoconjugate has good preventive and / or therapeutic effects on B7H6-mediated diseases, or B7H6 and CD3-mediated diseases.

[0073] In an eighth aspect, the present invention provides a composition comprising the aforementioned antibody or antigen-binding fragment, bispecific binding molecule, nucleic acid molecule, expression vector, or recombinant cell. As previously described, the antibody or antigen-binding fragment of some specific 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 the proliferation of tumor cells. Therefore, the composition containing the above substances can also effectively bind to human or monkey B7H6 protein, or to human or monkey B7H6 protein and CD3 protein, and has a good effect on the prevention and / or treatment of B7H6-mediated diseases. The type of composition is not particularly limited and can be a food composition or a pharmaceutical composition.

[0074] The compositions of the present invention can also be administered in combination with each other or in combination with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent. Therefore, the compositions may also contain a chemotherapeutic agent. The antibodies or antigen-binding fragments thereof, or immunoconjugates of the present invention can 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 upstream or downstream signal transduction of B7H6.

[0075] It should be noted that the compositions include combinations that are separate in time and / or space, as long as they can work together to achieve the objectives of the invention. For example, the components contained in the composition may be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0076] In a ninth aspect, the present invention provides a medicament comprising the aforementioned antibody or antigen-binding fragment, bispecific binding molecule, nucleic acid molecule, expression vector, recombinant cell, or composition. As previously described, the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to human or monkey B7H6 protein, and the bispecific binding molecule can bind to both human or monkey B7H6 protein and CD3 protein. Therefore, a medicament comprising an effective amount of the active ingredient of the antibody or antigen-binding fragment or the bispecific binding molecule, or a series thereof, can also effectively bind to human or monkey B7H6 protein, or to both human or monkey B7H6 protein and CD3 protein, exhibiting good preventive and / or therapeutic effects against B7H6-mediated diseases.

[0077] According to embodiments of the present invention, the above-mentioned drug may further include at least one of the following additional technical features:

[0078] According to embodiments of the present invention, the drug may further include a pharmaceutically acceptable carrier.

[0079] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0080] The effective amount of the antibody or antigen-binding fragment or the bispecific binding molecule described in this invention can vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0081] As used herein, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients and diluents.

[0082] The medicament of this invention contains a safe and effective amount of the active ingredient of this invention and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched with the route of administration, wherein the route of administration may be oral, nasal, intradermal, subcutaneous, intramuscular, intravenous, or intraperitoneal. The dosage forms of the medicament of this invention are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. For example, it is prepared using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The medicament is preferably manufactured under sterile conditions. The antibody or antigen-binding fragment can be administered by intravenous infusion or injection, or by intramuscular or subcutaneous injection.

[0083] Of course, the anti-B7H6 monoclonal antibody or bispecific binding molecule described in this article can also be made into kits or other diagnostic reagents as needed.

[0084] In a tenth aspect, the present invention provides a kit containing the aforementioned antibody or its antigen-binding fragment, a bispecific binding molecule, a nucleic acid molecule, an expression vector, or recombinant cells. As previously described, the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to human or monkey B7H6 protein, and the bispecific binding molecule can bind to both human or monkey B7H6 protein and CD3 protein. Therefore, kits containing the antibody or antigen-binding fragment can effectively perform qualitative or quantitative detection of human or monkey B7H6 protein, and kits containing the bispecific binding molecule can effectively perform qualitative or quantitative detection of both human or monkey B7H6 protein and CD3 protein. The kits provided by the present invention can be used, for example, in immunoblotting, immunoprecipitation, and other kits involving the detection of human or monkey B7H6 by utilizing the specific binding properties of antibodies. These kits may contain any one or more of the following: antagonist, anti-B7H6 antibody, or pharmaceutical reference material; protein purification column; immunoglobulin affinity purification buffer; cell assay diluent; instructions or literature, etc. Anti-B7H6 antibodies can be used in various diagnostic tests, such as detecting the presence of a wide range of diseases, drugs, toxins, or other proteins in vitro or in vivo. For example, they can be tested on the serum or blood of a subject to detect related diseases. They can also be used in scientific research, utilizing the kit to detect human or monkey B7H6 protein, or human or monkey B7H6 protein and CD3 protein, in a test sample. These related diseases may include B7H6-related diseases, such as cancer. Of course, the antibodies or antigen-binding fragments provided herein can also be used for radioimmunoassay and radioimmunotherapy for the aforementioned diseases. The bispecific binding molecules described above are also applicable to these applications and will not be elaborated upon here.

[0085] The kit may also include reagents commonly used for detecting B7H6, or B7H6 and CD3, such as coating solutions.

[0086] In an eleventh aspect, the present invention proposes the use of the aforementioned antibodies or antigen-binding fragments thereof, nucleic acid molecules, expression vectors, recombinant cells, or compositions in the preparation of pharmaceuticals for the prevention and / or treatment of B7H6-mediated diseases. As previously stated, the antibodies or antigen-binding fragments of some specific embodiments of the present invention can effectively bind to human or monkey B7H6 proteins. Therefore, pharmaceuticals containing an effective amount of the aforementioned antibodies or antigen-binding fragments or a series of substances thereof can also effectively bind to human or monkey B7H6 proteins, exhibiting good preventive and / or therapeutic effects against B7H6-mediated diseases.

[0087] According to embodiments of the present invention, the use of the above-mentioned prepared drug may further include at least one of the following additional technical features:

[0088] According to embodiments of the present invention, the B7H6-mediated diseases include cancer.

[0089] According to embodiments of the present invention, the cancer includes at least one of the following: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma.

[0090] In a twelfth aspect, the present invention provides for the use of the aforementioned bispecific binding molecules, nucleic acid molecules, expression vectors, recombinant cells, or compositions in the preparation of pharmaceuticals for the prevention and / or treatment of B7H6 and CD3-mediated diseases. As previously stated, the bispecific binding molecules of some specific embodiments of the present invention are capable of binding to human or monkey B7H6 and CD3 proteins. Therefore, pharmaceuticals containing an effective amount of the active ingredient of the bispecific binding molecule or a series thereof can also effectively bind to human or monkey B7H6 and CD3 proteins, exhibiting good preventive and / or therapeutic effects against B7H6 and CD3-mediated diseases.

[0091] According to embodiments of the present invention, the use of the above-mentioned prepared drug may further include at least one of the following additional technical features:

[0092] According to embodiments of the present invention, the B7H6 and CD3-mediated diseases include cancer.

[0093] According to embodiments of the present invention, the cancer includes at least one of the following: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma.

