Bispecific antibody targeting b7h7 and CD3 and use thereof

Abstract

The present application discloses a bispecific antibody targeting B7H7 and CD3 and a use thereof. The bispecific antibody comprises: a first antigen binding region comprising an anti-CD3 antibody and having CD3-binding activity; and a second antigen binding region comprising an anti-B7H7 antibody and having B7H7-binding activity. The bispecific antibody can bind to human and monkey B7H7 with high specificity and high affinity, and bind to human and monkey CD3 with low affinity and high specificity, promoting PBMC-mediated tumor cell killing.

Description

Bispecific antibodies targeting B7H7 and CD3 and uses thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and specifically relates to bispecific antibodies targeting B7H7 and CD3 and uses thereof. BACKGROUND

[0002] Bispecific antibodies are antibodies that can specifically bind to two antigenic sites at the same time.

[0003] Current treatments targeting B7H7 are limited to single antibody drugs, and there is a lack of bispecific or multispecific antibodies targeting this target, which limits the diversity and specificity of treatment strategies.

[0004] Current bispecific antibodies targeting CD3 can cause serious side effects such as cytokine storm, which limits their clinical application. Many CD3 antibodies have species specificity, and the feasibility of safety testing in non-human primates is low. Moreover, existing CD3 bispecific antibodies have problems such as too high cytokine toxicity and poor sequence stability.

[0005] Therefore, there is a need to develop new bispecific antibodies targeting B7H7 and CD3. SUMMARY

[0006] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes a high-safety bispecific antibody targeting B7H7 and CD3.

[0007] Specifically, the present application provides the following technical solutions:

[0008] In a first aspect, the present application provides a bispecific antibody. According to embodiments of the present application, the bispecific antibody comprises: a first antigen binding region having CD3 binding activity; and a second antigen binding region having B7H7 binding activity; wherein the first antigen binding region comprises an anti-CD3 antibody, the anti-CD3 antibody comprising a heavy chain complementarity determining region HCDR, the HCDR comprising at least one selected from the group consisting of SEQ ID NOs: 4-6, 66 or an amino acid sequence having at least 80% homology with SEQ ID NOs: 4-6, 66; the anti-CD3 antibody further comprising: a light chain complementarity determining region LCDR, the LCDR comprising at least one selected from the group consisting of SEQ ID NOs: 1-3 or an amino acid sequence having at least 80% homology with SEQ ID NOs: 1-3; the second antigen binding region comprises an anti-B7H7 antibody, the anti-B7H7 antibody comprising a heavy chain complementarity determining region HCDR, the HCDR of the anti-B7H7 antibody comprising at least one selected from the group consisting of SEQ ID NOs: 26-28 or an amino acid sequence having at least 80% homology with SEQ ID NOs: 26-28; the anti-B7H7 antibody further comprising: a light chain complementarity determining region LCDR, the LCDR comprising at least one selected from the group consisting of SEQ ID NOs: 23-25 or an amino acid sequence having at least 80% homology with SEQ ID NOs: 23-25. In some examples of the present application, the bispecific antibody with the foregoing sequences can specifically and highly bind to human and monkey B7H7, and specifically and lowly bind to human and monkey CD3, promote PBMC to kill tumor cells, have good anti-cancer activity and higher treatment safety, and effectively reduce the risk of cytokine storm caused by CD3 bispecific antibodies.

[0009] In a second aspect, the present application provides a nucleic acid molecule. According to embodiments of the present application, the nucleic acid molecule encodes the bispecific antibody of the first aspect of the present application. In some examples of the present application, the foregoing nucleic acid molecule can encode a bispecific antibody capable of simultaneously targeting B7H7 and CD3.

[0010] In a third aspect, the present application provides an expression vector. According to embodiments of the present application, the expression vector carries the nucleic acid molecule of the second aspect of the present application. In some examples of the present application, the foregoing expression vector can express a bispecific antibody capable of specifically and highly binding to human and monkey B7H7, and specifically and lowly binding to human and monkey CD3 in a suitable host cell.

[0011] In a fourth aspect, the present application provides a method for preparing the bispecific antibody of the first aspect. According to embodiments of the present application, the method comprises: introducing the expression vector of the third aspect into a cell; and culturing the cell under conditions suitable for protein expression and secretion, so as to obtain the bispecific antibody. In some examples of the present application, the bispecific antibody obtained based on the method can specifically and with high affinity bind to human and monkey B7H7, and can specifically and with low affinity bind to human and monkey CD3, promote PBMC to kill tumor cells, have good anti-cancer activity and higher safety, and effectively reduce the risk of cytokine storm caused by CD3 bispecific antibodies.

[0012] In a fifth aspect, the present application provides a recombinant cell. According to embodiments of the present application, the recombinant cell carries the bispecific antibody of the first aspect, the nucleic acid molecule of the second aspect, or the expression vector of the third aspect. The aforementioned recombinant cell is obtained by transfection or transformation of the expression vector. In some examples of the present application, the aforementioned recombinant cell can efficiently express the aforementioned bispecific antibody under suitable conditions.

[0013] In a sixth aspect, the present application provides a pharmaceutical composition. According to embodiments of the present application, the pharmaceutical composition comprises: the bispecific antibody of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, or the recombinant cell of the fifth aspect. In some examples of the present application, the aforementioned bispecific antibody can effectively promote PBMC to kill tumor cells, has good anti-cancer activity and higher safety, and effectively reduces the risk of cytokine storm caused by CD3 bispecific antibodies. Therefore, the pharmaceutical composition prepared based on the antibody can be further used for preventing or treating B7H7 and / or CD3 mediated related diseases.

[0014] In a seventh aspect, the present application provides use of the bispecific antibody of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, or the pharmaceutical composition of the sixth aspect in the preparation of a medicament for treating or preventing tumors. In some examples of the present application, the aforementioned bispecific antibody can effectively promote PBMC to kill tumor cells, has good anti-cancer activity and higher safety, and effectively reduces the risk of cytokine storm caused by CD3 bispecific antibodies. The medicament comprising the aforementioned bispecific antibody and other substances also has a significant effect on treating or preventing tumors.

[0015] In an eighth aspect, the present application provides use of the bispecific antibody of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the recombinant cell of the fifth aspect, or the pharmaceutical composition of the sixth aspect in the preparation of a kit for detecting B7H7 and / or CD3. Those skilled in the art can understand that the foregoing features and advantages of the bispecific antibody also apply to this use, which will not be repeated here.

[0016] In a ninth aspect, the present application provides a kit. According to embodiments of the present application, the kit comprises the bispecific antibody of the first aspect. In some examples of the present application, the foregoing bispecific antibody can specifically and highly bind to human and monkey B7H7, and specifically and lowly bind to human and monkey CD3. Therefore, the kit comprising the bispecific antibody can be used for detecting B7H7 and / or CD3 protein. The foregoing kit can be used for scientific research, such as qualitatively or quantitatively detecting B7H7 and CD3 protein in a biological sample, or can be used for judging the state of an individual, such as judging whether the B7H7 and CD3 protein level of the individual is higher or lower than the normal level after obtaining the B7H7 and CD3 protein level of the individual.

[0017] Additional aspects and advantages of the present application will be made apparent from the following description. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0019] FIG. 1 is a structural schematic diagram of B7H7xCD3 bispecific antibody provided by embodiments of the present application; wherein A is a structural schematic diagram of bispecific antibody BB1-T7-Z13; B is a structural schematic diagram of bispecific antibody BB3-T7-Z13; and C is a structural schematic diagram of bispecific antibody BB3-Tma-Z13;

[0020] FIG. 2 is a flow cytometry result schematic diagram of B7H7xCD3 bispecific antibody cell binding experiment provided by embodiments of the present application; wherein A is a result schematic diagram of bispecific antibody binding to human T cells; and B is a result schematic diagram of bispecific antibody binding to non-small cell lung cancer HCC827 cells overexpressing;

[0021] FIG. 3 is a result schematic diagram of B7H7xCD3 bispecific antibody binding to CD3 and B7H7 protein simultaneously provided by embodiments of the present application;

[0022] Figure 4 is a schematic diagram of the in vitro killing detection results of the CD3-B7H7 bispecific binding antibody provided in the embodiments of the present application; wherein A is a schematic diagram of the in vitro cytotoxicity experiment results of the bispecific antibody and PBMC on non-small cell lung cancer HCC827 cells; B is a schematic diagram of the in vitro cytotoxicity experiment results of the bispecific antibody and PBMC on lung adenocarcinoma NCI-H820 cells; C is a schematic diagram of the in vitro cytotoxicity experiment results of the bispecific antibody and PBMC on colorectal cancer LS180 cells;

[0023] Figure 5 is a schematic diagram of the results of the influence of different B7H7 expression abundance on the activity of B7H7xCD3 bispecific antibody provided in the embodiments of the present application; wherein A is the in vitro cytotoxicity experiment results of the bispecific antibody and PBMC on HCT-15-B7H7 high expression cells; B is a schematic diagram of the in vitro cytotoxicity experiment results of the bispecific antibody and PBMC on HCT-15-B7H7 low expression cells;

[0024] Figure 6 is a schematic diagram of the results of the influence of B7H7xCD3 bispecific antibody on PBMC cytokine secretion in the absence of target cells provided in the embodiments of the present application; wherein A is a schematic diagram of the influence of the bispecific binding antibody on the release of cytokine IL-2 by PBMC in the absence of target cells; B is a schematic diagram of the influence of the bispecific binding antibody on the release of cytokine IL-6 by PBMC in the absence of target cells; C is a schematic diagram of the influence of the bispecific binding protein on the release of cytokine IFN-γ by PBMC in the absence of target cells; D is a schematic diagram of the influence of the bispecific binding protein on the release of cytokine TNF by PBMC in the absence of target cells;

[0025] Figure 7 is a schematic diagram of the results of the influence of B7H7xCD3 bispecific antibody on PBMC cytokine secretion in the presence of target cells provided in the embodiments of the present application; wherein A is a schematic diagram of the influence of the bispecific binding antibody on the release of cytokine IL-2 by PBMC in the presence of target cells; B is a schematic diagram of the influence of the bispecific binding antibody on the release of cytokine IL-6 by PBMC in the presence of target cells; C is a schematic diagram of the influence of the bispecific binding protein on the release of cytokine IFN-γ by PBMC in the presence of target cells; D is a schematic diagram of the influence of the bispecific binding protein on the release of cytokine TNF by PBMC in the presence of target cells;

[0026] Figure 8 is a schematic diagram of the results of the in vivo anti-tumor activity of B7H7xCD3 bispecific antibodies on HCT-15-B7H7 tumor cells according to an embodiment of the present application; wherein A is a schematic diagram of the results of the in vivo anti-tumor activity of bispecific antibody BB1-T7-Z13 on B7H7+ tumor (HCT-15-B7H7); B is a schematic diagram of the results of the in vivo anti-tumor activity of bispecific antibody BB3-T7-Z13 on B7H7+ tumor (HCT-15-B7H7); C is a schematic diagram of the results of the in vivo anti-tumor activity of bispecific antibody BB3-Tma-Z13 on B7H7+ tumor (HCT-15-B7H7).

[0027] Figure 9 is a schematic diagram of the results of the influence of BB3-T7-Z13 and BB3-T8-Z13 bispecific antibodies on PBMC cytokine secretion in the presence of target cells (HCT-15-B7H7) according to an embodiment of the present application; wherein A is a schematic diagram of the results of the influence of bispecific binding antibodies on PBMC release of cytokine IL-2 in the presence of target cells; B is a schematic diagram of the results of the influence of bispecific binding antibodies on PBMC release of cytokine IL-6 in the presence of target cells; C is a schematic diagram of the results of the influence of bispecific binding proteins on PBMC release of cytokine TNF in the presence of target cells.

