Bispecific antibody targeting B7H7 and CD3 and application thereof

Abstract

The invention relates to a bispecific antibody targeting B7H7 and CD3 and application of the bispecific antibody. Wherein the 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 comprises a heavy chain complementarity determining region HCDR, and the HCDR comprises an amino acid sequence selected from at least one of SEQ ID NO: 4-6 and 66; the second antigen binding region comprises an anti-B7H7 antibody, the anti-B7H7 antibody comprises a heavy chain complementarity determining region HCDR, and the HCDR of the anti-B7H7 antibody comprises an amino acid sequence selected from at least one of SEQ ID NO: 26-28. The bispecific antibody with the sequence can be combined with human and monkey B7H7 with high specificity and high affinity, can be combined with human and monkey CD3 with low affinity and high specificity, can promote PBMC to kill tumor cells, and has good anti-cancer activity and higher treatment safety; the occurrence risk of cytokine storm caused by the CD3 bispecific antibody is effectively reduced.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to bispecific antibodies targeting B7H7 and CD3 and their applications. Background Technology

[0002] Bispecific antibodies are antibodies that can bind specifically to two antigen sites simultaneously.

[0003] Currently, treatment for B7H7 is 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] Currently, bispecific antibodies against CD3 may cause severe side effects such as cytokine storms, limiting their clinical application. Many CD3 antibodies are species-specific, making safety testing in non-human primates impractical. Furthermore, existing bispecific CD3 antibodies suffer from excessive cytokine toxicity and poor sequence stability.

[0005] Therefore, there is a need to develop new bispecific antibodies targeting B7H7 and CD3. Summary of the Invention

[0006] This application aims to address at least one of the technical problems existing in the prior art. To this end, this application proposes a highly safe bispecific antibody targeting B7H7 and CD3.

[0007] Specifically, this application provides the following technical solution:

[0008] In one aspect, this application proposes a bispecific antibody. According to an embodiment of this 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 containing at least one of SEQ ID NO:4-6, 66 or an amino acid sequence having at least 80% homology with SEQ ID NO:4-6, 66; the anti-CD3 antibody further comprises: a light chain complementarity-determining region (LCDR), the LCDR containing at least one of SEQ ID NO:1-3 or an amino acid sequence having at least 80% homology with SEQ ID NO: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 containing at least one of SEQ ID NO:26-28 or an amino acid sequence having at least 80% homology with SEQ ID NO:26-28. NO:26-28 have amino acid sequences with at least 80% homology; the anti-B7H7 antibody further comprises a light chain complementarity-determining region (LCDR), wherein the LCDR contains an amino acid sequence selected from at least one of SEQ ID NO:23-25 ​​or having at least 80% homology with SEQ ID NO:23-25. In some examples of this application, bispecific antibodies having the aforementioned sequences can bind human and monkey B7H7 with high specificity and high affinity, and bind human and monkey CD3 with low affinity and high specificity, promoting PBMC killing of tumor cells, exhibiting good anticancer activity and higher therapeutic safety; effectively reducing the risk of cytokine storm caused by CD3 bispecific antibodies.

[0009] Secondly, this application proposes a nucleic acid molecule. According to embodiments of this application, the nucleic acid molecule encodes the bispecific antibody described in the first aspect of this application. In some examples of this application, the aforementioned nucleic acid molecule may encode a bispecific antibody capable of simultaneously targeting B7H7 and CD3.

[0010] Thirdly, this application proposes an expression vector. According to embodiments of this application, the expression vector carries the nucleic acid molecule described in the second aspect of this application. In some examples of this application, the aforementioned expression vector can express in large quantities bispecific antibodies that bind human and monkey B7H7 with high specificity and high affinity, and bind human and monkey CD3 with low affinity and high specificity in suitable host cells.

[0011] Fourthly, this application proposes a method for preparing the bispecific antibody described in the first aspect. According to embodiments of this application, the method includes: introducing the expression vector described in the third aspect of this application into cells; culturing the cells under conditions suitable for protein expression and secretion to obtain the bispecific antibody. In some examples of this application, the bispecific antibody prepared based on this method can bind human and monkey B7H7 with high specificity and high affinity, and bind human and monkey CD3 with low affinity and high specificity, promoting PBMC killing of tumor cells, exhibiting good anti-cancer activity and higher safety; effectively reducing the risk of cytokine storm caused by CD3 bispecific antibodies.

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

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

[0014] Seventhly, this application discloses the use of the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cell described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect in the preparation of a drug for the treatment or prevention of tumors. In some examples of this application, the aforementioned bispecific antibody can effectively promote the killing of tumor cells by PBMCs, exhibiting good anti-cancer activity and higher safety; it effectively reduces the risk of cytokine storm caused by CD3 bispecific antibodies. Drugs containing the aforementioned bispecific antibody and other substances also have significant therapeutic or preventive effects on tumors.

[0015] Eighthly, this application discloses the use of the bispecific antibody described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the recombinant cells described in the fifth aspect, or the pharmaceutical composition described in the sixth aspect in the preparation of a kit for detecting B7H7 and / or CD3. Those skilled in the art will understand that the features and advantages of the bispecific antibody described above also apply to this use, and will not be repeated here.

[0016] Ninthly, this application provides a kit. According to embodiments of this application, the kit comprises the bispecific antibody described in the first aspect. In some examples of this application, the aforementioned bispecific antibody can bind human and monkey B7H7 with high specificity and high affinity, and bind human and monkey CD3 with low affinity and high specificity. Therefore, the kit containing the aforementioned bispecific antibody can be used to detect B7H7 and / or CD3 proteins. The aforementioned kit can be used for scientific research, such as qualitative or quantitative detection of B7H7 and CD3 proteins in biological samples, and can also be used to determine the status of an individual, such as determining whether the individual's B7H7 and CD3 protein levels are higher or lower than normal levels after obtaining the individual's B7H7 and CD3 protein levels.

[0017] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 is a schematic diagram of the structure of the B7H7×CD3 bispecific antibody provided in the embodiments of this application; wherein, A is a schematic diagram of the structure of the bispecific antibody BB1-T7-Z13; B is a schematic diagram of the structure of the bispecific antibody BB3-T7-Z13; and C is a schematic diagram of the structure of the bispecific antibody BB3-Tma-Z13.

[0020] Figure 2 is a schematic diagram of the flow cytometry results of the B7H7×CD3 bispecific antibody cell binding experiment provided in the embodiments of this application; wherein, A is a schematic diagram of the results of the bispecific antibody binding to human T cells; B is a schematic diagram of the results of the bispecific antibody binding to HCC827 cells overexpressing non-small cell lung cancer.

[0021] Figure 3A schematic diagram showing the simultaneous binding of the B7H7×CD3 bispecific antibody to CD3 and B7H7 proteins provided in this application embodiment;

[0022] Figure 4 is a schematic diagram of the in vitro killing effect detection results of the CD3-B7H7 bispecific binding antibody provided in the embodiments of this 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 showing the effect of different B7H7 expression levels on the activity of B7H7×CD3 bispecific antibody provided in the embodiments of this application; where A is the in vitro cytotoxicity experiment results of bispecific antibody and PBMC on HCT-15-B7H7 high-expressing cells; B is a schematic diagram showing the in vitro cytotoxicity experiment results of bispecific antibody and PBMC on HCT-15-B7H7 low-expressing cells.