[0094] In a thirteenth aspect of the invention, the present invention provides for the use of the aforementioned antibody or antigen-binding fragment, nucleic acid molecule, expression vector, or recombinant cell in the preparation of a kit for detecting B7H6. As previously described, the antibody or antigen-binding fragment of some specific embodiments of the present invention can effectively bind to human or monkey B7H6 protein and block the binding of the B7H6 protein to its receptor. Therefore, the antibody or antigen-binding fragment can be used to prepare a kit for detecting B7H6 protein, which can effectively perform qualitative or quantitative detection of human or monkey B7H6 protein.

[0095] In a fourteenth aspect, the present invention provides for the use of the aforementioned bispecific binding molecules, nucleic acid molecules, expression vectors, or recombinant cells in the preparation of kits for the detection of B7H6 and / or CD3. As previously described, the bispecific binding molecules of some specific embodiments of the present invention can effectively bind to human or monkey B7H6 and CD3 proteins. Therefore, the bispecific binding molecules can be used to prepare kits for the detection of B7H6 and / or CD3, which can effectively perform qualitative or quantitative detection of B7H6 and / or CD3 in humans or monkeys.

[0096] In a fifteenth aspect, the present invention provides a method for treating or preventing B7H6, or B7H6 and / or CD3-mediated diseases. According to embodiments of the invention, the method comprises administering to a subject at least one of the following: 1) the antibody or antigen-binding fragment described above; 2) the bispecific binding molecule described above; 3) the nucleic acid molecule described above; 4) the expression vector described above; 5) the recombinant cells described above; 6) the composition described above; and 7) the drug described above. As previously stated, the bispecific binding molecule is capable of effectively binding to human or monkey B7H6 and CD3 proteins, and the antibody or antigen-binding fragment is capable of binding to human or monkey B7H6 proteins, thus effectively treating or preventing B7H6, or B7H6 and / or CD3-mediated diseases, preferably cancer. Therefore, the method according to embodiments of the invention is capable of effectively treating or preventing B7H6, or B7H6 and / or CD3-mediated diseases, such as cancer.

[0097] According to embodiments of the present invention, the above-described method for treating or preventing diseases may further include at least one of the following additional technical features:

[0098] According to embodiments of the present invention, the B7H6-mediated diseases or the B7H6 and CD3-mediated diseases include cancer.

[0099] According to embodiments of the present invention, the cancer includes at least one of the following: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma.

[0100] In a sixteenth aspect, the present invention provides a method for diagnosing B7H6, or B7H6 and / or CD3-mediated diseases. According to embodiments of the invention, the method includes detecting B7H6, or B7H6 and / or CD3, in a test sample using at least one of the following: 1) the antibody or antigen-binding fragment described above; 2) the bispecific binding molecule described above; 3) the nucleic acid molecule described above; 4) the expression vector described above; and 5) the recombinant cells described above, and determining the content of B7H6, or B7H6 and / or CD3, in the test sample based on the detection results of the B7H6, or B7H6 and / or CD3. The antibody or antigen-binding fragments, or nucleic acid molecules, expression vectors, or recombinant cell-expressed antibody or antigen-binding fragments proposed in this application can all effectively bind to human or monkey B7H6 protein. The bispecific binding molecules, or nucleic acid molecules, expression vectors, or recombinant cell-expressed bispecific binding molecules can all effectively bind to B7H6, or B7H6 and / or CD3. Therefore, the method described in this application can effectively detect the content of B7H6, or B7H6 and / or CD3 in test samples derived from test individuals, and can effectively diagnose diseases related to B7H6, or B7H6 and / or CD3.

[0101] According to embodiments of the present invention, the above-described method for diagnosing diseases may further include at least one of the following additional technical features:

[0102] According to an embodiment of the present invention, the requirement that the content of B7H6, or B7H6 and / or CD3, in the test sample is not lower than the minimum standard for disease is an indication that the test sample originates from a patient suffering from a disease related to B7H6, or B7H6 and / or CD3. The value of the minimum standard can be determined by comparative analysis and verification of the content of B7H6, or B7H6 and / or CD3, in test samples from a large number of individuals suffering from diseases related to B7H6, or B7H6 and / or CD3, and a large number of healthy individuals.

[0103] According to embodiments of the present invention, the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cells, serum, plasma, feces, and urine.

[0104] According to embodiments of the present invention, the B7H6-mediated diseases and the B7H6 and CD3-mediated diseases include cancer.

[0105] According to embodiments of the present invention, the cancer includes at least one of the following: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma.

[0106] In a sixteenth aspect, the present invention provides a method for staging diseases mediated by B7H6, or B7H6 and / or CD3. According to embodiments of the invention, the method includes detecting B7H6, or B7H6 and / or CD3, in a test sample using at least one of the following: 1) the antibody or antigen-binding fragment described above; 2) the bispecific binding molecule described above; 3) the nucleic acid molecule described above; 4) the expression vector described above; and 5) the recombinant cells described above. Based on the detection results of B7H6, or B7H6 and / or CD3, the content of B7H6, or B7H6 and / or CD3, in the test sample is determined. The bispecific binding molecules, or nucleic acid molecules, expression vectors, and bispecific binding molecules expressed by recombinant cells, proposed in this application can all effectively bind to human or monkey B7H6 and / or CD3. The antibody or antigen-binding fragments, or nucleic acid molecules, expression vectors, and antibody or antigen-binding fragments expressed by recombinant cells, can all effectively bind to human or monkey B7H6 protein. Therefore, the method described in this application can effectively detect the content of B7H6, or B7H6 and / or CD3, in the test sample derived from the test individual, and assess the stage of the disease caused by B7H6, or B7H6 and / or CD3 based on the content of B7H6, or B7H6 and / or CD3.

[0107] According to embodiments of the present invention, the above-described method for staging diseases may further include at least one of the following additional technical features:

[0108] According to embodiments of the present invention, the presence of B7H6, or B7H6 and / or CD3, in the test sample at levels not lower than the standard level for stage IV tumor lesions indicates that the test sample originated from a patient with stage IV tumor lesions; the presence of B7H6, or B7H6 and / or CD3, in the test sample between the standard levels for stage IV and stage III tumor lesions indicates that the test sample originated from a patient with stage III tumor lesions; the presence of B7H6, or B7H6 and / or CD3, in the test sample between the standard levels for stage III and stage II tumor lesions indicates that the test sample originated from a patient with stage II tumor lesions; and the presence of B7H6, or B7H6 and / or CD3, in the test sample between the standard levels for stage I and stage II tumor lesions indicates that the test sample originated from a patient with stage I tumor lesions. Those skilled in the art will understand that the levels of B7H6, or B7H6 and / or CD3, in tumor stages I, II, III, and IV vary depending on the type of tumor. Determining the tumor stage can be achieved by comparing the content of B7H6, or B7H6 and / or CD3 in the test sample with the standard levels for that tumor stage, or by comparing the content of B7H6, or B7H6 and / or CD3 in the test sample with the content of B7H6, or B7H6 and / or CD3 in samples from individuals or groups with known disease stages. The standard levels for tumor stages I, II, III, and IV can be determined through comparative analysis and verification of the differences in B7H6, or B7H6 and / or CD3 content in test samples from a large number of individuals with angiogenesis-related diseases and a large number of healthy individuals.