[0028] Figure 10 is a schematic diagram of the results of the in vivo anti-tumor activity of BB3-T8-Z13 bispecific antibodies on HCT-15-B7H7 clone 43 tumor cells according to an embodiment of the present application. DETAILED DESCRIPTION

[0029] The embodiments of the present application are described in detail below with reference to the accompanying drawings. The embodiments described below are examples for explaining the present application and are not intended to be limiting of the present application.

[0030] In the present application, the terms "first", "second" are used only for the purpose of description and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Thus, the features defined with "first", "second" can explicitly or implicitly include at least one of the features. In the description of the present application, the meaning of "a plurality of" is at least two, for example, two, three, etc., unless otherwise explicitly and specifically limited.

[0031] In the present application, the term "antibody" refers to an antibody that can recognize one or more antigenic epitopes, including but not limited to monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain-only antibodies, triabodies, single-chain Fv (scFv), nanobodies, and the like, and also includes antibody fragments, as long as the desired biological activity is exhibited. Antibodies can be murine, human, humanized, chimeric, or derived from other species. Antibodies can refer to full-length heavy chains, full-length light chains, intact immunoglobulin molecules; or immunologically active portions of any of these polypeptides, i.e., molecules or portions thereof that include an antigen binding site that immunospecifically binds to a target antigen of interest, such targets including but not limited to cancer cells or cells producing autoantibodies associated with autoimmune diseases. In the present application, "bispecific antibodies" refer to antibodies that can recognize CD3 and B7H7 antigenic epitopes.

[0032] In the present application, certain regions in the variable region have a higher degree of variation in amino acid composition and sequence arrangement, referred to as "hypervariable regions (HVR)", which are the positions of antigen and antibody binding, and are also referred to as complementarity-determining regions (CDR). There are three CDR regions on the heavy chain variable region and the light chain variable region. For example, generally include: amino acid residues near 23-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable region, and 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, Bethesda, MD. (1991)); and / or from "hypervariable loops" (e.g., amino acid residues near 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the light chain variable region, and 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)).

[0033] As used herein, the term "human antibody or humanized antibody" refers to a recombinant antibody obtained by replacing the amino acid sequences of the constant region and the non-CDR (Fv framework region (FR)) amino acids of the variable region of a monoclonal antibody from one species (e.g., mouse) with the amino acid sequences of the constant region and the non-CDR amino acids of the variable region of an antibody from another species (e.g., human) using recombinant DNA technology. That is, when the constant region of an antibody is humanized, it is referred to as a chimeric antibody, and when the constant region and the non-CDR amino acids of the variable region are humanized, it is referred to as a humanized antibody. The method of humanization can be performed according to conventional antibody engineering techniques, which are not described herein.

[0034] As used herein, the term "chimeric antibody" refers to a recombinant antibody obtained by replacing the amino acid sequences of the constant region amino acids of a monoclonal antibody from one species (e.g., mouse) with the constant region of an antibody from another species (e.g., human) using recombinant DNA technology.

[0035] In the present application, the amino acid sequences of the listed CDRs are shown according to the IMGT definition rule. However, it is well known in the art that the CDRs of an antibody can be defined by various methods in the art, such as the Kabat rule, the Chothia rule, etc. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementary determining region" of a given antibody or region thereof (e.g., a variable region) are understood to encompass the complementary determining regions defined by any of the aforementioned known schemes described herein. Although the scope of protection claimed in the present application is based on the sequences shown according to the IMGT definition rule, the amino acid sequences corresponding to the CDRs according to other definition rules should also be included in the scope of protection of the present application.

[0036] In the present application, the term "amino acid" is represented by a single letter or a three-letter code, and has the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Asp (aspartic acid); C: Cys (cysteine); Q: Gin (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: His (histidine); I: Ile (isoleucine); L: Leu (leucine); K: Lys (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Ser (serine); T: Thr (threonine); W: Trp (tryptophan); Y: Tyr (tyrosine); V: Val (valine).

[0037] For nucleotides, the terms "homology," "identity," or "similarity" are used to describe or compare the degree of nucleotide similarity between two or more nucleotide sequences. The percent "sequence homology" between a first sequence and a second sequence can be calculated by dividing [the number of nucleotides in the first sequence that are identical to the nucleotides at the corresponding positions] by [the number of nucleotides in the second sequence]. The number of nucleotides in the second sequence] minus [the total number of nucleotides in the first sequence], then multiplied by [100%], wherein each deletion, insertion, substitution, or addition of a nucleotide in the second nucleotide sequence - relative to the first nucleotide sequence - is considered a difference at a single nucleotide (position). Alternatively, the degree of sequence identity between two or more nucleotide sequences can be calculated using known computer algorithms for sequence alignment, such as NCBI Blast v2.0, using standard settings. Some other techniques, computer algorithms, and settings for determining the degree of sequence identity are described, for example, in WO 04 / 037999, EP 0 967 284, EP 1 085 089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB 2357768-A.

[0038] For polypeptides, the terms "(substantial) homology," "identity," or "similarity" are used to describe or compare the degree of amino acid similarity between two or more polypeptides or specified sequences thereof when optimally aligned and compared (with appropriate insertions or deletions of nucleotides). The percent homology between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100), when the sequences are optimally aligned and compared. Optimal alignment of sequences for comparison can be achieved using various algorithms known in the art, such as those described in the following non-limiting examples.

[0039] In the present application, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids can be substituted, added and / or deleted from the sequences of the present application by one of skill in the art without materially affecting the activity of the antibody (retaining at least 95% activity) to obtain variants of the sequences of the antibody or functional fragments thereof. They are all considered to be included in the scope of protection of the present application. Amino acids with similar properties are substituted in the variable region. The variant sequences described in the present application can have at least 80% identity (or homology) with the reference sequences, which means at least 80%, which can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identity with each reference sequence. The sequence identity described in the present application can be measured using sequence analysis software. For example, using the computer program BLAST, especially BLASTP or TBLASTN with default parameters. The amino acid sequences referred to in the present application are all shown in the order of N-terminal to C-terminal.

[0040] In the present context, the term "vector" generally refers to a nucleic acid molecule capable of inserting itself into a suitable host and self-replicating, which transfers the inserted nucleic acid molecule to the host cell and / or between host cells. The vector can include a vector mainly for inserting DNA or RNA into a cell, a vector mainly for replicating DNA or RNA, and a vector mainly for expression of transcription and / or translation of DNA or RNA. The vector also includes a vector having a plurality of the aforementioned functions. The vector can be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, the vector can produce a desired expression product by culturing a suitable host cell containing the vector.

[0041] In the present context, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any one of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both.

[0042] In the present context, the term "pharmaceutically acceptable" ingredient is a substance that is suitable for use with humans and / or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio.

[0043] In the present context, the term "pharmaceutically acceptable excipient" can include any solvent, solid or other liquid excipient, etc., suitable for the particular target dosage form. Except insofar as any conventional excipient is incompatible with the compounds of the application, such as by producing any adverse biological effect or otherwise interacting in a deleterious manner with any other component(s) of a pharmaceutically acceptable composition, its use is contemplated to be within the scope of this application.

[0044] In the present context, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The antibodies or antigen binding fragments, recombinant proteins, multispecific antibodies, conjugates or pharmaceutical compositions of the application can be administered by any common route of administration, as long as it reaches the intended tissue. Various modes of administration are contemplated, including intraperitoneal, intravenous, intramuscular, subcutaneous, etc., but the application is not limited to these exemplified modes of administration. Preferably, the compositions of the application are administered by intravenous or subcutaneous injection.

[0045] In the present context, the term "treatment" refers to any action providing a desired pharmacological and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure of a disease and / or adverse effect attributable to the disease. "Treatment" as used herein covers the treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or condition from occurring in an individual which can be predisposed to the disease but has not yet developed the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing the partial or complete regression of the disease. "Treatment" as used herein covers any action providing a pharmacological and / or physiologic effect in an individual, including but not limited to the administration of a pharmaceutical comprising a compound as described herein to an individual in need thereof.

[0046] As used herein, the term "effective amount" or "effective dose" refers to an amount that is functional or active and acceptable to a human and / or animal.

[0047] In the present application, the amino acid sequences are shown in Table 1.

[0048] The present application provides a bispecific antibody targeting B7H7 and CD3, a nucleic acid molecule, an expression vector, a method of preparation, a recombinant cell, a pharmaceutical composition, a pharmaceutical use, a kit use, a kit, a method of treatment of a disease, and a use of a disease, which will be described in detail as follows, respectively:

[0049] Bispecific antibody

[0050] In a first aspect, the present application provides a bispecific antibody. According to embodiments of the present application, the aforementioned bispecific antibody comprises: a first antigen binding region, the first antigen binding region having CD3 binding activity; and a second antigen binding region, the second antigen binding region having B7H7 binding activity; wherein the first antigen binding region comprises an anti-CD3 antibody, the anti-CD3 antibody comprising a heavy chain complementarity determining region (HCDR), the HCDR comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 4-6, 66, or an amino acid sequence having at least 80% homology with SEQ ID NOs: 4-6, 66; the anti-CD3 antibody further comprising: a light chain complementarity determining region (LCDR), the LCDR comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1-3, or an amino acid sequence having at least 80% homology with SEQ ID NOs: 1-3; the second antigen binding region comprises an anti-B7H7 antibody, the anti-B7H7 antibody comprising a heavy chain complementarity determining region (HCDR), the HCDR of the anti-B7H7 antibody comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 26-28, or an amino acid sequence having at least 80% homology with SEQ ID NOs: 26-28; the anti-B7H7 antibody further comprising: a light chain complementarity determining region (LCDR), the LCDR comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 23-25, or an amino acid sequence having at least 80% homology with SEQ ID NOs: 23-25.

[0051] In some examples of the present application, the HCDR of the aforementioned anti-CD3 antibody comprises: an HCDR1, an HCDR2, and an HCDR3, wherein the HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 4 or having at least 80% homology with SEQ ID NO: 4, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 5 or having at least 80% homology with SEQ ID NO: 5, and the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 6, 66 or having at least 80% homology with SEQ ID NO: 6, 66. The anti-CD3 antibody based on the aforementioned HCDR sequence can bind to CD3 with low affinity and high specificity.

[0052] In some preferred examples of the present application, the aforementioned HCDR comprises: an HCDR1, an HCDR2, and an HCDR3, each as set forth in SEQ ID NO: 4-6, 66. It has been verified through experiments that the anti-CD3 antibody having the aforementioned HCDR sequence can bind to CD3 with low affinity and high specificity.

[0053] In some examples of the present application, the aforementioned anti-CD3 antibody further comprises: a light chain complementarity determining region (LCDR), the aforementioned LCDR comprising: an LCDR1, an LCDR2 and an LCDR3, wherein the aforementioned LCDR1 comprises an amino acid sequence as shown in SEQ ID NO: 1 or having at least 80% homology with SEQ ID NO: 1, the aforementioned LCDR2 comprises an amino acid sequence as shown in SEQ ID NO: 2 or having at least 80% homology with SEQ ID NO: 2, and the aforementioned LCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 3 or having at least 80% homology with SEQ ID NO: 3. Anti-CD3 antibodies based on the aforementioned LCDR sequences have higher binding affinity and specificity to CD3.

[0054] In some preferred examples of the present application, the aforementioned LCDR comprises: an LCDR1, an LCDR2 and an LCDR3, each having an amino acid sequence as shown in SEQ ID NO: 1-3. Anti-CD3 antibodies having the aforementioned LCDR sequences have been verified by experiments to have higher binding affinity and specificity to CD3.

[0055] It should be noted that one or more amino acid residues in the aforementioned anti-CD3 antibody HCDR and / or LCDR can be replaced by other amino acid residues from the same side chain family, and the retained function of the changed antibody can be tested using the functional assays described herein. Preferably, the number of amino acid substitutions is no more than 1 or 2.