[0024] Figure 6 is a schematic diagram showing the effect of the B7H7×CD3 bispecific antibody on PBMC cytokine secretion in the absence of target cells, as provided in the embodiments of this application; wherein, A is a schematic diagram showing the effect 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 showing the effect 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 showing the effect of the bispecific binding protein on the release of cytokine IFN-γ by PBMC in the absence of target cells; and D is a schematic diagram showing the effect 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 showing the effect of the B7H7×CD3 bispecific antibody on PBMC cytokine secretion in the presence of target cells, as provided in the embodiments of this application; wherein, A is a schematic diagram showing the effect of the bispecific binding antibody on the release of cytokine IL-2 from PBMC in the presence of target cells; B is a schematic diagram showing the effect of the bispecific binding antibody on the release of cytokine IL-6 from PBMC in the presence of target cells; C is a schematic diagram showing the effect of the bispecific binding protein on the release of cytokine IFN-γ from PBMC in the presence of target cells; and D is a schematic diagram showing the effect of the bispecific binding protein on the release of cytokine TNF from PBMC in the presence of target cells.

[0026] Figure 8 is a schematic diagram of the in vivo antitumor activity results of the B7H7×CD3 bispecific antibody against HCT-15-B7H7 tumor cells provided in the embodiments of this application; wherein, A is a schematic diagram of the in vivo antitumor activity results of the bispecific antibody BB1-T7-Z13 against B7H7+ tumor (HCT-15-B7H7); B is a schematic diagram of the in vivo antitumor activity results of the bispecific antibody BB3-T7-Z13 against B7H7+ tumor (HCT-15-B7H7); C is a schematic diagram of the in vivo antitumor activity results of the bispecific antibody BB3-Tma-Z13 against B7H7+ tumor (HCT-15-B7H7).

[0027] Figure 9 is a schematic diagram showing the effect of bispecific antibodies BB3-T7-Z13 and BB3-T8-Z13 on PBMC cytokine secretion in the presence of target cells (HCT-15-B7H7) provided in the embodiments of this application; wherein, A is a schematic diagram showing the effect of bispecific binding antibodies on PBMC cytokine IL-2 release in the presence of target cells; B is a schematic diagram showing the effect of bispecific binding antibodies on PBMC cytokine IL-6 release in the presence of target cells; and C is a schematic diagram showing the effect of bispecific binding proteins on PBMC cytokine TNF release in the presence of target cells.

[0028] Figure 10 This is a schematic diagram showing the in vivo antitumor activity of the BB3-T8-Z13 bispecific antibody against HCT-15-B7H7 clone 43 tumor cells, as provided in the embodiments of this application. Detailed Implementation

[0029] The embodiments of this application are described in detail below, with examples of these embodiments illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application.

[0030] In this application, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0031] In this application, the term "antibody" refers to an antibody that recognizes 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, triple-chain antibodies, single-chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, as long as they exhibit the desired biological activity. Antibodies can be mouse, human, humanized, chimeric, or derived from other species. Antibodies can refer to full-length heavy-chain, full-length light-chain, or intact immunoglobulin molecules; or the immunoactive portion of any of these polypeptides, i.e., a molecule or portion thereof containing an antigen-binding site that specifically binds to a target antigen of interest, such targets including but not limited to cancer cells or cells that produce autoantibodies associated with autoimmune diseases. In this application, "bispecific antibody" refers to an antibody that recognizes both CD3 and B7H7 antigenic epitopes.

[0032] In this application, certain regions within the variable region exhibit a higher degree of variation in amino acid composition and sequence, termed "hypervariable region (HVR)." The hypervariable region is the location where antigens and antibodies bind, and is therefore also called the complementarity-determining region (CDR). Both the heavy chain and light chain variable regions contain three CDR regions. For example, these typically include amino acid residues near 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and near 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)); and / or amino acid residues from “high-variable rings” (e.g., amino acid residues near 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable region, and near 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable region (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)).

[0033] In this article, the term "humanized antibody" or "humanized antibody" refers to a recombinant antibody obtained by replacing all non-CDR (Fv backbone region (FR)) amino acids in the constant and variable regions of a monoclonal antibody from one species (e.g., mouse) with the same amino acid sequences as those from the constant and variable regions of an antibody from another species (e.g., human). In other words, when the constant region of an antibody is humanized, it is called a chimeric antibody, and when all non-CDR amino acids in both the constant and variable regions are humanized, it is called a humanized antibody. The humanization method can be performed using conventional antibody engineering techniques and will not be elaborated upon here.

[0034] In this article, the term "chimeric antibody" refers to a recombinant antibody obtained by using recombinant DNA technology to replace the amino acid sequence of the constant region of a monoclonal antibody from one species (such as a mouse) with the constant region of an antibody from another species (such as a human).

[0035] In this application, the amino acid sequences of the listed CDRs are all as shown in the IMGT definition rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as the Kabat rule, the Chothia rule, etc. Those skilled in the art should understand that, unless otherwise specified, the terms "CDR" and "complementarity-determining region" for a given antibody or its region (e.g., variable region) should be understood to encompass complementarity-determining regions defined as in any of the aforementioned known schemes described in this invention. Although the scope of protection claimed in this application is based on the sequences shown in the IMGT definition rules, amino acid sequences corresponding to other CDR definition rules should also be included in the scope of protection of this application.

[0036] In this application, the term "amino acid" is represented by a single-letter or three-letter code, with the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Aspartic acid (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: Histidine; I: Ile (isoleucine); L: Leu (leucine); K: Lysine (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Proline (proline); S: Serine (serine); T: Threonine (threonine); W: Tryptophan (tryptophan); Y: Tyrosine (tyrosine); V: Valine (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 percentage of "sequence homology" between a first and a second sequence can be calculated by dividing the number of nucleotides in the first sequence that are identical to those at the corresponding positions by the number of nucleotides in the second sequence. This is calculated by subtracting the total number of nucleotides in the first sequence from the number of nucleotides in the second sequence and then multiplying by 100%, where each deletion, insertion, substitution, or addition of a nucleotide in the second sequence—relative to the first 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 standard settings and known computer algorithms for sequence alignment, such as NCBI Blast v2.0. Other techniques, computer algorithms, and settings used to determine the degree of sequence identity include, for example, those in WO 04 / 037999, EP 0967284, EP1085089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB 2357768-A.

[0038] For peptides, the terms "(substantial) homology," "identity," or "similarity" are used to describe or compare the degree of amino acid similarity between two or more peptides or their designated sequences at optimal alignment and comparison (where appropriate insertions or deletions of nucleotides are made). The percentage of homology between two sequences varies with the number of identical positions shared by these sequences at optimal alignment (i.e., homology % = number of identical positions / total number of positions × 100), where optimal alignment is determined taking into account the number of vacancies introduced to achieve optimal alignment of the two sequences and the length of each vacancy. Sequence comparison and identity percentage determination between two sequences can be performed using mathematical algorithms, as described in the non-limiting examples below.