[0109] According to embodiments of the present invention, the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cells, serum, plasma, feces, and urine.

[0110] According to embodiments of the present invention, the B7H6-mediated diseases and the B7H6 and CD3-mediated diseases include cancer.

[0111] According to embodiments of the present invention, the cancer includes at least one of the following: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma.

[0112] In a seventeenth aspect of the invention, a method for assessing the prognosis of diseases mediated by B7H6, or B7H6 and / or CD3, is provided. According to embodiments of the invention, the method includes detecting B7H6, or B7H6 and / or CD3, in a test sample using at least one of the following: 1) the aforementioned bispecific binding molecule; 2) the aforementioned antibody or antigen-binding fragment; 3) the aforementioned nucleic acid molecule; 4) the aforementioned expression vector; and 5) the aforementioned recombinant cells, and determining the content of B7H6, or B7H6 and / or CD3, in the test sample based on the detection results of the aforementioned B7H6, or B7H6 and / or CD3. As mentioned above, the levels of B7H6, or B7H6 and / or CD3, have a significant impact on cancer. After treatment, monitoring the levels of B7H6, or B7H6 and / or CD3 in tissues or excreta, such as peripheral blood and urine, can effectively assess the prognosis of this type of disease. For example, comparing the levels of B7H6, or B7H6 and / or CD3 in subjects before and after treatment, or comparing the levels of B7H6, or B7H6 and / or CD3 in subjects after treatment with the levels in normal or diseased individuals, etc., is how the dual-mode B7H6 proposed in this application is described. Specific binding molecules, or nucleic acid molecules, expression vectors, or bispecific binding molecules expressed by recombinant cells, can effectively bind to B7H6 and / or CD3. The antibodies or antigen-binding fragments, or nucleic acid molecules, expression vectors, or antibodies or antigen-binding fragments expressed by recombinant cells, can effectively bind to human or monkey B7H6. Therefore, the method described in this application can effectively detect the content of B7H6, or B7H6 and / or CD3, in test samples derived from test individuals, and assess the prognosis of diseases related to B7H6, or B7H6 and / or CD3, based on the content of B7H6, or B7H6 and / or CD3.

[0113] According to embodiments of the present invention, the above-described method for assessing disease prognosis may further include at least one of the following additional technical features:

[0114] According to an embodiment of the present invention, the test sample is derived from a patient with B7H6, or a B7H6 and / or CD3-mediated related disease, before or after treatment.

[0115] According to embodiments of the present invention, the sample to be tested includes at least one of the following: blood, saliva, sweat, tissue, cells, serum, plasma, feces, and urine.

[0116] According to embodiments of the present invention, the prognostic effect of B7H6 or B7H6 and / or CD3-mediated diseases is determined based on the content of B7H6 or B7H6 and / or CD3 in the test sample of a patient with B7H6 or B7H6 and / or CD3-mediated diseases before or after treatment.

[0117] According to embodiments of the present invention, a decrease in the levels of B7H6, or B7H6 and / or CD3, in the test sample of a patient with B7H6, or a related disease mediated by B7H6 and / or CD3, after treatment is an indicator of a good prognosis for the patient.

[0118] According to embodiments of the present invention, the B7H6-mediated diseases and the B7H6 and CD3-mediated diseases include cancer.

[0119] According to embodiments of the present invention, the cancer includes at least one of the following: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma.

[0120] In an eighteenth aspect, the present invention proposes the use of the aforementioned bispecific binding molecules, antibodies or antigen-binding fragments, nucleic acid molecules, expression vectors, recombinant cells, compositions, or drugs in the treatment or prevention of B7H6, or B7H6 and / or CD3-mediated diseases. As previously described, the bispecific binding molecules exhibit high binding activity to both B7H6 and / or CD3, and the antibodies or antigen-binding fragments can effectively bind to human or monkey B7H6, thereby effectively treating or preventing B7H6, or B7H6 and / or CD3-mediated diseases.

[0121] According to embodiments of the present invention, the above-described uses may further include at least one of the following additional technical features:

[0122] According to embodiments of the present invention, the B7H6-mediated diseases and the B7H6 and CD3-mediated diseases include cancer.

[0123] According to embodiments of the present invention, the cancer includes at least one of the following: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma.

[0124] In a nineteenth aspect of the invention, the invention proposes the use of the aforementioned bispecific binding molecules, antibodies or antigen-binding fragments, nucleic acid molecules, expression vectors or recombinant cells in the diagnosis of B7H6, or B7H6 and / or CD3, or related diseases, in the staging of B7H6, or B7H6 and / or CD3-mediated related diseases, or in assessing the prognosis of B7H6, or B7H6 and / or CD3-mediated related diseases. As previously stated, the bispecific binding molecules, or nucleic acid molecules, expression vectors, or bispecific binding molecules expressed by recombinant cells, proposed in this application can effectively bind to B7H6 and / or CD3. The antibody or antigen-binding fragments, or nucleic acid molecules, expression vectors, or antibody or antigen-binding fragments expressed by recombinant cells, can effectively bind to human or monkey B7H6. Therefore, the method described in this application can effectively detect the content of B7H6, or B7H6 and / or CD3, in test samples derived from test individuals, and can effectively diagnose, stage, and assess the prognosis of diseases mediated by B7H6, or B7H6 and / or CD3.

[0125] According to embodiments of the present invention, the above-described uses may further include at least one of the following additional technical features:

[0126] According to embodiments of the present invention, the B7H6-mediated diseases and the B7H6 and CD3-mediated diseases include cancer.

[0127] According to embodiments of the present invention, the cancer includes at least one of the following: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, rhabdomyosarcoma, astrocytoma, neuroblastoma, and glioma.

[0128] The term "subject" or "individual" in this invention generally refers to mammals, such as primates and / or rodents, particularly humans, monkeys, or rats.

[0129] The beneficial effects of this invention are:

[0130] 1) The mouse-derived anti-B7H6 monoclonal antibody obtained in this invention has higher B7H6 protein binding activity than existing B7H6 monoclonal antibodies, and the B7H6 protein includes human B7H6 protein and monkey B7H6 protein.

[0131] 2) The humanized antibody obtained by humanizing the mouse anti-B7H6 monoclonal antibody also has higher B7H6 protein binding activity than the existing B7H6 monoclonal antibody. The B7H6 protein includes human B7H6 protein and monkey B7H6 protein. Moreover, the humanized antibody has lower immunogenicity, higher safety and longer efficacy.