[0056] In some examples of the present application, the aforementioned anti-CD3 antibody further comprises: a heavy chain framework region (HFR), the aforementioned HFR comprising: an HFR1, an HFR2, an HFR3 and an HFR4, each having an amino acid sequence as shown in SEQ ID NO: 11-14, 19-22; wherein the aforementioned HFR1 comprises an amino acid sequence as shown in SEQ ID NO: 11, 19 or having at least 80% homology with SEQ ID NO: 11, 19, the aforementioned HFR2 comprises an amino acid sequence as shown in SEQ ID NO: 12, 20 or having at least 80% homology with SEQ ID NO: 12, 20, the aforementioned HFR3 comprises an amino acid sequence as shown in SEQ ID NO: 13, 21 or having at least 80% homology with SEQ ID NO: 13, 21, and the aforementioned HFR4 comprises an amino acid sequence as shown in SEQ ID NO: 14, 22 or having at least 80% homology with SEQ ID NO: 14, 22.

[0057] In some preferred examples of the present application, the aforementioned HFR comprises: HFR1, HFR2, HFR3 and HFR4 of the amino acid sequences shown in SEQ ID NOs: 7-14, respectively. The CD3 bispecific antibody based on the aforementioned framework region sequences has significantly improved stability.

[0058] In some more preferred examples of the present application, the aforementioned HFR comprises: HFR1, HFR2, HFR3 and HFR4 of the amino acid sequences shown in SEQ ID NOs: 11-14, respectively. The present inventors have modified the heavy chain framework region of the anti-CD3 antibody, and the CD3 bispecific antibody based on the aforementioned framework region sequences has significantly improved stability.

[0059] In some examples of the present application, the aforementioned anti-CD3 antibody further comprises: a light chain framework region LFR, the aforementioned LFR comprising: LFR1, LFR2, LFR3 and LFR4 of the amino acid sequences shown in SEQ ID NOs: 7-10, 15-18, respectively; wherein the aforementioned LFR1 comprises an amino acid sequence shown in SEQ ID NO: 7, 15, or has at least 80% homology with SEQ ID NO: 7, 15, the aforementioned LFR2 comprises an amino acid sequence shown in SEQ ID NO: 8, 16, or has at least 80% homology with SEQ ID NO: 8, 16, the aforementioned LFR3 comprises an amino acid sequence shown in SEQ ID NO: 9, 17, or has at least 80% homology with SEQ ID NO: 9, 17, and the aforementioned LFR4 comprises an amino acid sequence shown in SEQ ID NO: 10, 18, or has at least 80% homology with SEQ ID NO: 10, 18. The CD3 bispecific antibody based on the aforementioned framework region sequences has significantly improved stability.

[0060] In some preferred examples of the present application, the aforementioned LFR comprises: LFR1, LFR2, LFR3 and LFR4 of the amino acid sequences shown in SEQ ID NOs: 7-10, 15-18, respectively. The CD3 bispecific antibody based on the aforementioned framework region sequences has significantly improved stability.

[0061] In some more preferred examples of the present application, the aforementioned LFR comprises: LFR1, LFR2, LFR3 and LFR4 of the amino acid sequences shown in SEQ ID NOs: 7-10, respectively. The present inventors have modified the light chain framework region of the anti-CD3 antibody, and the CD3 bispecific antibody based on the aforementioned framework region sequences has significantly improved stability.

[0062] In some examples of the present application, the heavy chain variable region of the aforementioned anti-CD3 antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 38, 40, 67 or an amino acid sequence having at least 80% homology with SEQ ID NO: 38, 40, 67. Anti-CD3 antibodies based on the aforementioned heavy chain variable region sequence have low affinity, high specificity and stability.

[0063] In some preferred examples of the present application, the amino acid sequence of the heavy chain variable region of the aforementioned anti-CD3 antibody is as shown in SEQ ID NO: 38, 40, 67. Anti-CD3 antibodies based on the aforementioned heavy chain variable region sequence have low affinity, high specificity and stability.

[0064] In some more preferred examples of the present application, the amino acid sequence of the heavy chain variable region of the aforementioned anti-CD3 antibody is as shown in SEQ ID NO: 67. Anti-CD3 antibodies based on the aforementioned heavy chain variable region sequence have higher sequence stability.

[0065] In some preferred examples of the present application, the light chain variable region of the aforementioned anti-CD3 antibody comprises an amino acid sequence as shown in SEQ ID NO: 37, 39 or an amino acid sequence having at least 80% homology with SEQ ID NO: 37, 39. Anti-CD3 antibodies based on the aforementioned light chain variable region sequence have low affinity, high specificity and stability.

[0066] In some preferred examples of the present application, the amino acid sequence of the light chain variable region of the aforementioned anti-CD3 antibody is as shown in SEQ ID NO: 37, 39. Anti-CD3 antibodies based on the aforementioned light chain variable region sequence have low affinity, high specificity and stability.

[0067] In some more preferred examples of the present application, the amino acid sequence of the light chain variable region of the aforementioned anti-CD3 antibody is as shown in SEQ ID NO: 37. Anti-CD3 antibodies based on the aforementioned light chain variable region sequence have higher sequence stability.

[0068] In some examples of the present application, the HCDRs of the aforementioned anti-B7H7 antibody include: HCDR1, HCDR2 and HCDR3, wherein the aforementioned HCDR1 comprises an amino acid sequence as shown in SEQ ID NO: 26 or having at least 80% homology with SEQ ID NO: 26, the aforementioned HCDR2 comprises an amino acid sequence as shown in SEQ ID NO: 27 or having at least 80% homology with SEQ ID NO: 27, and the aforementioned HCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 28 or having at least 80% homology with SEQ ID NO: 28. Anti-B7H7 antibodies based on the aforementioned HCDR sequences have higher binding affinity and specificity to B7H7.

[0069] In some preferred examples of the present application, the aforementioned HCDRs include: HCDR1, HCDR2 and HCDR3 of the amino acid sequences as shown in SEQ ID NOs: 26-28, respectively. Anti-B7H7 antibodies having the amino acid sequences as shown in SEQ ID NOs: 26-28 have higher binding affinity and specificity to B7H7, which has been verified by experiments.

[0070] In some examples of the present application, the LCDRs of the aforementioned anti-B7H7 antibody include: LCDR1, LCDR2 and LCDR3, wherein the aforementioned LCDR1 comprises an amino acid sequence as shown in SEQ ID NO: 23 or having at least 80% homology with SEQ ID NO: 23, the aforementioned LCDR2 comprises an amino acid sequence as shown in SEQ ID NO: 24 or having at least 80% homology with SEQ ID NO: 24, and the aforementioned LCDR3 comprises an amino acid sequence as shown in SEQ ID NO: 25 or having at least 80% homology with SEQ ID NO: 25. Anti-B7H7 antibodies based on the aforementioned LCDR sequences have higher binding affinity and specificity to B7H7.

[0071] In some preferred examples of the present application, the aforementioned LCDRs include: LCDR1, LCDR2 and LCDR3 of the amino acid sequences as shown in SEQ ID NOs: 23-25, respectively. Anti-B7H7 antibodies having the amino acid sequences as shown in SEQ ID NOs: 23-25 have higher binding affinity and specificity to B7H7, which has been verified by experiments.

[0072] In some examples of the present application, the aforementioned anti-B7H7 antibody further comprises: a heavy chain framework region HFR, the aforementioned HFR comprising: HFR1, HFR2, HFR3 and HFR4 of the amino acid sequences shown in SEQ ID NOs: 33-36, respectively; wherein the aforementioned HFR1 comprises the amino acid sequence shown in SEQ ID NO: 33 or has at least 80% homology with SEQ ID NO: 33, the aforementioned HFR2 comprises the amino acid sequence shown in SEQ ID NO: 34 or has at least 80% homology with SEQ ID NO: 34, the aforementioned HFR3 comprises the amino acid sequence shown in SEQ ID NO: 35 or has at least 80% homology with SEQ ID NO: 35, and the aforementioned HFR4 comprises the amino acid sequence shown in SEQ ID NO: 36 or has at least 80% homology with SEQ ID NO: 36. The B7H7 bispecific antibody based on the aforementioned framework region sequence has high sequence stability.

[0073] In some preferred examples of the present application, the aforementioned HFR comprises: HFR1, HFR2, HFR3 and HFR4 of the amino acid sequences shown in SEQ ID NOs: 33-36, respectively. The B7H7 bispecific antibody based on the aforementioned framework region sequence has high sequence stability.

[0074] In some examples of the present application, the aforementioned anti-B7H7 antibody further comprises: a heavy chain framework region HFR, the aforementioned HFR comprising: HFR1, HFR2, HFR3 and HFR4 of the amino acid sequences shown in SEQ ID NOs: 33-36, respectively; wherein the aforementioned HFR1 comprises the amino acid sequence shown in SEQ ID NO: 33 or has at least 80% homology with SEQ ID NO: 33, the aforementioned HFR2 comprises the amino acid sequence shown in SEQ ID NO: 34 or has at least 80% homology with SEQ ID NO: 34, the aforementioned HFR3 comprises the amino acid sequence shown in SEQ ID NO: 35 or has at least 80% homology with SEQ ID NO: 35, and the aforementioned HFR4 comprises the amino acid sequence shown in SEQ ID NO: 36 or has at least 80% homology with SEQ ID NO: 36. The B7H7 bispecific antibody based on the aforementioned framework region sequence has high sequence stability.

[0075] In some preferred examples of the present application, the aforementioned HFR comprises: HFR1, HFR2, HFR3 and HFR4 of the amino acid sequences shown in SEQ ID NOs: 33-36, respectively. The B7H7 bispecific antibody based on the aforementioned framework region sequence has high sequence stability.

[0076] In some preferred examples of the present application, the heavy chain variable region of the aforementioned anti-B7H7 antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 42 and an amino acid sequence having at least 80% homology with SEQ ID NO: 42. Anti-B7H7 antibodies based on the aforementioned heavy chain variable region sequence have higher affinity, specificity and stability.

[0077] In some more preferred examples of the present application, the amino acid sequence of the heavy chain variable region of the aforementioned anti-B7H7 antibody is as set forth in SEQ ID NO: 42. It has been found through experimental verification that anti-B7H7 antibodies based on the aforementioned heavy chain variable region sequence have higher affinity, specificity and stability.

[0078] In some preferred examples of the present application, the light chain variable region of the aforementioned anti-B7H7 antibody comprises an amino acid sequence as set forth in SEQ ID NO: 41 or an amino acid sequence having at least 80% homology with SEQ ID NO: 41. Anti-B7H7 antibodies based on the aforementioned light chain variable region sequence have higher affinity, specificity and stability.

[0079] In some more preferred examples of the present application, the amino acid sequence of the light chain variable region of the aforementioned anti-B7H7 antibody is as set forth in SEQ ID NO: 41. It has been found through experimental verification that anti-B7H7 antibodies based on the aforementioned light chain variable region sequence have higher affinity, specificity and stability.

[0080] In some examples of the present application, the aforementioned first antigen binding region comprises a first anti-CD3 antibody scFv fragment, the aforementioned first anti-CD3 antibody scFv fragment comprising an anti-CD3 antibody heavy chain variable region, an anti-CD3 antibody light chain variable region and a connecting peptide 1.

[0081] In some examples of the present application, the C-terminus of the aforementioned anti-CD3 antibody light chain variable region is connected to the N-terminus of the aforementioned connecting peptide 1, and the C-terminus of the aforementioned connecting peptide 1 is connected to the N-terminus of the aforementioned anti-CD3 antibody heavy chain variable region.

[0082] In some examples of the present application, the C-terminus of the aforementioned anti-CD3 antibody light chain variable region is connected to the N-terminus of the aforementioned connecting peptide 1, and the C-terminus of the aforementioned connecting peptide 1 is connected to the N-terminus of the aforementioned anti-CD3 antibody heavy chain variable region.