[0039] In this application, without substantially affecting antibody activity (retaining at least 95% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids to obtain sequences of the antibody or its functional fragments. These are all considered to be included within the scope of protection of this invention. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences described in this application can have at least 80% identity (or homology) with the reference sequences, meaning 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%, or 99.9% identity with each reference sequence. The sequence consistency described in this application can be measured using sequence analysis software. For example, using the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The amino acid sequences described in this invention are shown from the N-terminus to the C-terminus.

[0040] In this document, the term "vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule to host cells and / or between host cells. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The vector also includes vectors having a variety of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, by culturing suitable host cells containing the vector, the vector can produce the desired expression product.

[0041] In this document, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely chopped solid carrier, or both.

[0042] In this article, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0043] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients, except those that are incompatible with the compounds of the present invention, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0044] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The antibodies or antigen-binding fragments, recombinant proteins, multispecific antibodies, conjugates, or pharmaceutical compositions of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the compositions of the present invention are administered via intravenous or subcutaneous injection.

[0045] In this document, the term "treatment" means used to refer to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" covers diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in individuals susceptible to disease but not yet diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0046] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0047] The amino acid sequences involved in this application are shown in Table 1.

[0048] This application discloses a bispecific antibody targeting B7H7 and CD3, a nucleic acid molecule, an expression vector, a preparation method, recombinant cells, a pharmaceutical composition, pharmaceutical uses, preparation kit uses, a kit, a disease treatment method, and a disease treatment use, which will be described in detail below:

[0049] Bispecific antibodies

[0050] In a first aspect, this application provides a bispecific antibody. According to an embodiment of this application, the aforementioned 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 of SEQ ID NO:4-6, 66 or an amino acid sequence having at least 80% homology with SEQ ID NO:4-6, 66; the anti-CD3 antibody further comprises: a light chain complementarity-determining region (LCDR), the LCDR comprising at least one of SEQ ID NO:1-3 or an amino acid sequence having at least 80% homology with SEQ ID NO: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 SEQ ID NO:26-28 or an amino acid sequence having at least 80% homology with SEQ ID NO:26-28. NO:26-28 have amino acid sequences with at least 80% homology; the aforementioned anti-B7H7 antibody further includes: a light chain complementarity-determining region LCDR, wherein the LCDR contains an amino acid sequence selected from at least one of SEQ ID NO:23-25 ​​or having at least 80% homology with SEQ ID NO:23-25.

[0051] In some examples of this application, the HCDRs of the aforementioned anti-CD3 antibodies include: HCDR1, HCDR2, and HCDR3, wherein HCDR1 contains an amino acid sequence as shown in SEQ ID NO:4 or having at least 80% homology with SEQ ID NO:4, HCDR2 contains an amino acid sequence as shown in SEQ ID NO:5 or having at least 80% homology with SEQ ID NO:5, and HCDR3 contains an amino acid sequence as shown in SEQ ID NO:6 and 66 or having at least 80% homology with SEQ ID NO:6 and 66. Anti-CD3 antibodies based on the aforementioned HCDR sequences can bind to CD3 with low affinity and high specificity.

[0052] In some preferred embodiments of this application, the aforementioned HCDR includes HCDR1, HCDR2, and HCDR3, respectively, with amino acid sequences as shown in SEQ ID NO:4-6 and 66. Experimental verification shows that anti-CD3 antibodies possessing the aforementioned HCDR sequences can bind to CD3 with low affinity and high specificity.

[0053] In some examples of this application, the aforementioned anti-CD3 antibody further includes a light chain complementarity-determining region (LCDR), wherein the LCDR includes LCDR1, LCDR2, and LCDR3, wherein LCDR1 contains an amino acid sequence as shown in SEQ ID NO:1 or having at least 80% homology with SEQ ID NO:1, LCDR2 contains an amino acid sequence as shown in SEQ ID NO:2 or having at least 80% homology with SEQ ID NO:2, and LCDR3 contains 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 exhibit high binding affinity and specificity to CD3.

[0054] In some preferred embodiments of this application, the aforementioned LCDR includes LCDR1, LCDR2, and LCDR3, respectively, with amino acid sequences as shown in SEQ ID NO:1–3. Experimental verification has shown that anti-CD3 antibodies possessing the aforementioned LCDR sequences exhibit high 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 may be replaced by other amino acid residues from the same side chain family, and the retained function of the modified antibody can be tested using the functional assay methods described herein. Preferably, the number of amino acid substitutions does not exceed one or two.

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

[0057] In some preferred embodiments of this application, the aforementioned HFRs include HFR1, HFR2, HFR3, and HFR4, respectively, as shown in the amino acid sequences of SEQ ID NO:7–14. The stability of the CD3 bispecific antibody based on the aforementioned frame region sequence is significantly improved.

[0058] In some preferred examples of this application, the aforementioned HFR includes HFR1, HFR2, HFR3, and HFR4, respectively, as shown in the amino acid sequences of SEQ ID NO:11-14. The inventors of this application modified the heavy chain framework region of the anti-CD3 antibody, resulting in a significantly improved stability of the CD3 bispecific antibody based on the aforementioned framework region sequence.

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

[0060] In some preferred embodiments of this application, the aforementioned LFRs include LFR1, LFR2, LFR3, and LFR4, respectively, with amino acid sequences as shown in SEQ ID NO:7–10 and 15–18. The stability of the CD3 bispecific antibody based on the aforementioned framework region sequence is significantly improved.

[0061] In some preferred embodiments of this application, the aforementioned LFRs include LFR1, LFR2, LFR3, and LFR4, respectively, as shown in the amino acid sequences of SEQ ID NO:7-10. The inventors of this application modified the light chain framework region of the anti-CD3 antibody, resulting in a significantly improved stability of the CD3 bispecific antibody based on the aforementioned framework region sequence.

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

[0063] In some preferred embodiments of this application, the amino acid sequences of the heavy chain variable region of the aforementioned anti-CD3 antibody are shown in SEQ ID NO: 38, 40, and 67. Experimental verification has shown that the anti-CD3 antibody based on the aforementioned heavy chain variable region sequence has low affinity, high specificity, and high stability.

[0064] In some preferred embodiments of this application, the amino acid sequence of the heavy chain variable region of the aforementioned anti-CD3 antibody is shown in SEQ ID NO:67. Experimental verification has shown that the anti-CD3 antibody based on the aforementioned heavy chain variable region sequence has higher sequence stability.

[0065] In some preferred embodiments of this 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 having at least 80% homology with SEQ ID NO:37, 39. Anti-CD3 antibodies based on the aforementioned light chain variable region sequence exhibit high affinity, specificity, and stability.

[0066] In some preferred embodiments of this application, the amino acid sequence of the light chain variable region of the aforementioned anti-CD3 antibody is shown in SEQ ID NO:37 and 39. Experimental verification has shown that the anti-CD3 antibody based on the aforementioned light chain variable region sequence has high affinity, specificity, and stability.

[0067] In some preferred embodiments of this application, the amino acid sequence of the light chain variable region of the aforementioned anti-CD3 antibody is shown in SEQ ID NO:37. Experimental verification has shown that the anti-CD3 antibody based on the aforementioned light chain variable region sequence has higher sequence stability.