[0132] 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. Attached Figure Description

[0133] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0134] Figure 1A This is an ELISA result diagram of the binding of human B7H6 antibody to B7H6 according to an embodiment of the present invention;

[0135] Figure 1B This is an ELISA result diagram of B7H6 antibody binding to monkey B7H6 according to an embodiment of the present invention;

[0136] Figure 2A This is a flow cytometry result of B7H6 antibody binding to CHO cells overexpressing human B7H6 according to an embodiment of the present invention;

[0137] Figure 2B This is a flow cytometry result of B7H6 antibody binding to CHO cells overexpressing monkey B7H6 according to an embodiment of the present invention.

[0138] Figure 3 This is a diagram showing the in vitro cytotoxicity experiment results of the B7H6 / CD3 bispecific binding protein and PBMC on HCT-15 cells according to an embodiment of the present invention. Detailed Implementation

[0139] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0140] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0141] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0142] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

[0143] The antibody or antigen-binding fragments described in this invention are typically prepared by biosynthetic methods. Based on the nucleotide sequence described in this invention, those skilled in the art can readily obtain the encoding nucleic acid of this invention using various known methods. These methods include, but are not limited to, PCR, artificial DNA synthesis, etc., and specific methods can be found in J. Sambrook, *Molecular Cloning: A Laboratory Manual*. As one embodiment of this invention, the encoding nucleic acid sequence of this invention can be constructed by synthesizing nucleotide sequences in segments and then performing overlap extension PCR. The antibody or antigen fragments are numbered and defined using the Kabat numbering system.

[0144] The antibodies of the present invention include murine antibodies, chimeric antibodies, and humanized antibodies, with humanized antibodies being preferred.

[0145] In this invention, the term "mouse antibody" refers to a monoclonal antibody against human B7H6 prepared according to the knowledge and skills in the art. Preparation involves injecting the test subject with an antigen, followed by isolating a hybridoma expressing an antibody with the desired sequence or functional characteristics. In a preferred embodiment of the invention, the mouse B7H6 antibody or its antigen-binding fragment may further comprise a light chain constant region of a mouse κ, λ chain or a variant thereof, or further comprise a heavy chain constant region of mouse IgG1, IgG2, IgG3 or a variant thereof.

[0146] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by murine antibodies. The variable region gene is cloned from mouse hybridoma cells, and then the constant region gene of a human antibody is cloned as needed. The mouse variable region gene and the human constant region gene are linked to form a chimeric gene, which is then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic expression system. In a preferred embodiment of the invention, the light chain of the B7H6 chimeric antibody further comprises the Fc region of a human κ, λ chain, or a variant thereof. The heavy chain of the B7H6 chimeric antibody further comprises the heavy chain constant region of human IgG1, IgG2, IgG3, IgG4, or a variant thereof.

[0147] The term "humanized antibody," also known as a CDR-grafted antibody, refers to an antibody produced by grafting a mouse CDR sequence into the variable region framework of a human antibody, i.e., a different type of human germline antibody framework sequence. Such framework sequences can be obtained from public DNA databases containing germline antibody gene sequences or from publicly available references. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the VBase human germline sequence database. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reversion mutations can be performed on the aforementioned human antibody variable region framework sequence to maintain activity.

[0148] In this article, "monoclonal antibody" refers to an antibody with a single antigen-binding site.

[0149] In this article, "dual antibody" refers to an antibody with two different antigen-binding sites.

[0150] In this document, the term "mutant" or "variant" may refer to a molecule obtained by mutating one or more nucleotides or amino acids into any naturally occurring or engineered molecule.

[0151] The terms "complementarity-determining region" or "CDR" or "CDR sequence" refer to the amino acid sequence in an antibody responsible for antigen binding. For example, these typically include amino acid residues near 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and near 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health). of Health, Bethesda, MD. (1991)); and / or amino acid residues from “high-variable rings” (e.g., amino acid residues near 26-32 (L1), 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 Lesk J. Mol. Biol. 196: 901-917 (1987)).

[0152] In this paper, the term "identity" is used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm by Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm by Smith et al. (1981) Adv. Appl. Math. 2: 482; the similarity search method by Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444; and the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997)). And the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, and include, but are not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266: 460-480 (1996)); or GAP, BESTFIT, BLASTAltschul et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0153] Without substantially affecting antibody activity (retaining at least 95% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequences of the present invention to obtain variants of the antibody or its functional fragments. These are all considered to be included within the scope of protection of the present invention. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences of the present invention can have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. Sequence identity described in the present invention can be measured using sequence analysis software, such as the computer program BLAST using default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in the present invention are shown in N-terminus to C-terminus arrangement.

[0154] As previously described, the monoclonal antibodies of the present invention may be full-length antibodies or may contain only their functional fragments (e.g., Fab, F(ab')2, or scFv fragments), or may be modified to affect function. The present invention includes anti-B7H6 antibodies with modified glycosylation patterns. In some applications, modification to remove undesirable glycosylation sites may be useful, or to antibodies lacking a fucose moiety on the oligosaccharide chain to, for example, enhance antibody-dependent cytotoxicity (ADCC) function. In other applications, galactosylation modification may be performed to alter complement-dependent cytotoxicity (CDC).

[0155] In this paper, the term "full-length antibody" refers to a tetrapeptide chain structure composed of two identical light chains and two identical heavy chains linked by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE). Immunoglobulins of the same class can also be divided into different subclasses based on their amino acid composition, such as IgG1, IgG2, IgG3, and IgG4. Immunoglobulin light chains are classified into κ chains or λ chains based on their constant regions.

[0156] As used herein, the term "functional fragment" specifically refers to antibody fragments such as CDR transplantation antibodies, Fab, Fab', F(ab')2, Fv, or scFv, nanobodies, or any fragment that should be able to increase its half-life through chemical modification or incorporation into liposomes, such as the addition of poly(alkylene) glycols, like polyethylene glycol ("PEGylated, PEGylated") (a PEGylated fragment referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" stands for polyethylene glycol), which have B7H6 binding activity. Preferably, the functional fragment will consist of or contain a portion of the heavy chain variable region or light chain variable region of its source antibody, the portion of which is sufficient to retain the same binding specificity and sufficient affinity as its source antibody, preferably at least 1 / 100 of the affinity for B7H6, and more preferably at least 1 / 10. This functional fragment will contain at least 3 amino acids, preferably 5, 10, 15, 25, 50, and 100 consecutive amino acids from the antibody sequence from which it is derived.

[0157] In this invention, unless otherwise stated, the term "antigen-binding fragment" as used generally refers to an antigen-binding antibody fragment, which may include a portion of a complete antibody, typically an antigen-binding region or a variable region, such as, for example, CDR transplanted antibodies, Fab, Fab', F(ab')2, Fv or scFv, nanobodies, etc.