[0083] In some examples of the present application, the aforementioned first antigen binding region further comprises a first Fc fragment.

[0084] In some examples of the present application, the C-terminus of the aforementioned anti-CD3 antibody light chain variable region is connected to the N-terminus of the aforementioned connecting peptide 1, the C-terminus of the aforementioned connecting peptide 1 is connected to the N-terminus of the aforementioned anti-CD3 antibody heavy chain variable region, and the C-terminus of the aforementioned anti-CD3 antibody heavy chain variable region is connected to the N-terminus of the aforementioned first Fc fragment.

[0085] In some examples of the application, the aforementioned second antigen binding region comprises a second anti-B7H7 antibody Fab fragment, the aforementioned second anti-B7H7 antibody Fab fragment comprising an anti-B7H7 antibody heavy chain variable region, a B7H7 antibody light chain variable region and a connecting peptide 1.

[0086] In some examples of the application, the C-terminus of the aforementioned anti-B7H7 antibody light chain variable region is connected to the N-terminus of the aforementioned connecting peptide 1, and the C-terminus of the aforementioned connecting peptide 1 is connected to the N-terminus of the aforementioned anti-B7H7 antibody heavy chain variable region.

[0087] In some examples of the application, the C-terminus of the aforementioned anti-B7H7 antibody light chain variable region is connected to the N-terminus of the aforementioned connecting peptide 1, and the C-terminus of the aforementioned connecting peptide 1 is connected to the N-terminus of the aforementioned anti-B7H7 antibody heavy chain variable region.

[0088] In some examples of the application, the aforementioned second antigen binding region further comprises a second Fc fragment.

[0089] In some examples of the application, the C-terminus of the aforementioned anti-B7H7 antibody light chain variable region is connected to the N-terminus of the aforementioned connecting peptide 1, the C-terminus of the aforementioned connecting peptide 1 is connected to the N-terminus of the aforementioned anti-B7H7 antibody heavy chain variable region, and the C-terminus of the aforementioned anti-B7H7 antibody heavy chain variable region is connected to the N-terminus of the aforementioned second Fc fragment.

[0090] In some examples of the application, the aforementioned first Fc fragment and the aforementioned second Fc fragment are connected by a knob-in-hole structure.

[0091] In a specific example of the application, the aforementioned bispecific antibody is composed and connected as shown in FIG. 1A.

[0092] In some examples of the application, the aforementioned first antigen binding region comprises a first anti-B7H7 antibody Fab fragment and a first anti-CD3 antibody scFv fragment, the aforementioned first anti-B7H7 antibody Fab fragment and the first anti-CD3 antibody scFv fragment being connected by a connecting peptide 2, wherein the aforementioned first anti-B7H7 antibody Fab fragment comprises an anti-B7H7 antibody heavy chain variable region, a B7H7 antibody light chain variable region, a first CL fragment and a first CH1 fragment; the aforementioned first anti-CD3 antibody scFv fragment comprises an anti-CD3 antibody heavy chain variable region, a CD3 antibody light chain variable region and a connecting peptide 1.

[0093] In some examples of the application, the C-terminus of the first anti-B7H7 antibody heavy chain variable region is connected to the N-terminus of the first CH1 fragment, the C-terminus of the first anti-B7H7 antibody light chain variable region is connected to the N-terminus of the first CL fragment, the first CL fragment and the first CH1 fragment are connected by a disulfide bond, the C-terminus of the first CH1 fragment is connected to the N-terminus of the connecting peptide 2, the C-terminus of the connecting peptide 2 is connected to the N-terminus of the first anti-CD3 antibody light chain variable region, the C-terminus of the first anti-CD3 antibody light chain variable region is connected to the N-terminus of the connecting peptide 1, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the first anti-CD3 antibody heavy chain variable region.

[0094] In some examples of the application, the first antigen binding region further comprises a first Fc fragment.

[0095] In some examples of the application, the first anti-CD3 antibody scFv fragment is connected to the first Fc fragment.

[0096] In some examples of the application, the second antigen binding region comprises a second anti-B7H7 antibody Fab fragment, the second anti-B7H7 antibody Fab fragment comprises an anti-B7H7 antibody heavy chain variable region, an anti-B7H7 antibody light chain variable region, a second CL fragment, and a second CH1 fragment.

[0097] In some examples of the application, the C-terminus of the second anti-B7H7 antibody heavy chain variable region is connected to the N-terminus of the second CH1 fragment, the C-terminus of the second anti-B7H7 antibody light chain variable region is connected to the N-terminus of the second CL fragment, and the second CL fragment and the second CH1 fragment are connected by a disulfide bond.

[0098] In some examples of the application, the second antigen binding region further comprises a second Fc fragment.

[0099] In some examples of the application, the second anti-B7H7 antibody Fab fragment is connected to the second Fc fragment.

[0100] In some examples of the application, the first Fc fragment and the second Fc fragment are connected by a knob-in-hole structure.

[0101] In some specific examples of the application, the bispecific antibody is composed and connected as shown in FIG. IB.

[0102] In some examples of the application, the first antigen binding region comprises a first anti-B7H7 antibody Fab fragment and a first anti-CD3 antibody scFv fragment, wherein the first anti-B7H7 antibody Fab fragment and the first anti-CD3 antibody scFv fragment are connected by a connecting peptide 2, and wherein the first anti-B7H7 antibody Fab fragment comprises an anti-B7H7 antibody heavy chain variable region, a B7H7 antibody light chain variable region, a first CL fragment, and a first CH1 fragment; and the first anti-CD3 antibody scFv fragment comprises an anti-CD3 antibody heavy chain variable region, a CD3 antibody light chain variable region, and a connecting peptide 1.

[0103] In some examples of the application, the C-terminus of the first anti-B7H7 antibody heavy chain variable region is connected to the N-terminus of the first CH1 fragment, the C-terminus of the first anti-B7H7 antibody light chain variable region is connected to the N-terminus of the first CL fragment, the first CL fragment and the first CH1 fragment are connected by a disulfide bond, the C-terminus of the first CH1 fragment is connected to the N-terminus of the connecting peptide 2, the C-terminus of the connecting peptide 2 is connected to the N-terminus of the first anti-CD3 antibody heavy chain variable region, the C-terminus of the first anti-CD3 antibody heavy chain variable region is connected to the N-terminus of the connecting peptide 1, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the first anti-CD3 antibody light chain variable region.

[0104] In some examples of the application, the first antigen binding region further comprises a first Fc fragment.

[0105] In some examples of the application, the first anti-CD3 antibody scFv fragment is connected to the first Fc fragment.

[0106] In some examples of the application, the second antigen binding region comprises a second anti-B7H7 antibody Fab fragment, and the second anti-B7H7 antibody Fab fragment comprises an anti-B7H7 antibody heavy chain variable region, a B7H7 antibody light chain variable region, a second CL fragment, and a second CH1 fragment.

[0107] In some examples of the application, the C-terminus of the second anti-B7H7 antibody heavy chain variable region is connected to the N-terminus of the second CH1 fragment, the C-terminus of the second anti-B7H7 antibody light chain variable region is connected to the N-terminus of the second CL fragment, and the second CL fragment and the second CH1 fragment are connected by a disulfide bond.

[0108] In some examples of the application, the second antigen binding region further comprises a second Fc fragment.

[0109] In some examples of the application, the second anti-B7H7 antibody Fab fragment is connected to the second Fc fragment.

[0110] In some examples of the present application, the first Fc fragment and the second Fc fragment are connected by a Knob-in-hole structure.

[0111] In one specific example of the present application, the aforementioned bispecific antibody is composed of and connected as shown in FIG. 1C.

[0112] In some examples of the present application, the aforementioned first CH1 and second CH1 are each independently selected from human antibody IgG1.

[0113] In some examples of the present application, the aforementioned human antibody IgG1 has L234A / L235A mutations. Those skilled in the art can understand that the bioavailability of the antibody can be improved by humanizing the antibody.

[0114] In some examples of the present application, the aforementioned connecting peptide 1 has an amino acid sequence as shown in SEQ ID NO: 63.

[0115] In some examples of the present application, the aforementioned connecting peptide 2 has an amino acid sequence as shown in SEQ ID NO: 64.

[0116] In some examples of the present application, the aforementioned first antigen binding region of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO: 47, and the aforementioned second antigen binding region of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO: 48 (BB1-T7-Z13, FIG. 1A). It has been verified by experiments that the aforementioned bispecific antibody can specifically and highly bind to human and monkey B7H7, and can specifically and weakly bind to human and monkey CD3, and has excellent anti-cancer activity.

[0117] In some examples of the present application, the aforementioned first antigen binding region of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO: 51 and SEQ ID NO: 53, and the aforementioned second antigen binding region of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO: 52 and SEQ ID NO: 53 (BB3-T7-Z13, FIG. 1B). It has been verified by experiments that the aforementioned bispecific antibody has better sequence stability, can specifically and highly bind to human and monkey B7H7, and can specifically and weakly bind to human and monkey CD3, and has excellent anti-cancer activity and good treatment safety.

[0118] In some examples of the present application, the first antigen binding region of the aforementioned bispecific antibody has an amino acid sequence as shown in SEQ ID NO: 68 and SEQ ID NO: 53, and the second antigen binding region of the aforementioned bispecific antibody has an amino acid sequence as shown in SEQ ID NO: 52 and SEQ ID NO: 53 (BB3-T8-Z13, FIG. IB). It has been verified through experiments that the aforementioned bispecific antibody sequence is more stable, can bind to human and monkey B7H7 with high specificity and high affinity, can bind to human and monkey CD3 with low affinity and high specificity, has excellent anti-cancer activity and excellent treatment safety.

[0119] In some examples of the present application, the first antigen binding region of the aforementioned bispecific antibody has an amino acid sequence as shown in SEQ ID NO: 68 and SEQ ID NO: 53, and the second antigen binding region of the aforementioned bispecific antibody has an amino acid sequence as shown in SEQ ID NO: 52 and SEQ ID NO: 53 (BB3-T8-Z13, FIG. IB). It has been verified through experiments that the aforementioned bispecific antibody sequence is more stable, can bind to human and monkey B7H7 with high specificity and high affinity, can bind to human and monkey CD3 with low affinity and high specificity, has excellent anti-cancer activity and excellent treatment safety.

[0120] The aforementioned bispecific antibody has a binding arm that binds to CD3 on T cells and a binding arm that binds to B7H7 on the cell surface of tumor cells. By facilitating the simultaneous binding of T cells and tumor cells, the bispecific antibody facilitates the formation of a cell synapse between the two cells and thus selectively reorients the activity of T cells against the targeted tumor cells, achieving the killing of tumor cells.

[0121] Nucleic acid molecule

[0122] In another aspect of the present application, the present application provides a nucleic acid molecule, wherein the aforementioned nucleic acid molecule encodes the aforementioned bispecific antibody.

[0123] In some examples of the present application, the aforementioned nucleic acid molecule is DNA.

[0124] It should be noted that for the nucleic acid molecules mentioned in the specification and claims of the present application, those skilled in the art should understand that any one of the complementary double strands or both are actually included. For the sake of convenience, in the specification and claims, although only one strand is given in most cases, the other complementary strand is actually disclosed. In addition, the nucleic acid sequences in the present application include DNA or RNA forms, and the disclosure of one means the disclosure of the other.

[0125] The nucleic acid molecule obtained based on the above-mentioned method can encode a bispecific antibody targeting B7H7 and CD3.

[0126] Expression vector

[0127] In another aspect of the present application, an expression vector is provided. According to embodiments of the present application, the aforementioned expression vector carries the aforementioned nucleic acid molecule. The aforementioned expression vector comprises optional control amino acid sequences, such as the aforementioned control amino acid sequences operably linked to the aforementioned nucleic acid molecule. Among them, the aforementioned control amino acid sequences can be one or more control amino acid sequences that direct the expression of the aforementioned nucleic acid molecule in a host. The vector thus constructed can effectively express the aforementioned bispecific antibody.