[0068] In some examples of this application, the HCDRs of the aforementioned anti-B7H7 antibodies include: HCDR1, HCDR2, and HCDR3, wherein HCDR1 contains an amino acid sequence as shown in SEQ ID NO:26 or having at least 80% homology with SEQ ID NO:26, HCDR2 contains an amino acid sequence as shown in SEQ ID NO:27 or having at least 80% homology with SEQ ID NO:27, and HCDR3 contains 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 exhibit high binding affinity and specificity to B7H7.

[0069] In some preferred embodiments of this application, the aforementioned HCDR includes HCDR1, HCDR2, and HCDR3, respectively, with amino acid sequences as shown in SEQ ID NO:26-28. Experimental verification has shown that anti-B7H7 antibodies having amino acid sequences as shown in SEQ ID NO:26-28 exhibit high binding affinity and specificity to B7H7.

[0070] In some examples of this application, the LCDRs of the aforementioned anti-B7H7 antibodies include: LCDR1, LCDR2, and LCDR3, wherein LCDR1 contains an amino acid sequence as shown in SEQ ID NO:23 or having at least 80% homology with SEQ ID NO:23, LCDR2 contains an amino acid sequence as shown in SEQ ID NO:24 or having at least 80% homology with SEQ ID NO:24, and LCDR3 contains 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 exhibit high binding affinity and specificity to B7H7.

[0071] In some preferred embodiments of this application, the aforementioned LCDR includes LCDR1, LCDR2, and LCDR3, respectively, with amino acid sequences as shown in SEQ ID NO:23-25. Experimental verification has shown that anti-B7H7 antibodies having amino acid sequences as shown in SEQ ID NO:23-25 ​​exhibit high binding affinity and specificity to B7H7.

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

[0073] In some preferred embodiments of this application, the aforementioned HFRs include HFR1, HFR2, HFR3, and HFR4, respectively, as shown in the amino acid sequences of SEQ ID NO:33-36. The B7H7 bispecific antibody based on the aforementioned frame region sequence exhibits high sequence stability.

[0074] In some examples of this application, the aforementioned anti-B7H7 antibody further comprises a light chain framework region (LFR), wherein the LFR comprises LFR1, LFR2, LFR3, and LFR4, respectively, having amino acid sequences as shown in SEQ ID NO:29–32; wherein LFR1 comprises an amino acid sequence as shown in SEQ ID NO:29 or having at least 80% homology with SEQ ID NO:29, LFR2 comprises an amino acid sequence as shown in SEQ ID NO:30 or having at least 80% homology with SEQ ID NO:30, LFR3 comprises an amino acid sequence as shown in SEQ ID NO:31 or having at least 80% homology with SEQ ID NO:31, and LFR4 comprises an amino acid sequence as shown in SEQ ID NO:32 or having at least 80% homology with SEQ ID NO:32. The B7H7 bispecific antibody based on the aforementioned framework region sequence exhibits high sequence stability.

[0075] In some preferred embodiments of this application, the aforementioned LFRs include LFR1, LFR2, LFR3, and LFR4, respectively, as shown in the amino acid sequences of SEQ ID NO:29–32. The B7H7 bispecific antibody based on the aforementioned frame region sequence exhibits high sequence stability.

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

[0077] In some preferred embodiments of this application, the amino acid sequence of the heavy chain variable region of the aforementioned anti-B7H7 antibody is shown in SEQ ID NO:42. Experimental verification has shown that the anti-B7H7 antibody based on the aforementioned heavy chain variable region sequence exhibits high affinity, specificity, and stability.

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

[0079] In some preferred embodiments of this application, the amino acid sequence of the light chain variable region of the aforementioned anti-B7H7 antibody is shown in SEQ ID NO:41. Experimental verification has shown that the anti-B7H7 antibody based on the aforementioned light chain variable region sequence exhibits high affinity, specificity, and stability.

[0080] In some examples of this application, the aforementioned first antigen-binding region includes a first anti-CD3 antibody scFv fragment, which includes an anti-CD3 antibody heavy chain variable region, a CD3 antibody light chain variable region, and a linker peptide 1.

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

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

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

[0084] In some examples of this application, the C-terminus of the aforementioned anti-CD3 antibody light chain variable region is connected to the N-terminus of the aforementioned linker peptide 1, the C-terminus of the aforementioned linker 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 this application, the aforementioned second antigen-binding region includes a second anti-B7H7 antibody Fab fragment, which includes an anti-B7H7 antibody heavy chain variable region, a B7H7 antibody light chain variable region, and a linker peptide 1.

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

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

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

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

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

[0091] In a specific example of this application, the composition and conjugation of the aforementioned bispecific antibody are as follows: Figure 1A As shown.

[0092] In some examples of this application, the aforementioned first antigen-binding region includes a first anti-B7H7 antibody Fab fragment and a first anti-CD3 antibody scFv fragment, which are linked by a linker peptide 2. The first anti-B7H7 antibody Fab fragment includes 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 includes an anti-CD3 antibody heavy chain variable region, a CD3 antibody light chain variable region, and a linker peptide 1.

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

[0094] In some examples of this application, the aforementioned first antigen-binding region further includes a first Fc fragment.

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

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

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

[0098] In some examples of this application, the aforementioned second antigen-binding region further includes a second Fc fragment.

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

[0100] In some examples of this application, the aforementioned first Fc segment and the aforementioned second Fc segment are connected by a Knob-in-hole structure.

[0101] In some specific examples of this application, the composition and conjugation of the aforementioned bispecific antibodies are as follows: Figure 1B As shown.

[0102] In some examples of this application, the aforementioned first antigen-binding region includes a first anti-B7H7 antibody Fab fragment and a first anti-CD3 antibody scFv fragment, which are linked by a linker peptide 2. The first anti-B7H7 antibody Fab fragment includes 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 includes an anti-CD3 antibody heavy chain variable region, a CD3 antibody light chain variable region, and a linker peptide 1.

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

[0104] In some examples of this application, the aforementioned first antigen-binding region further includes a first Fc fragment.

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

[0106] In some examples of this application, the aforementioned second antigen-binding region includes a second anti-B7H7 antibody Fab fragment, which includes 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 this application, the C-terminus of the aforementioned second anti-B7H7 antibody heavy chain variable region is connected to the N-terminus of the aforementioned second CH1 fragment, the C-terminus of the aforementioned second anti-B7H7 antibody light chain variable region is connected to the N-terminus of the aforementioned second CL fragment, and the aforementioned second CL fragment and the second CH1 fragment are connected by disulfide bonds.

[0108] In some examples of this application, the aforementioned second antigen-binding region further includes a second Fc fragment.

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

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

[0111] In a specific example of this application, the composition and conjugation of the aforementioned bispecific antibody are as follows: Figure 1C As shown.

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

[0113] In some examples of this application, the aforementioned human antibody IgG1 has an L234A / L235A mutation. Those skilled in the art will understand that humanization of the antibody can improve its bioacceptability.

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

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

[0116] In some examples of this application, the first antigen-binding region of the aforementioned bispecific antibody has the amino acid sequence shown in SEQ ID NO:47, and the second antigen-binding region of the aforementioned bispecific antibody has the amino acid sequence shown in SEQ ID NO:48 (BB1-T7-Z13). Figure 1A Experimental results show that the aforementioned bispecific antibodies 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, exhibiting excellent anticancer activity.