[0158] In this paper, the term "CDR-transplanted antibody" refers to the transplantation of the CDR of a monoclonal antibody from one species into the variable region of an antibody from another species. For example, the CDR of a murine monoclonal antibody can be transplanted into the variable region of a human antibody to replace the human antibody's CDR, thereby giving the human antibody the antigen-binding specificity of the murine monoclonal antibody while reducing its heterologous nature.

[0159] In this article, the term "Fab antibody" or "Fab" generally refers to an antibody containing only Fab molecules, which consist of the VH and CH1 of the heavy chain and the complete light chain, linked by a disulfide bond.

[0160] In this paper, the term “nanobody” (single-domain antibody or VHH antibody), which was originally described as an antigen-binding immunoglobulin (variable) domain of “heavy chain antibody” (i.e., “antibody lacking light chains”) (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363, 446-448 (1993)), contains only the heavy chain variable region (VH) and the conventional CH2 and CH3 regions, through which it specifically binds to the antigen.

[0161] In this article, the term "Fv antibody" generally refers to an antibody composed only of a light chain variable region (VL) and a heavy chain variable region (VH) linked by non-covalent bonds. It is the smallest functional fragment of an antibody that retains the complete antigen-binding site.

[0162] In this paper, the term "single-chain antibody" or "scFv" refers to a fragment consisting of the variable regions of the antibody heavy chain and light chain linked by a short peptide.

[0163] In this paper, the "knob into hole structure" refers to the formation of a button (hole) mutation in the CH3 region of the constant region of the antibody heavy chain, facilitating heavy chain interlocking and the formation of a heterodimer. For example, in this application, this is achieved by mutating the amino acids in the CH3 domain of the constant region of the human IgG1 heavy chain (T366S, L368A, Y407V, Y349C mutations in one chain, i.e., "hole"; and T366W, S354C mutations in the other chain, i.e., "knob"). The amino acid numbers here are based on the Kabat numbering system. For example, "T366S" means that the T amino acid at position 366 according to the Kabat numbering system is replaced by an S amino acid.

[0164] The amino acid or nucleic acid sequences involved in this invention are detailed in Table 1.

[0165] Table 1:

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174] The present invention will be described in detail below through examples. In the examples or test cases, experimental methods without specific conditions are performed under conventional conditions.

[0175] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0176] Example 1: Preparation of anti-human B7H6 hybridoma monoclonal antibody

[0177] This embodiment was used to obtain a murine monoclonal antibody against human B7H6. The immunogenic antigens were human B7H6 (ACRO, B76-H52H8, SEQ ID NO:37) and monkey B7H6 protein (ACRO, B76-C52Ha, SEQ ID NO:38). C57bl / 6 mice (9 weeks old, purchased from Shanghai Lexco, weighing about 20g) were immunized.

[0178] Mice were immunized three times intraperitoneally with purified antigen and complete Freund's adjuvant, and the immune response was assessed after blood collection via tail vein. Serum was screened using ELISA and flow cytometry following standard procedures to obtain mice with anti-human B7H6 immunoglobulin. Spleen cells from mice with the highest anti-B7H6 immunoglobulin levels were fused with mouse myeloma cells SP2 / 0 (ATCC code CRL-1581). The fused hybridoma cells were resuspended in HAT complete medium (RPMI-1640 medium containing 20% ​​FBS, 1×HAT, and 1×OPI) and aliquoted into 96-well cell culture plates, incubated at 37°C and 5% CO2. On day 5 post-fusion, 50 μL of HAT complete medium was added per well. On days 7–8 post-fusion, the medium was completely replaced with HT complete medium (RPMI-1640 medium containing 20% ​​FBS, 1×HT, and 1×OPI) at 200 μL per well, based on cell growth density.

[0179] On days 10-11 post-fusion, flow cytometry was performed to detect cell conjugation based on cell growth density. Positive wells were replaced with new medium, and the cells were promptly expanded to 24-well plates according to density. Cell lines transferred to 24-well plates were retested and then preserved for the first subcloning. Cells showing positive results in the first subcloning were preserved and subjected to a second subcloning. Cells showing positive results in the second subcloning were preserved and used for protein expression. Antibodies were further prepared using serum-free cell culture and purified by protein G affinity chromatography for subsequent functional activity assays.

[0180] Example 2: Hybridoma cell sequencing

[0181] The total number of candidate hybridoma cells screened in Example 1 above was cultured to 10. 6 Cells were collected by centrifugation at 800 rpm for 10 minutes, and total RNA was extracted using the Trizol kit (Invitrogen). Using the total RNA as a template, a cDNA library was synthesized by reverse transcription (Invitrogen). The cDNA was then used as a template for PCR amplification of the corresponding B7H6 antibody variable region nucleic acid sequence in hybridoma cells. The primer sequences used in the PCR amplification reaction were complementary to the first frame region or signal peptide region and constant region of the antibody variable region (for specific sequence selection, refer to Larrick, JW, et al., (1990) Scand. J. Immunol., 32, 121-128 and Coloma, JJ et al., (1991) BioTechniques, 11, 152-156). PCR amplification was performed in a 50 μL reaction system, with the following components added: 2 μL cDNA, 5 μL 10× PCR buffer, 2 μL (5 μM) upstream and downstream primers, 2 μL dNTPs, 1 μL Taq enzyme (Takara, ExTaq), and 38 μL H2O. The mixture was pre-denatured at 95℃ for 5 min, followed by temperature cycling for PCR amplification. The reaction conditions were: denaturation at 94℃ for 30 s, annealing at 58℃ for 45 s, extension at 72℃ for 50 s, for a total of 32 cycles, followed by a final extension at 72℃ for 7 min. Sequencing of the amplified products yielded the heavy chain variable region (SEQ ID NO: 23) and light chain variable region (SEQ ID NO: 24) sequences of the anti-B7H6 mouse monoclonal antibody 4-99.

[0182] Example 3: Humanization of Anti-human B7H6 Monoclonal Antibody

[0183] Based on Example 2, by comparing the IMGT human antibody heavy and light chain variable region germline gene database and MOE software, heavy chain or light chain variable region germline genes with high homology to murine monoclonal antibodies were selected as templates. The CDRs of murine monoclonal antibodies were then transplanted into the corresponding human templates, forming variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The amino acid residues were determined and annotated using the Kabat numbering system.

[0184] To maintain the conformation of the CDR region, residues at the VL and VH binding interfaces, residues close to the CDR and embedded inside the protein, and residues that directly interact with the CDR are reverse-mutated to ensure that the activity of the variable region is not affected.