[0128] It should be noted that when the aforementioned nucleic acid molecule is linked to the vector, the aforementioned nucleic acid molecule 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 aforementioned nucleic acid molecule, etc. Of course, these control elements can be directly from the vector itself, or can be exogenous, i.e., not from the vector itself. Of course, the aforementioned nucleic acid molecule can be operably linked to the control elements.

[0129] According to embodiments of the present application, the aforementioned vector can refer to a cloning vector or an expression vector, which can be obtained by operably linking the aforementioned nucleic acid to a commercially available vector (such as a plasmid or a viral vector). The vector in the present application is not particularly limited, and common plasmids can be used, such as pSeTag2, PEE14, pMH3, etc.

[0130] In this context, the term "operably linked" means that the exogenous gene is linked to the vector, so that the control elements in the vector, such as transcription control amino acid sequences, such as and translation control amino acid sequences, etc., can exert their expected functions of regulating the transcription and translation of the exogenous gene. Common vectors can be viral vectors, plasmids, bacteriophages, etc. According to some specific embodiments of the present application, the expression vector introduced into a suitable recipient cell can effectively realize the expression of the aforementioned nucleic acid molecule under the mediation of the regulation system, and further realize the in vitro mass acquisition of the protein encoded by the aforementioned nucleic acid molecule.

[0131] In some examples of the present application, the aforementioned vector is a eukaryotic vector or a prokaryotic vector.

[0132] In some examples of the present application, the aforementioned vector comprises at least one selected from a plasmid vector, an adenovirus vector, a lentivirus vector, and an adeno-associated virus vector.

[0133] Method for preparing bispecific antibody

[0134] In another aspect of the present application, the present application provides a method for preparing the aforementioned bispecific antibody, comprising: introducing the aforementioned expression vector into a cell; culturing the aforementioned cell under conditions suitable for protein expression and secretion, so as to obtain the aforementioned bispecific antibody. Based on the method, the aforementioned bispecific antibody can be expressed in vitro in large quantities.

[0135] In some examples of the present application, the aforementioned cell is a eukaryotic cell.

[0136] Recombinant cell

[0137] In another aspect of the present application, the present application provides a recombinant cell carrying the aforementioned nucleic acid molecule or expression vector. In some examples of the present application, the aforementioned recombinant cell is obtained by transfecting or transforming the aforementioned expression vector, and the aforementioned recombinant cell can efficiently express the aforementioned bispecific antibody under suitable conditions.

[0138] In some examples of the present application, the aforementioned recombinant cell is a prokaryotic cell, a eukaryotic cell or a bacteriophage. It should be noted that the aforementioned eukaryotic cell does not include an animal reproductive cell, a fertilized egg or an embryonic stem cell.

[0139] In some examples of the present application, the aforementioned prokaryotic cell is Escherichia coli, Bacillus subtilis, Streptomyces or Giardia lamblia.

[0140] In some examples of the present application, the aforementioned eukaryotic cell is a fungus, an insect cell, a plant cell or a mammalian cell.

[0141] In some examples of the present application, the aforementioned fungus is Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe or Trichoderma.

[0142] In some examples of the present application, the aforementioned insect cell is a Spodoptera frugiperda cell; in some examples of the present application, the aforementioned plant cell is a tobacco plant cell; in some examples of the present application, the aforementioned mammalian cell is a BHK cell, a CHO cell, a COS cell, a myeloma cell or a human embryonic kidney 293 cell; and does not include an animal reproductive cell, a fertilized egg or an embryonic stem cell.

[0143] In some examples of the present application, the aforementioned recombinant cell is a mammalian cell. When the cell is a mammalian cell, the expression efficiency of the aforementioned antibody or antigen-binding fragment thereof is higher.

[0144] In some examples of the present application, the aforementioned recombinant cell is a BHK cell, a CHO cell, a COS cell or an NSO cell.

[0145] It should be noted that the "suitable condition" mentioned in the specification of the present application refers to the condition suitable for the expression of the bispecific antibody of the present application. It is easily understood by those skilled in the art that the condition suitable for the expression of the bispecific antibody includes but is not limited to suitable transformation or transfection method, suitable transformation or transfection condition, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The "suitable condition" is not particularly limited, and those skilled in the art can optimize the most suitable condition for the expression of the bispecific antibody according to the specific environment of the laboratory.

[0146] Pharmaceutical composition

[0147] In another aspect of the present application, a pharmaceutical composition is provided, comprising the aforementioned bispecific antibody, nucleic acid molecule, expression vector, or recombinant cell. The aforementioned pharmaceutical composition can effectively promote the killing of tumor cells by PBMCs, has good anti-cancer activity and higher safety, and can also reduce the risk of cytokine storm caused by CD3 bispecific antibodies.

[0148] In some examples of the present application, a pharmaceutically acceptable excipient is further included.

[0149] In some examples of the present application, the aforementioned excipient includes one or more pharmaceutically acceptable excipients, diluents, stabilizers, or carriers.

[0150] In some examples of the present application, the aforementioned pharmaceutical composition is an injection.

[0151] It should be noted that the aforementioned pharmaceutical composition includes combinations separated in time and / or space as long as they can act together to achieve the purpose of the present application. For example, the components contained in the aforementioned pharmaceutical composition can be administered to the subject as a whole, or administered to the subject separately. When the components contained in the aforementioned pharmaceutical composition are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0152] The pharmaceuticals of the present application contain a safe and effective amount of the active ingredient (bispecific antibody) of the present application and pharmaceutically acceptable excipients. Such excipients include (but are not limited to) saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should be matched with the administration method, and the dosage form of the pharmaceuticals of the present application is injection, oral preparation (tablet, capsule, oral liquid), transdermal preparation, sustained-release preparation. For example, it is prepared by conventional methods with normal saline or aqueous solution containing glucose and other auxiliary agents. The aforementioned pharmaceuticals are preferably manufactured under sterile conditions.

[0153] The effective amount of the aforementioned active ingredient of the present application can vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by one of ordinary skill in the art (e.g., through clinical trials) according to various factors. The aforementioned factors include, but are not limited to, the pharmacokinetic parameters of the aforementioned active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, several separate doses can be administered daily, or the dose can be proportionally reduced, as required by the exigencies of the therapeutic situation.

[0154] The aforementioned pharmaceutically acceptable excipient of the present application includes (but is not limited to) water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptide substances, cellulose, nanogels, or combinations thereof. The selection of the carrier should be matched with the mode of administration, which is well known to one of ordinary skill in the art.

[0155] Use in the preparation of a medicament

[0156] In yet another aspect of the present application, the present application proposes the use of the aforementioned bispecific antibody, nucleic acid molecule, expression vector, recombinant cell or pharmaceutical composition in the preparation of a medicament for treating or preventing a tumor. Based on the aforementioned bispecific antibody and the corresponding nucleic acid molecule, vector, recombinant cell or pharmaceutical composition can be further prepared into a medicament which can be clinically used for preventing or treating diseases B7H7 and / or CD3 mediated related diseases.

[0157] In some examples of the present application, the aforementioned tumor includes at least one of the following: lung cancer and colorectal cancer.

[0158] Use in the preparation of a kit

[0159] In yet another aspect of the present application, the present application proposes the use of the aforementioned bispecific antibody, nucleic acid molecule, expression vector, recombinant cell or pharmaceutical composition in the preparation of a kit for detecting B7H7 and / or CD3.

[0160] As previously described, the bispecific antibody of the embodiments of the present application can specifically bind to B7H7 and CD3, and thus the aforementioned bispecific antibody can be used to detect B7H7 and / or CD3. Further, it can be used to prepare a B7H7 and / or CD3 related kit and used for scientific research, such as qualitative or quantitative detection of B7H7 and / or CD3 protein molecules in a biological sample. More specifically, it can be used for a kit and the like related to immunoblotting, immunoprecipitation and the like involving the use of the specific binding performance of B7H7 and / or CD3 and the antibody for detection. These kits can contain any one or more of the following: antagonists, bispecific antibodies of the present application or pharmaceutical reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents. The bispecific antibodies of the present application can be used for different types of diagnostic tests, for example, various diseases or the presence of drugs, toxins or other proteins and the like can be detected in vitro or in vivo. For example, the presence of B7H7 and / or CD3 mediated related diseases can be tested by detecting the serum or blood of the subject.

[0161] Kit

[0162] In yet another aspect of the present application, a kit comprising the aforementioned bispecific antibody is provided. The aforementioned kit can be used to conveniently and accurately detect B7H7 and CD3 proteins in a sample.

[0163] In some examples of the present application, the aforementioned kit is used to detect at least one of B7H7 and CD3.

[0164] Disease treatment method

[0165] In yet another aspect of the present application, a method for preventing and / or treating a B7H7 and / or CD3 mediated disease is provided. According to the embodiments of the present application, the aforementioned method comprises administering a pharmaceutically acceptable amount of a bispecific antibody, a nucleic acid molecule, a vector or transformant, a recombinant cell or a pharmaceutical composition to a subject.

[0166] It should be noted that the terms "subject", "individual" and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and / or being treated. In one embodiment, the mammal is a human. The terms "subject", "individual" and "patient" include, but are not limited to, an individual having cancer, an individual having an autoimmune disease, an individual having a pathogen infection, and the like. The subject can be a human, but also includes other mammals, particularly mammals useful as laboratory models of human disease, such as mice, rats, and the like.

[0167] The effective amount of the aforementioned bispecific antibody, nucleic acid molecule, vector or transformant, recombinant cell or pharmaceutical composition of the present application can vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by one of ordinary skill in the art (e.g., through clinical trials) according to various factors. The aforementioned factors include, but are not limited to, the pharmacokinetic parameters of the aforementioned active ingredients, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, several separate doses can be administered daily, or the dose can be proportionally reduced, as required by the urgency of the therapeutic situation.

[0168] In some examples of the present application, the aforementioned disease is a tumor, including lung cancer and colorectal cancer.

[0169] Disease treatment use

[0170] In still another aspect of the present application, the present application proposes the use of the aforementioned bispecific antibody, nucleic acid molecule, expression vector, recombinant cell or pharmaceutical composition in the treatment or prevention of a disease. In some examples of the present application, the administration of an effective dose of the bispecific antibody, nucleic acid molecule, vector or transformant, recombinant cell or pharmaceutical composition to a subject can effectively treat and / or prevent the disease.

[0171] In some examples of the present application, the aforementioned disease is a tumor, including lung cancer and colorectal cancer.

[0172] Table 1 Amino acid sequences and corresponding nucleotide sequences involved in the present application

[0173] The solutions of the present application will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustration of the present application and should not be regarded as limiting the scope of the present application. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are followed. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased on the market.

[0174] Example 1 Design and construction of bispecific antibody targeting CD3-B7H7

[0175] To better compare the function of CD3-B7H7 bispecific antibodies of different configurations and sequences, the present application designs three different configurations of CD3-B7H7 bispecific antibodies. The first antibody is named BB1-T7-Z13 (Figure 1A), which contains two monovalent units, one of which is an anti-CD3 scFv-Fc form, and the other is an anti-B7H7 scFv-Fc form. The second antibody is named BB3-T7-Z13 (Figure IB), which contains three monovalent units, one of which is an anti-CD3 scFv form, and the other two are anti-B7H7 Fab forms. The third antibody is named BB3-Tma-Z13 (Figure 1C), which contains three monovalent units, one of which is an anti-CD3 scFv form, and the other two are anti-B7H7 Fab forms. The antibody configuration is similar to BB3-T7-Z13, but the CD3 variable region sequence is different. The heavy chain constant region of the first and second polypeptide chains of the two antibodies is from human antibody IgG1, due to the special asymmetric structure of the molecule, in order to reduce the production of homodimers, different amino acid mutations are introduced into the constant region of the first and second polypeptide chains to form a knob-in-hole structure. At the same time, in order to prevent cross-linking activation caused by Fc gamma receptor, (L234A / L235A) mutations are also introduced into the constant region of the first and second polypeptide chains.