[0117] In some examples of this application, the first antigen-binding region of the aforementioned bispecific antibody has the amino acid sequence shown in SEQ ID NO:51 and SEQ ID NO:53, and the second antigen-binding region of the aforementioned bispecific antibody has the amino acid sequence shown in SEQ ID NO:52 and SEQ ID NO:53 (BB3-T7-Z13, Figure 1B Experimental results show that the aforementioned bispecific antibody sequences have better stability, 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, exhibiting excellent anticancer activity and good therapeutic safety.

[0118] In some examples of this application, the first antigen-binding region of the aforementioned bispecific antibody has the amino acid sequence shown in SEQ ID NO:68 and SEQ ID NO:53, and the second antigen-binding region of the bispecific antibody has the amino acid sequence shown in SEQ ID NO:52 and SEQ ID NO:53 (BB3-T8-Z13). Figure 1B Experimental results show that the aforementioned bispecific antibody sequences have better stability, 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, exhibiting excellent anticancer activity and excellent therapeutic safety.

[0119] In some examples of this application, the first antigen-binding region of the aforementioned bispecific antibody has the amino acid sequence shown in SEQ ID NO:57 and SEQ ID NO:59, and the second antigen-binding region of the aforementioned bispecific antibody has the amino acid sequence shown in SEQ ID NO:58 and SEQ ID NO:59 (BB3-Tma-Z13, Figure 1C Experimental results show that the aforementioned bispecific antibodies 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, exhibiting good anti-cancer activity.

[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 surface of tumor cells. By inducing simultaneous binding of T cells and tumor cells, the bispecific antibody promotes the formation of synapses between the two cells and thus selectively redirects T cell activity to target tumor cells, thereby achieving the killing of tumor cells.

[0121] Nucleic acid molecules

[0122] In another aspect of this application, a nucleic acid molecule is proposed that encodes the aforementioned bispecific antibody.

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

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

[0125] The nucleic acid molecules obtained in the above manner can encode bispecific antibodies targeting B7H7 and CD3.

[0126] expression carrier

[0127] In another aspect of this application, an expression vector is proposed. According to an embodiment of this application, the aforementioned expression vector carries the aforementioned nucleic acid molecule. The aforementioned expression vector includes an optional controlled amino acid sequence, such as the aforementioned controlled amino acid sequence being operatively linked to the aforementioned nucleic acid molecule. The aforementioned controlled amino acid sequence can direct one or more controlled amino acid sequences of the aforementioned nucleic acid molecule to be expressed in a host. The vector thus constructed can effectively express the aforementioned bispecific antibody.

[0128] It should be noted that when linking the aforementioned nucleic acid molecules to the vector, the nucleic acid molecules and the control elements on the vector can be directly or indirectly connected, as long as these control elements can control the translation and expression of the aforementioned nucleic acid molecules. These control elements can originate directly from the vector itself or be exogenous, i.e., not derived from the vector itself. Of course, the connection between the aforementioned nucleic acid molecules and the control elements must be operably established.

[0129] According to embodiments of the present invention, the aforementioned vector may refer to a cloning vector or an expression vector, which can be obtained by operatively ligating the aforementioned nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vectors used in this invention are not particularly limited; commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used.

[0130] In this document, the term "operably ligated" refers to ligating a foreign gene to a vector such that the control elements within the vector, such as amino acid sequences controlling transcription and amino acid sequences controlling translation, can perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors include viral vectors, plasmids, bacteriophages, etc. After the expression vectors of some specific embodiments of the present invention are introduced into suitable recipient cells, the expression of the aforementioned nucleic acid molecules can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro production of proteins encoded by the aforementioned nucleic acid molecules.

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

[0132] In some examples of this application, the aforementioned vector includes at least one selected from plasmid vectors, adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors.

[0133] Bispecific antibody preparation method

[0134] In another aspect of this application, a method for preparing the aforementioned bispecific antibody is proposed, comprising: introducing the aforementioned expression vector into cells; culturing the aforementioned cells under conditions suitable for protein expression and secretion to obtain the aforementioned bispecific antibody. This method enables the large-scale expression of the aforementioned bispecific antibody in vitro.

[0135] In some examples of this application, the aforementioned cells are eukaryotic cells.

[0136] Recombinant cells

[0137] In another aspect of this application, a recombinant cell carrying the aforementioned nucleic acid molecule or expression vector is proposed. In some examples of this 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 this application, the aforementioned recombinant cells are prokaryotic cells, eukaryotic cells, or bacteriophages. It should be noted that the aforementioned eukaryotic cells do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0139] In some examples of this application, the aforementioned prokaryotic cells are Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.

[0140] In some examples of this application, the aforementioned eukaryotic cells are fungal, insect, plant, or mammalian cells.

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

[0142] In some examples of this application, the aforementioned insect cells are grass armyworm cells; in some examples of this application, the aforementioned plant cells are tobacco plant cells; in some examples of this application, the aforementioned mammalian cells are BHK cells, CHO cells, COS cells, myeloma cells, or human embryonic kidney 293 cells; and do not include animal germ cells, fertilized eggs, or embryonic stem cells.

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

[0144] In some examples of this application, the aforementioned recombinant cells are BHK cells, CHO cells, COS cells, or NSO cells.

[0145] It should be noted that the "suitable conditions" mentioned above in this application refer to conditions suitable for the expression of the bispecific antibody of this application. Those skilled in the art will readily understand that suitable conditions for bispecific antibody expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell state, suitable host cell density, suitable cell culture environment, and suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for bispecific antibody expression based on the specific environment of their laboratory.

[0146] Pharmaceutical Composition

[0147] In another aspect of this application, a pharmaceutical composition is proposed, comprising: the aforementioned bispecific antibody, nucleic acid molecule, expression vector, or recombinant cells. The aforementioned pharmaceutical composition can effectively promote PBMC killing of tumor cells, exhibiting good anti-cancer activity and higher safety; it can also reduce the risk of cytokine storm induced by CD3 bispecific antibodies.

[0148] In some examples of this application, pharmaceutically acceptable excipients are further included.

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

[0150] In some examples of this application, the aforementioned pharmaceutical composition is an injectable formulation.

[0151] It should be noted that the aforementioned pharmaceutical compositions include combinations that are separate in time and / or space, as long as they can work together to achieve the objectives of the present invention. For example, the components contained in the aforementioned pharmaceutical compositions may be administered to the subject as a whole or separately. When the components contained in the aforementioned pharmaceutical compositions are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

[0152] The drug of this application contains a safe and effective amount of the active ingredient (bispecific antibody) of this application, as well as pharmaceutically acceptable excipients. These excipients include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be matched to the route of administration; the dosage form of the drug of this application is an injection, an oral formulation (tablet, capsule, oral liquid), a transdermal formulation, or a sustained-release formulation. For example, it may be prepared using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The aforementioned drug is preferably manufactured under aseptic conditions.

[0153] The effective amount of the active ingredient described above may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, immune status, and route of administration. For example, due to the urgency of the treatment, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0154] The pharmaceutically acceptable excipients mentioned above in this application include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be compatible with the route of administration, as is well known to those skilled in the art.