[0185] Example 4: Transient expression of B7H6 humanized antibody

[0186] After selecting and reverting mutations to the variable region framework of the above-mentioned B7H6 monoclonal antibody, the heavy chain variable region sequence of the obtained humanized B7H6 antibody is shown in SEQ ID NO:25, and the light chain variable region sequence is shown in SEQ ID NO:26. The nucleotide sequences encoding the humanized antibody heavy chain (SEQ ID NO:39) and the nucleotide sequences encoding the humanized antibody light chain (SEQ ID NO:40) were recombined into the pTT5 plasmid using molecular biology techniques. The B7H6 antibody was prepared by transiently transfecting ExpiCHO-S cells (Gibco, catalog number A29127) with the pTT5 vector carrying the humanized anti-human B7H6 antibody light and heavy chains. The specific experimental procedures are as follows:

[0187] One day before transfection, the cell density of ExpiCHO-S cells was adjusted to (3-4) × 10⁻⁶. 6 Incubate overnight at 37°C, 8% CO2, with shaking at 120 rpm. On the day of transfection, cells grew to 7 × 10⁹ / mL. 6 ~1×10 7 When the cell viability is greater than 95%, prepare for transfection by diluting the cells to 6 × 10⁶ / mL using fresh, pre-warmed ExpiCHO medium (Gibco, catalog number A2910002). 6The pTT5 plasmid carrying the humanized anti-human B7H6 antibody light and heavy chains was transfected into ExpiCHO-S cells at a 2:1 molar ratio using ExpiFectamine CHO transfection reagent (Gibco, catalog number A29129). Cells were incubated at 37°C, 8% CO2, and 120 rpm with shaking. 18-22 h post-transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed were mixed and immediately added to the transfected cells. Cells were then incubated at 32°C, 5% CO2, and 120 rpm with shaking. On day 5 post-transfection, 8 mL of ExpiCHO Feed was added to the cells, mixed, and the cells were cultured further. Cell count and viability were observed daily. Cells were harvested by centrifugation when viability dropped below 80% or after 10-14 days of culture. The expression supernatant was filtered through a 0.22 μm filter membrane. Antibodies with Fc domains were captured from the expression supernatant using a Mabselect Prism A affinity chromatography column (GE, catalog number 17549854). After equilibrating the column with phosphate buffer at pH 7.2, the supernatant was passed through the affinity chromatography column and eluted with elution buffer (100 mM citric acid, pH 2.7). Finally, the elution was concentrated and replaced with PBS buffer. The purified antibody was identified by SDS-PAGE and its purity was above 95%, thus obtaining the humanized B7H6 antibody.

[0188] Example 5: Mouse B7H6 Antibody ELISA Binding Assay

[0189] The ELISA assay was used to detect the binding characteristics of the murine B7H6 antibody 4-99 obtained in Example 1. Human B7H6 protein (ACRO biosystems, B76-H52H8) and monkey B7H6 protein (ACRO biosystems, B76-C52Ha) were diluted to 2 μg / mL with coating buffer (35 mM NaHCO3, 15 mM Na2CO3, pH 9.6), and 100 μL was added to each well of the ELISA plate and incubated overnight at 4°C. The plate was then washed three times with PBST (0.05% Tween 20-PBS, pH 7.2). 300 μL of blocking buffer (1% BSA, 0.05% Tween 20-PBS, pH 7.2) was added to the plate, and the plate was incubated at room temperature for 2 h, followed by three washes with PBST. Murine antibody 4-99 was added to each well and incubated at room temperature for 1 h. The plate was then washed three times with PBST. Add 100 μL of HRP-goat anti-mouse IgG secondary antibody (boster, catalog number BA1051) diluted with blocking buffer to each well and incubate at room temperature for 1 hour. Wash three times with PBST, add TMB to each well, and incubate at room temperature in the dark for 2-5 minutes. Terminate the reaction with 2M sulfuric acid in each well. Finally, read the OD450 value using a microplate reader. Figure 1AThis indicates that the murine anti-B7H6 antibody (4-99) of the present invention can bind to human B7H6 protein. Figure 1B This indicates that the murine anti-B7H6 antibody (4-99) of the present invention can also bind to monkey B7H6 protein.

[0190] Example 6: Construction of CHO-K1 cells overexpressing human and cynomolgus monkey B7H6

[0191] HEK293T cells were seeded in T150 culture flasks and cultured in DMEM complete medium. After overnight culture, endotoxin-free psPAX2, pMD2.G, and pCDH-CMV-FCGR3A-IRES-puro vectors (pCDH-CMV-MCS-IRES-puro vectors with an inserted human B7H6 protein coding sequence (SEQ ID NO:54) or cynomolgus monkey protein coding sequence (SEQ ID NO:55)) were added to 1.5 mL of Opti-MEM (Gibco, catalog number 31985070) medium at a ratio of 7:3:10, followed by 100 μL of Lipofectamine 3000 transfection reagent. 100 μL of Lipofectamine 3000 (thermo, catalog number L3000008) transfection reagent was then added to 1.5 mL of Opti-MEM medium and mixed thoroughly. Mix DNA dilution buffer and liposome dilution buffer at a volume ratio of 1:1, incubate at room temperature for 5-10 min, then add to 293T cells. After culturing for 48 hours, harvest the viral supernatant. Centrifuge the viral supernatant at 2000g for 10 min at 4℃, collect the supernatant, filter through a 0.45μm filter, add PEG8000 solution (Shanghai Sangon Biotech), mix thoroughly, incubate at 4℃ overnight, then centrifuge at 2200g for 90 min. A white precipitate will appear at the bottom of the centrifuge tube. Resuspend the virus in sterile PBS buffer.

[0192] Polybrene (Sigma, TR-1003) (final concentration 8 μg / mL) was added to DME / F12 medium, mixed well, and then an appropriate amount of virus solution was added. 2E5 CHO-K1 cells were placed in 24-well plates, and virus-containing medium was added. After 8 hours in an incubator, the medium was replaced with fresh medium. After 48 hours, the B7H6 expression level on the surface of CHO-K1 cells was detected by flow cytometry. Once a positive cell population appeared, a limiting dilution was performed (digested cells diluted to a density of 4 cells / mL), and then seeded into 96-well plates at a density of 200 μL per well. After culturing for 2 weeks until the cells clearly formed single cell clusters, the B7H6 expression level on the surface of each clone was detected by flow cytometry. All positive cells were identified as CHO-K1 cells overexpressing human B7H6 or cynomolgus monkey CD16.