[0176] Using molecular biology techniques, the polypeptide chains were cloned into the expression vector pcDNA3.4 vector respectively, and in order to make them highly expressed in CHO cells and secreted into the culture medium, the lead peptide of the murine antibody heavy chain was selected as the secretion signal peptide and inserted into the expression vector, and the signal peptide (SEQ ID NO: 65) is located at the N-terminus of the antibody variable region.

[0177] Example 2 Targeting CD3-B7H7 bispecific antibody expression and purification

[0178] The CD3-B7H7 bispecific molecules were prepared by transiently transfecting ExpiCHO-S cells (Gibco, Cat No. A29127) with pcDNA3.4 vectors carrying the CD3-B7H7 chain encoding genes. One day before transfection, the ExpiCHO-S cells were adjusted to a cell density of (3-4) x 10 6 / ml, 37°C, 8% CO2, 120 rpm shaking culture overnight. On the day of transfection, the cells were grown to 7 x 10 6 -1 x 10 7 / ml, and the viability was greater than 95%, the cells were diluted to 6 x 10 6 / ml, and the plasmids containing polypeptide chains were taken at a mass ratio of 1:1 (BB1-T7-Z13) or 1:1:2 (BB3-T7-Z13 or BB3-Tma-Z13) and transfected into ExpiCHO-S cells using ExpiFectamine CHO transfection reagent (Gibco, item number A29129) and cultured at 37°C, 8% CO2, and 120 rpm shaking. Eighteen to twenty-two hours after transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed were mixed and immediately added to the cells after transfection, mixed, and cultured at 32°C, 5% CO2, and 120 rpm shaking. On the fifth day after transfection, 8 ml of ExpiCHO Feed was added to the cells, mixed, and continued to be cultured. The number of cells and the cell viability were observed daily, and the cells were harvested by centrifugation after the cell viability decreased to below 80% or after 10-14 days of culture. The supernatant was purified or frozen at -80°C.

[0179] The expressed supernatant was filtered with a 0.22 μm filter, and the antibody with an Fc domain was captured from the expression supernatant using a Mabselect prism A affinity chromatography column (cytiva company, item number 17549854). After equilibrating the chromatography column with a phosphate buffer at pH 7.2, the supernatant was passed through the affinity chromatography column, eluted with elution buffer (100 mM citric acid, pH 2.7), and finally concentrated and replaced with PBS buffer. The purified antibody was identified by SDS-PAGE to have a purity of more than 90%, and finally a CD3-B7H7 bispecific binding antibody was obtained.

[0180] Example 3 Flow cytometry binding experiment of CD3-B7H7 bispecific antibody

[0181] Test Example 1: Bispecific antibody binding to human T cells

[0182] The human T cells were adjusted to a cell density of 5 x 10 5 / mL, and added to 1.5 ml EP tubes at a volume of 100 μL / tube. Gradient concentrations of bispecific antibodies were added, and incubated at 4°C for 30 min. 1 mL of PBS was added to the EP tubes, centrifuged at 3500 rpm for 5 min at 4°C, and the supernatant was discarded. Then, the cells were washed once with PBS. Goat anti-human antibody secondary antibody (Jackson ImmunoResearch, 109-606-170) was added at 100 μL per well to the cell wells, mixed, and incubated at 4°C for 30 min. Then, the cells were washed twice with PBS, and the supernatant was discarded after centrifugation. The cells were resuspended with 200 μL / tube of PBS, and detected by flow cytometry. The results in FIG. 2A show that the bispecific antibody BB1-T7-Z13 has the strongest ability to bind T cells, followed by BB3-T7-Z13, and BB3-Tma-Z13 has the weakest ability.

[0183] Test Example 2: Bispecific antibody binding to HCC827 tumor cells

[0184] HCC827 non-small cell lung cancer cells were diluted to 2 × 10⁻⁶ with PBS. 6 Add 100 μL of the sample to a 1.5 mL EP tube, along with 10 μL of goat serum, and block at 4°C for 30 min. Add a gradient concentration of bispecific antibody and incubate at 4°C for 30 min. Add 1 mL of PBS to the EP tube, centrifuge at 3500 rpm for 5 min at 4°C, discard the supernatant, and wash once with PBS. After centrifugation, discard the supernatant again, resuspend the cells in 100 μL of PBS, add 0.1 μL of Alexa 647-labeled goat anti-human antibody secondary antibody (Jackson ImmunoResearch, 109-606-170), and incubate at 4°C in the dark for 30 min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μL of PBS and analyze using flow cytometry. Figure 2B shows that all three bispecific antibodies can bind to B7H7+ HCC827 cells, and the EC50 values ​​of the three are similar, while the MFI peak of BB1-T7-Z13 is higher.

[0185] Example 4: Assay of the binding activity of CD3-B7H7 bispecific antibody to two antigens (ELISA)

[0186] To detect whether the bispecific antibody can bind to both CD3 and B7H7 antigens to achieve the bridging effect, human CD3 antigen (ACRO biosystems, item number CDD-H52W1) was diluted to 1 pg / mL with coating buffer (35 mM NaHCO3, 15 mM Na2CO3, pH 9.6) to 100 pL per well of enzyme-linked plate, and incubated at 4°C overnight. Then wash 3 times with PBST (0.05% Tween 20-PBS, pH 7.2). Add 300 pL blocking buffer (1% BSA, 0.05% Tween 20-PBS, pH 7.2) to the plate, and incubate at room temperature for 2 h. Wash 3 times with PBST again. Add different concentrations of bispecific antibody to each well, and incubate at room temperature for 1 h. Wash 3 times with PBST again. Add 100 pL of biotinylated human B7H7 antigen (ACRO biosystems, item number B77-H82E3) diluted with blocking buffer to each well, and incubate at room temperature for 1 h. Wash 3 times with PBST again. Add 100 pL of HRP-labeled streptavidin secondary antibody (SouthernBiotech, item number 7105-05) diluted with blocking buffer to each well, and incubate at room temperature for 1 h. Wash 3 times with PBST, add TMB to each well, and react at room temperature for 2-5 min. Stop the reaction with 2M sulfuric acid in each well, and finally read the OD450 value with a microplate reader. The specific results are shown in Figure 3, which show that the OD450 reading of the bispecific antibody gradually increases with increasing concentration of the bispecific antibody molecule, indicating that the three bispecific antibodies can simultaneously bind to human CD3 and B7H7, and the binding capacity of BB1-T7-Z13 is slightly stronger.

[0187] Example 5 Affinity verification of CD3-B7H7 bispecific antibody targeting

[0188] The Biacore 1K platform was used to test the kinetic and affinity performance of the antibody. According to the system instructions and indications, the antibody was diluted to 1 pg / mL with running buffer (HBS-EP), and the antibody was coupled to the protein A chip at a flow rate of 10 pL / min. The kinetic and affinity data of antigen binding to the antibody were detected at a flow rate of 30 pL / min, and the binding time was set to 90 s and the dissociation time was set to 300 s. The results are shown in Table 2, which show that the three bispecific antibodies can bind to human B7H7 and monkey B7H7, and the affinity with B7H7 is much higher than that with CD3.

[0189] Table 2

[0190] Example 6 In vitro killing detection of CD3-B7H7 bispecific binding antibody targeting

[0191] Adherent tumor cells were counted after digestion and the cell density was adjusted to 2 x 10 5 / ml. The RTCA instrument (Agilent) was opened, the experiment mode was selected, and the cell information and drug information were filled in. The experiment steps were set in schedule, the plate was added with 50 μΐ^ of fresh medium (89% RPMI 1640 medium + 10% fetal bovine serum + 1% penicillin-streptomycin) and then placed in the instrument, closed, and the first step was started. After completion, the plate was taken out, 100 μΐ^ of cell suspension was added, and the plate was placed in the instrument after incubation at room temperature for 15-30 min to prevent edge effect. After the cells grew to the logarithmic phase, the instrument was paused, 50 μΐ^ of PBMC (1 x 10 6 / ml) was added, and the bispecific antibodies at gradient concentrations were added. After a period of time, the analysis was performed.

[0192] With the increase of the bispecific antibody concentration, the killing efficiency of PBMC on non-small cell lung cancer HCC827 cells (Fig. 4A), lung adenocarcinoma NCI-H820 cells (Fig. 4B), and colorectal cancer LS180 (Fig. 4C) gradually increased. In HCC827 and NCI-H820, the BB1-T7-Z13 and BB3-Tma-Z13 had significantly higher killing-promoting activity than BB3-T7-Z13. In LS180 cells, BB3-Tma-Z13 had the strongest killing-promoting activity, and BB3-T7-Z13 had the weakest killing-promoting activity.

[0193] Example 7: Effect of different B7H7 expression levels on the activity of bispecific antibodies

[0194] To further analyze the effect of different B7H7 expression levels on the activity of the three bispecific antibodies, colorectal cancer HCT-15 cells were used to overexpress B7H7 molecules at different levels on the cell surface. By using the killing mode shown in Example 6, the results shown in Fig. 5A show that in B7H7-high-expressing HCT-15 cells, BB1-T7-Z13 has the strongest killing-promoting effect on tumor cells, followed by BB3-Tma-Z13, and BB3-T7-Z13 has the weakest killing-promoting effect. Fig. 5B shows that in B7H7-low-expressing HCT-15 cells, the three bispecific antibodies have little difference in killing-promoting activity. In combination with the results shown in Fig. 5A and Fig. 5B, the EC50 of the same antibody varies greatly in different B7H7 expression levels, indicating that the activity of the bispecific antibody depends on the B7H7 expression level on the tumor cell surface.

[0195] Example 8: Detection of cytokine secretion

[0196] Test Example 1: Effect of CD3-B7H7 on PBMC cytokine secretion in the absence of target cells

[0197] 1 x 10 5One PBMC was added to a 96-well plate, with a total volume of 200 μL. Then, three bispecific binding antibodies of varying concentrations as shown in the figure were added and mixed thoroughly. After incubation at 37°C for 48 hours, the supernatant was collected by centrifugation. The concentrations of various cytokines in the supernatant were detected using a CBA cytokine assay kit (BD, catalog numbers 560111, 558276, 560112, 558270). Finally, the analysis was performed using flow cytometry.

[0198] Figures 6A-D show the concentrations of IL-2, IL-6, IFN-γ, and TNF cytokines in the supernatant during co-incubation in the absence of target cells. As the concentration of CD3-B7H7 antibody increased, the levels of these four cytokines secreted by PBMCs remained almost unchanged, indicating that the CD3-B7H7 bispecific antibody does not nonspecifically activate PBMCs in the absence of target cells, suggesting that this bispecific antibody has potentially good safety.

[0199] Test Example 2: Effect of CD3-B7H7 on PBMC cytokine secretion in the presence of target cells

[0200] Take 1×10 5 One PBMC was added to a 96-well plate, with 1 × 10⁻⁶ ppm added. 4 Lung adenocarcinoma NCI-H820 cells, total volume 200 μL. CD3-B7H7 bispecific binding antibody at gradient concentrations as shown in the figure was added and mixed well. After incubation at 37°C for 48 hours, the supernatant was collected by centrifugation. The concentrations of various cytokines in the supernatant were detected using a CBA cytokine assay kit (BD, catalog numbers 560111, 558276, 560112, 558270). Finally, flow cytometry was used for analysis.