[0155] Uses in drug preparation

[0156] In another aspect of this application, the use of the aforementioned bispecific antibody, nucleic acid molecule, expression vector, recombinant cell, or pharmaceutical composition in the preparation of a drug for the treatment or prevention of tumors is proposed. Based on the aforementioned bispecific antibody and corresponding nucleic acid molecule, vector, recombinant cell, or pharmaceutical composition, a drug can be further prepared, which can be clinically used to prevent or treat diseases B7H7 and / or CD3-mediated diseases.

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

[0158] Uses in the preparation kit

[0159] In another aspect of this application, the 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 the detection of B7H7 and / or CD3.

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

[0161] Reagent test kit

[0162] In another aspect, this application provides a kit comprising the aforementioned bispecific antibody. The kit enables portable and accurate detection of B7H7 and CD3 proteins in a sample.

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

[0164] Disease treatment methods

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

[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 implementation, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infections, etc. Subjects can be humans, but also include other mammals, particularly mammals that can be used as laboratory models of human diseases, such as mice, rats, etc.

[0167] The effective amount of the aforementioned bispecific antibody, nucleic acid molecule, vector or transformant, recombinant cell, or pharmaceutical composition may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: the pharmacokinetic parameters of the aforementioned active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

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

[0169] Uses in disease treatment

[0170] In another aspect of this application, the use of the aforementioned bispecific antibodies, nucleic acid molecules, expression vectors, recombinant cells, or pharmaceutical compositions in the treatment or prevention of disease is proposed. In some examples of this application, 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 disease.

[0171] In some examples of this 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 this application.

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0186] Example 1: Design and Construction of a Bispecific Antibody Targeting CD3-B7H7

[0187] To better compare the functions of CD3-B7H7 bispecific antibodies with different conformations and sequences, this application designed three CD3-B7H7 bispecific antibodies with different conformations. The first antibody was named BB1-T7-Z13. Figure 1A This antibody 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 1B This antibody 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 This antibody contains three monovalent units, one of which is an anti-CD3 scFv form, and the other two are anti-B7H7 Fab forms. The antibody conformation is similar to BB3-T7-Z13, but the CD3 variable region sequence is different. The heavy chain constant regions of the first and second polypeptide chains of both antibodies are derived from the human antibody IgG1. Due to the molecule's unique asymmetric structure, to reduce the formation of homodimers, different amino acid mutations were introduced into the constant regions of the first and second polypeptide chains, forming a knock-in-hole structure. Simultaneously, to prevent cross-linking activation caused by the Fcγ receptor, (L234A / L235A) mutations were also introduced into the constant regions of the first and second polypeptide chains.

[0188] Using molecular biology techniques, the polypeptide chains were cloned into the expression vector pcDNA3.4. To enable efficient expression and secretion into the culture medium in CHO cells, the leader peptide of the mouse antibody heavy chain was selected as the secretion signal peptide and inserted into the expression vector. The signal peptide (SEQ ID NO:65) is located at the N-terminus of the antibody variable region.

[0189] Example 2: Expression and purification of CD3-B7H7 bispecific antibody

[0190] A bispecific molecule binding to CD3 and B7H7 was prepared by transiently transfecting ExpiCHO-S cells (Gibco, catalog number A29127) with the pcDNA3.4 vector carrying the CD3-B7H7 chain encoding gene. One day prior to transfection, the ExpiCHO-S cells were adjusted to a cell density of (3-4) × 10⁻⁶ cells. 6 Incubate overnight at 37°C, 8% CO2, with shaking at 120 rpm, using a 1 / ml incubator. On the day of transfection, cells grew to 7 × 10⁹ cells / ml. 6 –1×10 7 When the cell viability is greater than 95%, prepare for transfection by diluting the cells to 6 × 10⁶ / ml using fresh, pre-warmed ExpiCHO medium (Gibco, catalog number A2910002). 6 / ml, then take plasmids containing polypeptide chains and transfect them into ExpiCHO-S cells at a mass ratio of 1:1 (BB1-T7-Z13) or 1:1:2 (BB3-T7-Z13 or BB3-Tma-Z13) using ExpiFectamine CHO transfection reagent (Gibco, catalog number A29129). Incubate at 37℃, 8% CO2, and 120 rpm with shaking. 18-22 h post-transfection, immediately add ExpiFectamine CHO Enhancer and ExpiCHO Feed to the transfected cells, mix well, and incubate at 32℃, 5% CO2, and 120 rpm with shaking. On day 5 post-transfection, add another 8 ml of ExpiCHO Feed to the cells, mix well, and continue culturing. Observe cell count and cell viability changes daily. When cell viability drops below 80% or after 10-14 days of culture, centrifuge to harvest cells, collect the supernatant for purification, or freeze at -80℃.

[0191] The expression supernatant was filtered through a 0.22 μm filter membrane. Antibodies with Fc domains were captured from the expression supernatant using a Mabselect Prism A affinity chromatography column (Cytiva, catalog number 17549854). After equilibrating the column with phosphate buffer at pH 7.2, the supernatant was passed through the affinity chromatography column and eluted with elution buffer (100 mM citric acid, pH 2.7). Finally, the elution was concentrated and replaced with PBS buffer. The purified antibody was identified by SDS-PAGE and its purity was above 90%, thus obtaining the CD3-B7H7 bispecific binding antibody.

[0192] Example 3: Flow cytometry assay using a CD3-B7H7 bispecific antibody.

[0193] Test Example 1: Bispecific antibody binding to human T cells

[0194] Human T cells were adjusted to a cell density of 5 × 10⁶. 5Add 100 μL of bispecific antibody to each 1.5 mL EP tube, 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. Add 100 μL of goat anti-human secondary antibody (Jackson ImmunoResearch, 109-606-170) to each well, mix well, and incubate at 4 °C for 30 min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200 μL of PBS per tube and analyze using flow cytometry. Figure 2A The results showed that the bispecific antibody BB1-T7-Z13 had the strongest binding ability to T cells, followed by BB3-T7-Z13, while BB3-Tma-Z13 had the weakest ability.

[0195] Test Example 2: Bispecific antibody binding to HCC827 tumor cells

[0196] HCC827 non-small cell lung cancer cells were diluted to 2 × 10⁻⁶ with PBS. 6 / mL, add 100μL / tube to a 1.5ml EP tube, add 10μL / tube of goat serum, and block at 4℃ for 30min. Add gradient concentrations of bispecific antibody and incubate at 4℃ for 30min. Add 1mL of PBS to the EP tube, centrifuge at 3500rpm×5min at 4℃, discard the supernatant, and wash once with PBS. After centrifugation, discard the supernatant, resuspend the cells in 100μL / tube of PBS, add 0.1μL / tube of Alexa-647-labeled goat anti-human antibody secondary antibody (Jackson ImmunoResearch, 109-606-170), and incubate at 4℃ in the dark for 30min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in 200μL / tube of PBS and analyze using flow cytometry. Figure 2B The results showed that all three bispecific antibodies could bind to B7H7+ HCC827 cells, and the EC50 values ​​of the three were similar, while the MFI peak of BB1-T7-Z13 was higher.