[0193] Example 7: Flow Cytometry Combination Experiment with Mouse B7H6 Antibody

[0194] The CHO-K1 cells overexpressing human B7H6 or CHO-K1 cells overexpressing cynomolgus monkey CD16 obtained in Example 6 were diluted with PBS to a concentration of 2 × 10⁻⁶. 6 / mL, add 100μL / tube to a 1.5mL EP tube, add 10μL / tube of goat serum, and block at 4℃ for 30min. Add graded concentrations (5-fold dilution, final concentration up to 10μg / mL) of B7H6 antibody (4-99), control antibody 12G4 (CN114395045A), and control antibody B7H6#14 / CD3#1 (US20210107983A1) separately, and incubate at 4℃ for 30min. Add 1mL of PBS to the EP tube, centrifuge at 3500rpm×5min at 4℃, discard the supernatant, and wash once with PBS. After centrifugation, discard the supernatant, resuspend the cells in 100μL / tube of PBS, add 0.1μL / tube of Alexa-647-labeled goat anti-mouse antibody secondary antibody (Invitrogen), and incubate at 4℃ in the dark for 30min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μL / tube of PBS and analyze using flow cytometry. Figure 2A and Figure 2B The results show that the B7H6 antibody 4-99 of the present invention can bind to human B7H6 and monkey B7H6, and has a higher affinity than the control.

[0195] Example 8: Preparation of bispecific antibody against B7H6×CD3 and detection of its binding activity and cell-killing ability.

[0196] Based on the experimental results of Examples 1-7, the inventors designed a bispecific antibody B7H6 / CD3 and tested its binding activity and cell-killing ability.

[0197] 8.1 Design and preparation of bispecific antibodies against B7H6 / CD3

[0198] This antibody contains two monovalent units. One monovalent unit is an anti-CD3 scFv-Fc form, with variable region amino acids derived from CD3 clone UCHT-1 (SEQ ID NO: 41, 42). The other monovalent unit is a humanized anti-B7H6 IgG-like form (one heavy chain + one light chain, sequences from SEQ ID NO: 49, SEQ ID NO: 50 of this invention). This bispecific antibody is named B7H6 / CD3. This bispecific antibody contains three polypeptide chains: the heavy chain of the anti-B7H6 IgG-like antibody, the light chain of the anti-B7H6 IgG-like antibody, and the heavy chain containing the CD3 single-chain antibody scFv (SEQ ID NO: 46). The constant regions of the heavy chains of the two heavy chains are derived from the human antibody IgG. Due to the molecule's unique asymmetric structure, different amino acid mutations were introduced into the constant regions of the two chains to reduce the formation of homodimers. Simultaneously, to prevent cross-linking activation caused by the Fcγ receptor, (L234A / L235A) mutations were also introduced into the constant region of the heavy chain.

[0199] The bispecific antibody was prepared according to the method described in Example 6.2, and the bispecific antibody was prepared by combining the plasmid ratio (1:1 or other ratio) and undergoing one-step affinity purification. The relevant sequence of the bispecific antibody B7H6 / CD3 is shown in Table 3.

[0200] Table 3

[0201] B7H6 heavy chain B7H6 Light Chain CD3 Rechain 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

[0202] 8.2 Cell killing experiment

[0203] HCT-15 colorectal cancer cells were digested, counted, and the cell density was adjusted to 2 × 10⁻⁶. 5 / mL. Turn on the RTCA instrument (Agilent), select instrument type DP, select experimental mode, and fill in cell and drug information. Enter the schedule to set the experimental steps. Add 50μL of fresh culture medium (89% RPMI 1640 medium + 10% fetal bovine serum + 1% penicillin and streptomycin) to the plate and place it in the instrument. Close the instrument and click Start. After completion, remove the plate, add 100μL of cell suspension, incubate at room temperature for 15-30 minutes to prevent edge effects, place it in the instrument, and click Start. After growth reaches the logarithmic growth phase, pause and add 50μL of LPBMC (4×10⁻⁶). 6 Add the sample ( / mL) and a gradient concentration of bispecific antibody, click start, and analyze after a period of time. Figure 3 The results showed that with increasing concentrations of the B7H6 / CD3 bispecific antibody, the killing efficiency of PBMCs against colorectal cancer HCT-15 cells gradually increased, indicating that B7H6 / CD3 can effectively promote the specific killing of B7H6 cells by PBMCs.+ Tumor cells.

[0204] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0205] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A B7H6 antibody or its antigen-binding fragment, characterized in that, include: The heavy chain variable regions CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NO: 1, 2, and 3, respectively; The light chain variable regions CDR1, CDR2, and CDR3 sequences are shown in the amino acid sequences of SEQ ID NO: 4, 5, and 6, respectively.

2. The B7H6 antibody or its antigen-binding fragment according to claim 1, characterized in that, include: Heavy chain FR region and light chain FR region.

3. The B7H6 antibody or its antigen-binding fragment according to claim 2, characterized in that, The heavy chain FR region and the light chain FR region are derived from primate-derived antibodies or murine antibodies.

4. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The heavy chain FR region and the light chain FR region are derived from human antibodies.

5. The B7H6 antibody or its antigen-binding fragment according to claim 2, characterized in that, include: The heavy chain frame regions HFR1, HFR2, HFR3, and HFR4 sequences as shown in SEQ ID NO: 7-10 respectively; the light chain frame regions LFR1, LFR2, LFR3, and LFR4 sequences as shown in SEQ ID NO: 11-14 respectively; or The heavy chain frame regions HFR1, HFR2, HFR3 and HFR4 sequences as shown in SEQ ID NO: 15~18 respectively; and the light chain frame regions LFR1, LFR2, LFR3 and LFR4 sequences as shown in SEQ ID NO: 19~22 respectively.

6. The B7H6 antibody or its antigen-binding fragment according to claim 1, characterized in that, include: 1) The heavy chain variable region as shown in SEQ ID NO: 23, and the light chain variable region as shown in SEQ ID NO: 24; or 2) The heavy chain variable region as shown in SEQ ID NO:25, and the light chain variable region as shown in SEQ ID NO:

26.

7. The B7H6 antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or antigen-binding fragment contains a heavy chain constant region and a light chain constant region, which are derived from primate-derived antibodies or murine antibodies.

8. The B7H6 antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or antigen-binding fragment contains a heavy chain constant region and a light chain constant region, which are derived from human antibodies.

9. The B7H6 antibody or its antigen-binding fragment according to claim 7, characterized in that, Both the light chain constant region and the heavy chain constant region are derived from mouse IgG antibodies or human IgG antibodies.

10. The B7H6 antibody or its antigen-binding fragment according to claim 7, characterized in that, Both the light chain constant region and the heavy chain constant region are derived from mouse IgG1 antibody or human IgG1 antibody.

11. The B7H6 antibody or its antigen-binding fragment according to claim 7, characterized in that, The antibody has a heavy chain constant region as shown in SEQ ID NO:27 and a light chain constant region as shown in SEQ ID NO:28; or The antibody has a heavy chain constant region as shown in SEQ ID NO:29 and a light chain constant region as shown in SEQ ID NO:

30.