[0201] Figures 7A-D show the concentrations of IL-2, IL-6, IFN-γ, and TNF cytokines in the supernatant of co-incubation with NCI-H820 lung adenocarcinoma cells. When target cells are present, the levels of these four cytokines secreted by PBMCs are significantly higher than those without target cells as the concentration of CD3-B7H7 antibody increases, but the magnitude is controllable. The BB3-T7-Z13 conformation and sequence composition produces the fewest cytokines.

[0202] Example 9: Study on the efficacy of in vivo xenotransplantation

[0203] To detect the in vivo antitumor activity of CD3-B7H7 bispecific binding antibody against HCT-15-B7H7 tumor cells, PBMCs were intravenously injected into female NOD.Cg-Prkdc cells. scid Il2rg em1Smoc IL15em1(hIL15)Smoc mice, 4 days later HCT-15-B7H7 cells (4×10⁻⁶) were added. 6s.c. injection (s.c.) to the subcutis of mice, and tumors were allowed to grow to 50-110 mm 3 After grouping, the mice were then injected intravenously with different doses of CD3-B7H7 bispecific antibodies (0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg) or control PBS solvent twice a week for a total of 3 injections. Tumor length and width were measured by external caliper and tumor volume was calculated using the standard formula.

[0204] Figures 8A-C show the in vivo anti-tumor activity of CD3-B7H7 bispecific antibodies against HCT-15-B7H7 cells. Figure 8A shows that treatment with BB1-T7-Z13 bispecific antibodies can significantly inhibit tumor growth, and complete tumor clearance can be achieved even at a dose of 0.03 mg / kg. Figure 8B shows that BB3-T7-Z13 also has strong anti-tumor activity, and complete tumor clearance can be achieved at a dose of 0.1 mg / kg, and significant tumor inhibition can be achieved at a dose of 0.03 mg / kg. Figure 8C shows that BB3-Tma-Z13 has relatively weak anti-tumor activity, while previous examples show that this antibody has strong in vitro killing activity, and the reason for this difference can be that the CD3 antibody can cause T cell exhaustion or that the CD3 sequence Tma has poor serum stability.

[0205] Examples 8 and 9 collectively suggest that the CD3 variable region sequence of T7 has better biological activity and lower potential toxicity, and the BB3 bispecific antibody configuration with the T7 CD3 variable region sequence (BB3-T7-Z13) has the best potential safety.

[0206] Example 10: Bispecific antibody safety optimization

[0207] Further studies found that too high CD3 affinity in bispecific antibodies can cause a higher incidence of cytokine storm (CRS) and lower safety, and BB3-T7-Z13 was found to cause a more severe cytokine storm in cynomolgus monkeys at a dose of 0.1 mg / kg. Therefore, in order to further improve the therapeutic window of bispecific antibodies, the CD3 antibody affinity was further reduced to improve the potential safety. The second antibody configuration mentioned in Example 1 (Figure IB) was used in this application, and the CD3 sequence was replaced with the affinity-optimized sequence, and the molecule constructed was named BB3-T8-Z13. The method mentioned in Example 2 was used to express and purify the bispecific antibody, and finally the CD3-B7H7 bispecific antibody BB3-T8-Z13 was obtained.

[0208] The affinity of the BB3-T8-Z13 antibody was determined using the SPR method described in Example 5, and the results are shown in Table 3, which shows that the CD3 affinity is only 1.14E-6M, indicating that the CD3 affinity of BB3-T8-Z13 is lower than that of BB3-T7-Z13.

[0209] Table 3

[0210] In vitro cytokine release is generally tested as a method for predicting potential safety, and the cytokine release of BB3-T7-Z13 and BB3-T8-Z13 was compared after PBMCs were co-incubated with colorectal cancer HCT-15-B7H7 cells using the method in Example 8. Figures 9A-C show the concentrations of IL-2, IL-6 and TNF cytokines in the supernatant of the co-incubation in the presence of colorectal cancer HCT-15-B7H7 cells, and the cytokine release level of BB3-T8-Z13 with low CD3 affinity is significantly lower than that of BB3-T7-Z13 as the concentration of CD3-B7H7 antibody increases, suggesting that BB3-T8-Z13 has potentially higher safety. Meanwhile, BB3-T8-Z13 was found to have no obvious clinical toxicity in cynomolgus monkeys at 0.3 mg / kg.

[0211] BB3-T8-Z13 was tested using the in vivo pharmacodynamic evaluation method described in Example 9, and Figure 10 shows that complete tumor clearance can be achieved at 0.3 mg / kg, and significant tumor inhibition can also be achieved at 0.1 mg / kg. The above results collectively suggest that BB3-T8-Z13 has good anti-tumor activity and better safety, and has a larger clinical treatment window.

[0212] The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be considered as disclosed by the present application and fall within the protection scope of the present application.

[0213] In the description of the specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are contained in at least one embodiment or example of the present application. In the specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any suitable manner in any one or more embodiments or examples. In addition, the person skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples without contradiction.

[0214] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary and are not to be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A bispecific antibody, characterized in that, Comprise: a first antigen binding region having CD3 binding activity; and a second antigen binding region having B7H7 binding activity; wherein the first antigen binding region comprises an anti-CD3 antibody comprising heavy chain complementarity determining regions HCDRs comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 4-6, 66 or an amino acid sequence having at least 80% homology with SEQ ID NOs: 4-6, 66; the anti-CD3 antibody further comprises light chain complementarity determining regions LCDRs comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1-3 or an amino acid sequence having at least 80% homology with SEQ ID NOs: 1-3; the second antigen binding region comprises an anti-B7H7 antibody comprising heavy chain complementarity determining regions HCDRs comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 26-28 or an amino acid sequence having at least 80% homology with SEQ ID NOs: 26-28; the anti-B7H7 antibody further comprises light chain complementarity determining regions LCDRs comprising at least one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 23-25 or an amino acid sequence having at least 80% homology with SEQ ID NOs: 23-25.

2. The bispecific antibody of claim 1, wherein the HCDRs of the anti-CD3 antibody comprise: an HCDR1, an HCDR2 and an HCDR3, wherein the HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 4 or an amino acid sequence having at least 80% homology with SEQ ID NO: 4, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 5 or an amino acid sequence having at least 80% homology with SEQ ID NO: 5, the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 6, 66 or an amino acid sequence having at least 80% homology with SEQ ID NO: 6, 66; preferably, the HCDRs comprise: an HCDR1, an HCDR2 and an HCDR3, respectively, of the amino acid sequences as set forth in SEQ ID NOs: 4-6, 66; optionally, the LCDRs comprise: an LCDR1, an LCDR2 and an LCDR3, wherein the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 1 or an amino acid sequence having at least 80% homology with SEQ ID NO: 1, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 2 or an amino acid sequence having at least 80% homology with SEQ ID NO: 2, the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 3 or an amino acid sequence having at least 80% homology with SEQ ID NO: 3; preferably, the LCDRs comprise: an LCDR1, an LCDR2 and an LCDR3, respectively, of the amino acid sequences as set forth in SEQ ID NOs: 1-3. Optionally, the anti-CD3 antibody further comprises: a heavy chain framework region HFR comprising: HFR1, HFR2, HFR3 and HFR4 of the amino acid sequence as shown in SEQ ID NO: 11-14, 19-22, respectively; wherein, the HFR1 comprises an amino acid sequence as shown in SEQ ID NO: 11, 19, or has at least 80% homology with SEQ ID NO: 11, 19, the HFR2 comprises an amino acid sequence as shown in SEQ ID NO: 12, 20, or has at least 80% homology with SEQ ID NO: 12, 20, the HFR3 comprises an amino acid sequence as shown in SEQ ID NO: 13, 21, or has at least 80% homology with SEQ ID NO: 13, 21, the HFR4 comprises an amino acid sequence as shown in SEQ ID NO: 14, 22, or has at least 80% homology with SEQ ID NO: 14, 22; Preferably, the HFR comprises: HFR1, HFR2, HFR3 and HFR4 of the amino acid sequence as shown in SEQ ID NO: 7-14, respectively; More preferably, the HFR comprises: HFR1, HFR2, HFR3 and HFR4 of the amino acid sequence as shown in SEQ ID NO: 11-14, respectively; Optionally, the anti-CD3 antibody further comprises: a light chain framework region LFR comprising: LFR1, LFR2, LFR3 and LFR4 of the amino acid sequence as shown in SEQ ID NO: 7-10, 15-18, respectively; wherein, the LFR1 comprises an amino acid sequence as shown in SEQ ID NO: 7, 15, or has at least 80% homology with SEQ ID NO: 7, 15, the LFR2 comprises an amino acid sequence as shown in SEQ ID NO: 8, 16, or has at least 80% homology with SEQ ID NO: 8, 16, the LFR3 comprises an amino acid sequence as shown in SEQ ID NO: 9, 17, or has at least 80% homology with SEQ ID NO: 9, 17, the LFR4 comprises an amino acid sequence as shown in SEQ ID NO: 10, 18, or has at least 80% homology with SEQ ID NO: 10, 18; Preferably, the LFR comprises: LFR1, LFR2, LFR3 and LFR4 of the amino acid sequence as shown in SEQ ID NO: 7-10, 15-18, respectively; More preferably, the LFR comprises: LFR1, LFR2, LFR3 and LFR4 of the amino acid sequence as shown in SEQ ID NO: 7-10, respectively; Optionally, the heavy chain variable region of the anti-CD3 antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 38, 40, 67 or an amino acid sequence having at least 80% homology with SEQ ID NO: 38, 40, 67; preferably, the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody is as set forth in SEQ ID NO: 38, 40, 67; more preferably, the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody is as set forth in SEQ ID NO:

67. Preferably, the light chain variable region of the anti-CD3 antibody comprises an amino acid sequence as set forth in SEQ ID NO: 37, 39 or an amino acid sequence having at least 80% homology with SEQ ID NO: 37, 39; preferably, the amino acid sequence of the light chain variable region of the anti-CD3 antibody is as set forth in SEQ ID NO: 37, 39; more preferably, the amino acid sequence of the light chain variable region of the anti-CD3 antibody is as set forth in SEQ ID NO:

37.

3. The bispecific antibody of claim 1, wherein The HCDR of the anti-B7H7 antibody comprises: HCDR1, HCDR2 and HCDR3, wherein, the HCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 26 or an amino acid sequence having at least 80% homology with SEQ ID NO: 26, the HCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 27 or an amino acid sequence having at least 80% homology with SEQ ID NO: 27, the HCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 28 or an amino acid sequence having at least 80% homology with SEQ ID NO: 28; Preferably, the HCDR comprises: HCDR1, HCDR2 and HCDR3, respectively, of the amino acid sequences as set forth in SEQ ID NO: 26-28. The LCDR of the anti-B7H7 antibody comprises: LCDR1, LCDR2 and LCDR3, wherein, the LCDR1 comprises an amino acid sequence as set forth in SEQ ID NO: 23 or an amino acid sequence having at least 80% homology with SEQ ID NO: 23, the LCDR2 comprises an amino acid sequence as set forth in SEQ ID NO: 24 or an amino acid sequence having at least 80% homology with SEQ ID NO: 24, the LCDR3 comprises an amino acid sequence as set forth in SEQ ID NO: 25 or an amino acid sequence having at least 80% homology with SEQ ID NO: 25; Preferably, the LCDR comprises: LCDR1, LCDR2 and LCDR3, respectively, of the amino acid sequences as set forth in SEQ ID NO: 23-25. Optionally, the anti-B7H7 antibody further comprises: a heavy chain framework region HFR, the HFR comprising: HFR1, HFR2, HFR3 and HFR4, respectively, of the amino acid sequences as set forth in SEQ ID NO: 33-36; wherein, the HFR1 comprises an amino acid sequence as set forth in SEQ ID NO: 33 or an amino acid sequence having at least 80% homology with SEQ ID NO: 33, the HFR2 comprises an amino acid sequence as set forth in SEQ ID NO: 34 or an amino acid sequence having at least 80% homology with SEQ ID NO: 34, the HFR3 comprises an amino acid sequence as set forth in SEQ ID NO: 35 or an amino acid sequence having at least 80% homology with SEQ ID NO: 35, the HFR4 comprises an amino acid sequence as set forth in SEQ ID NO: 36 or an amino acid sequence having at least 80% homology with SEQ ID NO:

36. the HFR2 comprises an amino acid sequence as set forth in SEQ ID NO: 34 or has at least 80% homology with SEQ ID NO: 34, the HFR3 comprises an amino acid sequence as set forth in SEQ ID NO: 35 or has at least 80% homology with SEQ ID NO: 35, the HFR4 comprises an amino acid sequence as set forth in SEQ ID NO: 36 or has at least 80% homology with SEQ ID NO: 36; Preferably, the HFR comprises: HFR1, HFR2, HFR3 and HFR4 having amino acid sequences as set forth in SEQ ID NOs: 33-36, respectively; Optionally, the anti-B7H7 antibody further comprises: a light chain framework region (LFR) comprising: LFR1, LFR2, LFR3 and LFR4 having amino acid sequences as set forth in SEQ ID NOs: 29-32, respectively; wherein, the LFR1 comprises an amino acid sequence as set forth in SEQ ID NO: 29 or has at least 80% homology with SEQ ID NO: 29, the LFR2 comprises an amino acid sequence as set forth in SEQ ID NO: 30 or has at least 80% homology with SEQ ID NO: 30, the LFR3 comprises an amino acid sequence as set forth in SEQ ID NO: 31 or has at least 80% homology with SEQ ID NO: 31, the LFR4 comprises an amino acid sequence as set forth in SEQ ID NO: 32 or has at least 80% homology with SEQ ID NO: 32; Preferably, the LFR comprises: LFR1, LFR2, LFR3 and LFR4 having amino acid sequences as set forth in SEQ ID NOs: 29-32, respectively; Preferably, the heavy chain variable region of the anti-B7H7 antibody comprises an amino acid sequence selected from the group consisting of: an amino acid sequence as set forth in SEQ ID NO: 42 or has at least 80% homology with SEQ ID NO: 42; more preferably, the amino acid sequence of the heavy chain variable region of the anti-B7H7 antibody is as set forth in SEQ ID NO: 42; Preferably, the light chain variable region of the anti-B7H7 antibody comprises an amino acid sequence as set forth in SEQ ID NO: 41 or has at least 80% homology with SEQ ID NO: 41; more preferably, the amino acid sequence of the light chain variable region of the anti-B7H7 antibody is as set forth in SEQ ID NO:

41.

4. The bispecific antibody of claim 1, wherein the first antigen binding region comprises a first anti-CD3 antibody scFv fragment comprising an anti-CD3 antibody heavy chain variable region, an anti-CD3 antibody light chain variable region and a connecting peptide 1; Optionally, the C-terminus of the anti-CD3 antibody light chain variable region is connected to the N-terminus of the connecting peptide 1, and the C-terminus of the connecting peptide 1 is connected to the N-terminus of the anti-CD3 antibody heavy chain variable region; Optionally, the first antigen binding region further comprises a first Fc fragment; optionally, a C-terminus of the anti-B7H7 antibody light chain variable region is connected to an N-terminus of the connecting peptide 1, a C-terminus of the connecting peptide 1 is connected to an N-terminus of the anti-B7H7 antibody heavy chain variable region, a C-terminus of the anti-B7H7 antibody heavy chain variable region is connected to an N-terminus of the second Fc fragment; optionally, the second antigen binding region comprises a second anti-B7H7 antibody Fab fragment, the second anti-B7H7 antibody Fab fragment comprises an anti-B7H7 antibody heavy chain variable region, a B7H7 antibody light chain variable region, and a connecting peptide 1; optionally, a C-terminus of the anti-B7H7 antibody light chain variable region is connected to an N-terminus of the connecting peptide 1, a C-terminus of the connecting peptide 1 is connected to an N-terminus of the anti-B7H7 antibody heavy chain variable region, a C-terminus of the anti-B7H7 antibody heavy chain variable region is connected to an N-terminus of the second Fc fragment; optionally, the second antigen binding region further comprises a second Fc fragment; optionally, a C-terminus of the anti-B7H7 antibody light chain variable region is connected to an N-terminus of the connecting peptide 1, a C-terminus of the connecting peptide 1 is connected to an N-terminus of the anti-B7H7 antibody heavy chain variable region, a C-terminus of the anti-B7H7 antibody heavy chain variable region is connected to an N-terminus of the second Fc fragment; optionally, the connecting peptide 1 has an amino acid sequence as set forth in SEQ ID NO:

63.

5. The bispecific antibody of claim 1, wherein the first antigen binding region comprises a first anti-B7H7 antibody Fab fragment and a first anti-CD3 antibody scFv fragment, the first anti-B7H7 antibody Fab fragment and the first anti-CD3 antibody scFv fragment are connected by a connecting peptide 2, wherein the first anti-B7H7 antibody Fab fragment comprises an anti-B7H7 antibody heavy chain variable region, a B7H7 antibody light chain variable region, a first CL fragment, and a first CH1 fragment; the first anti-CD3 antibody scFv fragment comprises an anti-CD3 antibody heavy chain variable region, a CD3 antibody light chain variable region, and a connecting peptide 1; optionally, a C-terminus of the first anti-B7H7 antibody heavy chain variable region is connected to an N-terminus of the first CH1 fragment, a C-terminus of the first anti-B7H7 antibody light chain variable region is connected to an N-terminus of the first CL fragment, the first CL fragment and the first CH1 fragment are connected by a disulfide bond, a C-terminus of the first CH1 fragment is connected to an N-terminus of the connecting peptide 2, a C-terminus of the connecting peptide 2 is connected to an N-terminus of the first anti-CD3 antibody light chain variable region, a C-terminus of the first anti-CD3 antibody light chain variable region is connected to an N-terminus of the connecting peptide 1, a C-terminus of the connecting peptide 1 is connected to an N-terminus of the first anti-CD3 antibody heavy chain variable region; optionally, the C-terminus of the first anti-B7H7 antibody heavy chain variable region is connected to the N-terminus of the first CH1 fragment, the C-terminus of the first anti-B7H7 antibody light chain variable region is connected to the N-terminus of the first CL fragment, the first CL fragment and the first CH1 fragment are connected by a disulfide bond, the C-terminus of the first CH1 fragment is connected to the N-terminus of the connecting peptide 2, the C-terminus of the connecting peptide 2 is connected to the N-terminus of the first anti-CD3 antibody heavy chain variable region, the C-terminus of the first anti-CD3 antibody heavy chain variable region is connected to the N-terminus of the connecting peptide 1, the C-terminus of the connecting peptide 1 is connected to the N-terminus of the first anti-CD3 antibody light chain variable region; optionally, the first antigen binding region further comprises a first Fc fragment; optionally, the first anti-CD3 antibody scFv fragment is connected to the first Fc fragment; optionally, the connecting peptide 2 has an amino acid sequence as set forth in SEQ ID NO:

64.

6. The bispecific antibody of claim 5, wherein The second antigen binding region comprises a second anti-B7H7 antibody Fab fragment, the second anti-B7H7 antibody Fab fragment comprises a second anti-B7H7 antibody heavy chain variable region, a second anti-B7H7 antibody light chain variable region, a second CL fragment and a second CH1 fragment; optionally, the C-terminus of the second anti-B7H7 antibody heavy chain variable region is connected to the N-terminus of the second CH1 fragment, the C-terminus of the second anti-B7H7 antibody light chain variable region is connected to the N-terminus of the second CL fragment, the second CL fragment and the second CH1 fragment are connected by a disulfide bond; optionally, the second antigen binding region further comprises a second Fc fragment; optionally, the second anti-B7H7 antibody Fab fragment is connected to the second Fc fragment; optionally, the connecting peptide 2 has an amino acid sequence as set forth in SEQ ID NO:

64.

7. The bispecific antibody according to any one of claims 4 to 6, characterized in that The first CH1 and the second CH1 are independently selected from human antibody IgG1; Preferably, the human antibody IgG1 has L234A / L235A mutations; optionally, the first Fc fragment and the second Fc fragment are connected by a Knob-in-hole structure.

8. The bispecific antibody of claim 1, wherein, The first antigen binding region of the bispecific antibody has an amino acid sequence as set forth in SEQ ID NO: 47, the second antigen binding region of the bispecific antibody has an amino acid sequence as set forth in SEQ ID NO: 48; optionally, the first antigen binding region of the bispecific antibody has an amino acid sequence as set forth in SEQ ID NO: 51 and SEQ ID NO: 53, the second antigen binding region of the bispecific antibody has an amino acid sequence as set forth in SEQ ID NO: 52 and SEQ ID NO: 53; optionally, the first antigen binding region of the bispecific antibody has an amino acid sequence as set forth in SEQ ID NO: 68 and SEQ ID NO: 53, the second antigen binding region of the bispecific antibody has an amino acid sequence as set forth in SEQ ID NO: 52 and SEQ ID NO: 53; Optionally, the first antigen-binding region of the bispecific antibody has an amino acid sequence as set forth in SEQ ID NO: 57 and SEQ ID NO: 59, and the second antigen-binding region of the bispecific antibody has an amino acid sequence as set forth in SEQ ID NO: 58 and SEQ ID NO:

59.

9. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the bispecific antibody of any one of claims 1-8; Optionally, the nucleic acid molecule is selected from DNA.

10. An expression vector, characterized in that, Carrying the nucleic acid molecule of claim 9.

11. A method of producing a bispecific antibody according to any one of claims 1 to 8, characterized in that, Comprising: Introducing the expression vector of claim 10 into a cell; Culturing the cell under conditions suitable for protein expression and secretion so as to obtain the bispecific antibody; Optionally, the cell is a eukaryotic cell.

12. A recombinant cell, wherein, The recombinant cell carries the bispecific antibody of any one of claims 1-8, the nucleic acid molecule of claim 9, or the expression vector of claim 10.

13. A pharmaceutical composition, characterized by, Comprising: The bispecific antibody of any one of claims 1-8, the nucleic acid molecule of claim 9, the expression vector of claim 10, or the recombinant cell of claim 12; Optionally, further comprising a pharmaceutically acceptable excipient; Optionally, the excipient comprises one or more pharmaceutically acceptable excipients, diluents, stabilizers, or carriers; Optionally, the pharmaceutical composition is an injection.

14. Use of the bispecific antibody of any one of claims 1-8, the nucleic acid molecule of claim 9, the expression vector of claim 10, the recombinant cell of claim 12, or the pharmaceutical composition of claim 13 in the manufacture of a medicament for treating or preventing a tumor.

15. Use according to claim 14, characterized in that, The tumor comprises at least one of lung cancer and colorectal cancer.

16. Use of the bispecific antibody of any one of claims 1-8, the nucleic acid molecule of claim 9, the expression vector of claim 10, the recombinant cell of claim 12, or the pharmaceutical composition of claim 13 in the manufacture of a kit for detecting B7H7 and / or CD3.

17. A kit comprising, The kit comprises the bispecific antibody of any one of claims 1-8.

18. The kit of claim 17, wherein The kit is for detecting at least one of B7H7 and CD3.

19. A method of preventing and / or treating a B7H7 and CD3 mediated disease, characterized in that, Comprising: Administering to a subject a pharmaceutically acceptable amount of the antibody or antigen-binding fragment thereof of any one of claims 1-8, the expression vector of claim 10, the recombinant cell of claim 12, or the pharmaceutical composition of claim 13.

20. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-8, the nucleic acid molecule of claim 9, the expression vector of claim 10, the recombinant cell of claim 12, or the pharmaceutical composition of claim 13 in treating or preventing a disease; Optionally, the disease is a tumor, comprising lung cancer and colorectal cancer.

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