[0197] Example 4: Assay of the binding activity of CD3-B7H7 bispecific antibody to two antigens (ELISA)

[0198] To test whether the bispecific antibody could simultaneously bind to both CD3 and B7H7 antigens to achieve bridging, human CD3 antigen (ACRO biosystems, catalog number CDD-H52W1) was diluted to 1 μg / mL with coating buffer (35 mM NaHCO3, 15 mM Na2CO3, pH 9.6), and 100 μL was added to each well of an ELISA plate and incubated overnight at 4°C. The plate was then washed three times with PBST (0.05% Tween 20-PBS, pH 7.2). 300 μL of blocking buffer (1% BSA, 0.05% Tween 20-PBS, pH 7.2) was added to the plate and incubated at room temperature for 2 hours. The plate was then washed three more times with PBST. Different concentrations of bispecific antibody were added to each well and incubated at room temperature for 1 hour. The plate was then washed three more times with PBST. Add 100 μL of biotinylated human B7H7 antigen (ACRO biosystems, catalog number B77-H82E3) diluted with blocking buffer to each well and incubate at room temperature for 1 hour. Wash three times with PBST. Add 100 μL of HRP-labeled streptavidin secondary antibody (SouthernBiotech, catalog number 7105-05) diluted with blocking buffer to each well and incubate at room temperature for 1 hour. Wash three times with PBST, add TMB to each well, and incubate at room temperature in the dark for 2-5 minutes. Terminate the reaction with 2M sulfuric acid in each well. Finally, read the OD450 value using a microplate reader. Specific results are as follows: Figure 3 As shown, the results indicate that the OD450 reading gradually increases with the increase of the concentration of bispecific antibody molecules, indicating that all three bispecific antibodies can bind to human CD3 and B7H7 simultaneously, while BB1-T7-Z13 has a slightly stronger binding ability.

[0199] Example 5: Validation of the affinity of the CD3-B7H7 bispecific antibody

[0200] The Biacore 1K platform was used to test the kinetics and affinity properties of the antibodies. Following the system instructions, the antibodies were diluted to 1 μg / mL with run buffer (HBS-EP) and conjugated to the protein A chip at a flow rate of 10 μL / min. The kinetics and affinity data of antigen-antibody binding were then measured at a flow rate of 30 μL / min, with a binding time of 90 s and a dissociation time of 300 s. The results are shown in Table 2, indicating that all three bispecific antibodies can bind to human B7H7 and monkey B7H7, with a significantly higher affinity for B7H7 than for CD3.

[0201] Table 2

[0202]

[0203]

[0204] Example 6: In vitro killing assay of CD3-B7H7 bispecific binding antibody

[0205] Adherent tumor cells were digested, counted, and the cell density was adjusted to 2 × 10⁻⁶. 5 / ml. Turn on the RTCA instrument (Agilent), select the experimental mode, and fill in the cell and drug information. Enter the schedule to set the experimental steps. Add 50μL of fresh culture medium (89% RPMI 1640 medium + 10% fetal bovine serum + 1% penicillin-streptomycin) to the plate, place it in the instrument, close it, and click "Start" (Step 1). After completion, remove the plate, add 100μL of cell suspension, incubate at room temperature for 15-30 minutes to prevent edge effects, place it in the instrument, and click "Start". Once growth reaches the logarithmic growth phase, pause the process and add 50μL of LPBMC (1×10⁻⁶). 6 Add the sample ( / ml) and a gradient concentration of bispecific antibody, click Start, and analyze after a period of time.

[0206] With increasing bispecific antibody concentration, PBMCs showed improved activity against non-small cell lung cancer HCC827 cells (…). Figure 4A ), lung adenocarcinoma NCI-H820 cells ( Figure 4B ) and colorectal cancer LS180 ( Figure 4C The killing efficiency of BB1-T7-Z13 and BB3-Tma-Z13 gradually increased, and the killing ability of BB1-T7-Z13 and BB3-Tma-Z13 was significantly higher than that of BB3-T7-Z13 in HCC827 and NCI-H820. In LS180 cells, BB3-Tma-Z13 had the strongest killing activity and BB3-T7-Z13 had the weakest killing activity.

[0207] Example 7: Effect of different B7H7 expression levels on bispecific antibody activity

[0208] To further analyze the effect of different B7H7 expression levels on the activity of three bispecific antibodies, different levels of B7H7 molecules were overexpressed on the surface of colorectal cancer HCT-15 cells. Using the killing method shown in Example 6, Figure 5A The results showed that in HCT-15 cells with high B7H7 expression, BB1-T7-Z13 had the strongest tumor cell killing effect, followed by BB3-Tma-Z13, and BB3-T7-Z13 had the weakest effect. Figure 5B The study showed that in HCT-15 cells with low B7H7 expression, the three drugs exhibited little difference in their cytotoxic abilities. Figure 5A and Figure 5B As a result, the EC50 of the same antibody varied greatly at different B7H7 expression levels, indicating that the activity of bispecific antibodies depends on the B7H7 expression level on the surface of tumor cells.

[0209] Example 8 Cytokine Secretion Detection

[0210] Test Example 1: Effect of CD3-B7H7 on PBMC cytokine secretion in the absence of target cells

[0211] Take 1×10 5 One 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.

[0212] Figure 6A -D shows 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.

[0213] Test Example 2: Effect of CD3-B7H7 on PBMC cytokine secretion in the presence of target cells

[0214] 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.

[0215] Figure 7A -D shows the concentrations of IL-2, IL-6, IFN-γ, and TNF cytokines in the supernatant after 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.

[0216] Example 9: Study on the efficacy of in vivo xenotransplantation

[0217] 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 Il15 em1(hIL15)Smoc Mice, 4 days later, HCT-15-B7H7 cells (4×10⁻⁶) were injected. 6 Subcutaneous injection (sc) was administered to mice until the tumor grew to 50-110 mm. 3 Mice were then grouped and subsequently administered intravenous injections twice weekly of different doses of the CD3-B7H7 bispecific antibody (0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg) or control PBS solution for a total of three injections. Tumor length and width were measured using external calipers, and tumor volume was calculated using a standard formula.

[0218] Figure 8A -C demonstrates the in vivo antitumor activity of the CD3-B7H7 bispecific binding antibody against HCT-15-B7H7 cells. Figure 8A The results showed that treatment with the BB1-T7-Z13 bispecific antibody could significantly inhibit tumor growth, and complete tumor clearance could be achieved even at a dose of 0.03 mg / kg. Figure 8B The results showed that BB3-T7-Z13 also had strong anti-tumor activity, achieving complete tumor clearance at 0.1 mg / kg and significant tumor inhibition at 0.03 mg / kg. Figure 8C The results showed that BB3-Tma-Z13 had relatively weak anti-tumor activity, while previous examples showed that the antibody had strong in vitro killing activity. The reason for this difference may be that the CD3 antibody can lead to T cell exhaustion or that the CD3 sequence Tma has poor serum stability.

[0219] Examples 8 and 9 suggest that the CD3 variable region sequence of T7 has better biological activity and lower potential toxicity, while the BB3 bispecific antibody configuration paired with the T7 CD3 variable region sequence (BB3-T7-Z13) has the best potential safety.

[0220] Example 10: Safety Optimization of Bispecific Antibody

[0221] Further research revealed that excessively high CD3 affinity in bispecific antibodies leads to a higher incidence of cytokine storm (CRS) and lower safety. BB3-T7-Z13 was found to cause a severe cytokine storm in cynomolgus monkeys at 0.1 mg / kg. Therefore, to further improve the therapeutic window of bispecific antibodies, the CD3 antibody affinity was further reduced to enhance potential safety. This application utilizes the second antibody configuration mentioned in Example 1 (…). Figure 1B The CD3 sequence was replaced with an affinity-optimized sequence, and the constructed molecule was named BB3-T8-Z13. The bispecific antibody was expressed and purified using the method described in Example 2, ultimately yielding the CD3-B7H7 bispecific antibody BB3-T8-Z13.