12. The B7H6 antibody or its antigen-binding fragment according to claim 1, characterized in that, 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 of the amino acid sequence shown in SEQ ID NO:33 and a light chain of the amino acid sequence shown in SEQ ID NO:34; or The antibody or antigen-binding fragment has a heavy chain of the amino acid sequence shown in SEQ ID NO:35 and a light chain of the amino acid sequence shown in SEQ ID NO:

36.

13. The B7H6 antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or antigen-binding fragment includes monoclonal antibodies.

14. The B7H6 antibody or its antigen-binding fragment according to claim 13, characterized in that, The monoclonal antibody includes at least one of full-length antibody, Fv, single-chain antibody, and Fab.

15. The B7H6 antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment can bind to the amino acid sequence shown in SEQ ID NO: 37 and / or 38.

16. A B7H6 / CD3 bispecific binding molecule, characterized in that, include: A first binding region, comprising peptide chain 1, peptide chain 2, a first heavy chain constant region, and a light chain constant region; wherein peptide chain 1 includes the heavy chain variable region as described in claim 6, peptide chain 2 includes the light chain variable region as described in claim 6, the first heavy chain constant region and the light chain constant region are derived from a human antibody, the N-terminus of the first heavy chain constant region is connected to the C-terminus of the heavy chain variable region, the N-terminus of the light chain constant region is connected to the C-terminus of the light chain variable region, and peptide chain 1 and peptide chain 2 are linked by disulfide bonds; and The second binding region, having CD3 binding activity, comprises an anti-CD3 single-chain antibody and a second heavy chain constant region. The anti-CD3 single-chain antibody includes an anti-CD3 antibody light chain variable region, an anti-CD3 antibody heavy chain variable region, and a linker peptide. The N-terminus of the linker peptide is connected to the C-terminus of the anti-CD3 antibody heavy chain variable region, and the C-terminus of the linker peptide is connected to the N-terminus of the anti-CD3 antibody light chain variable region; or the N-terminus of the linker peptide is connected to the C-terminus of the anti-CD3 antibody light chain variable region, and the C-terminus of the linker peptide is connected to the N-terminus of the anti-CD3 antibody heavy chain variable region. The anti-CD3 antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO:41, and the anti-CD3 antibody light chain variable region has the amino acid sequence shown in SEQ ID NO:

42. The second heavy chain constant region is derived from a human antibody. The first heavy chain constant region and the second heavy chain constant region are connected by a knot-into-hole structure.

17. The bispecific binding molecule according to claim 16, characterized in that, The linker peptide has an amino acid sequence (GGGGS)n, where n is an integer greater than or equal to 1.

18. The bispecific binding molecule according to claim 17, characterized in that, n can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

19. The bispecific binding molecule according to claim 16, characterized in that, The linker peptide has the amino acid sequence shown in SEQ ID NO:

44.

20. The bispecific binding molecule of claim 16, characterized in that, The anti-CD3 single-chain antibody has the amino acid sequence shown in SEQ ID NO:

43.

21. The bispecific binding molecule according to claim 16, characterized in that, Both the first heavy chain constant region and the light chain constant region are derived from human IgG antibodies.

22. The bispecific binding molecule according to claim 16, characterized in that, Both the first heavy chain constant region and the light chain constant region are derived from human IgG1 antibody.

23. The bispecific binding molecule according to claim 16, characterized in that, 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.

24. The bispecific binding molecule according to claim 16, characterized in that, 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.

25. The bispecific binding molecule according to claim 16, characterized in that, The second heavy chain constant region is derived from human IgG antibody.

26. The bispecific binding molecule according to claim 16, characterized in that, The second heavy chain constant region is derived from human IgG1 antibody.

27. The bispecific binding molecule according to claim 16, characterized in that, The second heavy chain constant region has the amino acid sequence shown in SEQ ID NO:

45.

28. The bispecific binding molecule according to claim 16, characterized in that, The second binding region has the amino acid sequence shown in SEQ ID NO:

46.

29. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the B7H6 antibody or its antigen-binding fragment as described in any one of claims 1 to 15, or the bispecific binding molecule as described in any one of claims 16 to 28.

30. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 29.

31. A recombinant cell, characterized in that, The recombinant cell carries the nucleic acid molecule of claim 29, the expression vector of claim 30, or is capable of expressing the B7H6 antibody or its antigen-binding fragment of any one of claims 1 to 15, or the bispecific binding molecule of any one of claims 16 to 28, wherein the recombinant cell is a prokaryotic cell or a eukaryotic cell, and the eukaryotic cell is a mammalian cell.

32. The recombinant cell according to claim 31, characterized in that, The recombinant cells are obtained by introducing the expression vector of claim 30 into host cells.

33. A composition, characterized in that, It includes the B7H6 antibody or its antigen-binding fragment as described in any one of claims 1 to 15, the bispecific binding molecule as described in any one of claims 16 to 28, the nucleic acid molecule as described in claim 29, the expression vector as described in claim 30, or the recombinant cell as described in any one of claims 31 to 32.

34. A drug, characterized in that, It includes the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 15, the bispecific binding molecule as described in any one of claims 16 to 28, the nucleic acid molecule as described in claim 29, the expression vector as described in claim 30, the recombinant cell as described in any one of claims 31 to 32, or the composition as described in claim 33.

35. A reagent kit, characterized in that, The kit contains the B7H6 antibody or its antigen-binding fragment as described in any one of claims 1 to 15, the bispecific binding molecule as described in any one of claims 16 to 28, the nucleic acid molecule as described in claim 29, the expression vector as described in claim 30, or the recombinant cells as described in any one of claims 31 to 32.

36. Use of the B7H6 antibody or its antigen-binding fragment according to any one of claims 1 to 15, the nucleic acid molecule according to claim 29, the expression vector according to claim 30, the recombinant cell according to any one of claims 31 to 32, or the composition according to claim 33 in the preparation of a medicament for killing colon cancer cells.

37. Use of the bispecific binding molecule of any one of claims 16-28, the nucleic acid molecule of claim 29, the expression vector of claim 30, the recombinant cell of any one of claims 31-32, or the composition of claim 33 in the preparation of a medicament for the treatment of colon cancer.

38. Use of the B7H6 antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, the nucleic acid molecule according to claim 29, the expression vector according to claim 30, or the recombinant cells according to any one of claims 31 to 32 in the preparation of a kit for detecting B7H6.

39. Use of the bispecific binding molecule of any one of claims 16-28, the nucleic acid molecule of claim 29, the expression vector of claim 30, or the recombinant cells of any one of claims 31-32 in the preparation of a kit for detecting B7H6 and / or CD3.

Images