[0222] The affinity of the BB3-T8-Z13 antibody was determined using the SPR method described in Example 5. The results are shown in Table 3, indicating that the CD3 affinity is only 1.14E-6M, which means that the CD3 affinity in BB3-T8-Z13 is lower than that in BB3-T7-Z13.

[0223] Table 2

[0224]

[0225] Testing in vitro cytokine release is a common method for predicting potential safety. Using the method in Example 8, PBMCs were co-incubated with colorectal cancer HCT-15-B7H7 cells, and the cytokine release of BB3-T7-Z13 and BB3-T8-Z13 cells was compared. Figure 9A -C shows the concentrations of IL-2, IL-6, and TNF cytokines in the supernatant after co-incubation with HCT-15-B7H7 colorectal cancer cells. With increasing CD3-B7H7 antibody concentration, the release level of BB3-T8-Z13, a low-affinity CD3 cytokine, was significantly lower than that of BB3-T7-Z13, suggesting that BB3-T8-Z13 has potentially higher safety. Furthermore, BB3-T8-Z13 was found to have no significant clinical toxicity in cynomolgus monkeys at 0.3 mg / kg.

[0226] BB3-T8-Z13 was tested using the in vivo pharmacodynamic evaluation method described in Example 9. Figure 10 The results showed that complete tumor clearance could be achieved at 0.3 mg / kg, while significant tumor inhibition could be achieved at 0.1 mg / kg. These results collectively suggest that BB3-T8-Z13 possesses good antitumor activity and better safety, with a wider clinical therapeutic window.

[0227] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

[0228] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0229] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A bispecific antibody, characterized in that, include: The first antigen-binding region has CD3 binding activity; and The second antigen-binding region has B7H7 binding activity; Wherein, the first antigen-binding region includes an anti-CD3 antibody, the anti-CD3 antibody includes a heavy chain complementarity-determining region (HCDR), and the HCDR contains at least one of SEQ ID NO:4-6, 66 or an amino acid sequence having at least 80% homology with SEQ ID NO:4-6, 66; The anti-CD3 antibody further comprises: a light chain complementarity-determining region (LCDR), wherein the LCDR contains an amino acid sequence selected from at least one of SEQ ID NO:1-3 or having at least 80% homology with SEQ ID NO:1-3; The second antigen-binding region includes an anti-B7H7 antibody, the anti-B7H7 antibody including a heavy chain complementarity-determining region (HCDR), the HCDR of the anti-B7H7 antibody containing at least one of SEQ ID NO:26-28 or an amino acid sequence having at least 80% homology with SEQ ID NO:26-28; The anti-B7H7 antibody further comprises a light chain complementarity-determining region (LCDR), wherein the LCDR contains at least one of SEQ ID NO:23-25 ​​or an amino acid sequence having at least 80% homology with SEQ ID NO:23-25.

2. The bispecific antibody according to claim 1, characterized in that, The HCDRs of the anti-CD3 antibody include: HCDR1, HCDR2, and HCDR3, wherein, The HCDR1 contains an amino acid sequence as shown in SEQ ID NO:4 or having at least 80% homology with SEQ ID NO:

4. The HCDR2 contains an amino acid sequence as shown in SEQ ID NO:5 or having at least 80% homology with SEQ ID NO:

5. The HCDR3 comprises an amino acid sequence as shown in SEQ ID NO:6, 66 or having at least 80% homology with SEQ ID NO:6, 66; Preferably, the HCDR comprises: HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:4-6 and 66, respectively; Optionally, the LCDR includes: LCDR1, LCDR2, and LCDR3, wherein, The 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 LCDR2 comprises an amino acid sequence as shown in SEQ ID NO:2 or having at least 80% homology with SEQ ID NO:

2. The LCDR3 comprises an amino acid sequence as shown in SEQ ID NO:3 or having at least 80% homology with SEQ ID NO:3; Preferably, the LCDR comprises: LCDR1, LCDR2 and LCDR3, respectively, having amino acid sequences as shown in SEQ ID NO:1 to 3; Optionally, the anti-CD3 antibody further comprises a heavy chain framework region (HFR), wherein the HFR comprises HFR1, HFR2, HFR3, and HFR4, respectively, as shown in the amino acid sequences of SEQ ID NO: 11–14 and 19–22; wherein, The 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 HFR2 contains 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 HFR3 contains an amino acid sequence as shown in SEQ ID NO:13, 21 or having 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 having at least 80% homology with SEQ ID NO:14, 22; Preferably, the HFR includes HFR1, HFR2, HFR3 and HFR4, respectively, as shown in the amino acid sequences of SEQ ID NO:7-14; More preferably, the HFR includes HFR1, HFR2, HFR3 and HFR4, respectively, as shown in the amino acid sequences of SEQ ID NO:11-14; Optionally, the anti-CD3 antibody further comprises: a light chain framework region (LFR), wherein the LFR comprises: LFR1, LFR2, LFR3, and LFR4, respectively, as shown in SEQ ID NO:7–10 and 15–18; wherein, The LFR1 comprises an amino acid sequence as shown in SEQ ID NO:7, 15 or having at least 80% homology with SEQ ID NO:7, 15. The LFR2 contains an amino acid sequence as shown in SEQ ID NO:8, 16 or having at least 80% homology with SEQ ID NO:8, 16. The LFR3 contains an amino acid sequence as shown in SEQ ID NO:9, 17 or having 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 having at least 80% homology with SEQ ID NO:10, 18; Preferably, the LFR includes LFR1, LFR2, LFR3 and LFR4, respectively, with amino acid sequences as shown in SEQ ID NO:7-10 and 15-18; More preferably, the LFR includes LFR1, LFR2, LFR3 and LFR4, respectively, as shown in the amino acid sequences of SEQ ID NO:7 to 10; Optionally, the heavy chain variable region of the anti-CD3 antibody comprises an amino acid sequence selected from or 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 shown 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 shown in SEQ ID NO:

67. Preferably, the light chain variable region of the anti-CD3 antibody comprises an amino acid sequence as shown in SEQ ID NO:37, 39 or 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 shown 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 shown in SEQ ID NO:

37.

3. The bispecific antibody according to claim 1, characterized in that, The HCDRs of the anti-B7H7 antibody include: HCDR1, HCDR2, and HCDR3, wherein, The HCDR1 contains an amino acid sequence as shown in SEQ ID NO:26 or having at least 80% homology with SEQ ID NO:

26. The HCDR2 contains an amino acid sequence as shown in SEQ ID NO:27 or having at least 80% homology with SEQ ID NO:

27. The HCDR3 contains an amino acid sequence as shown in SEQ ID NO:28 or having at least 80% homology with SEQ ID NO:28; Preferably, the HCDR comprises: HCDR1, HCDR2 and HCDR3, respectively, as shown in the amino acid sequences of SEQ ID NO:26-28; Preferably, the LCDRs of the anti-B7H7 antibody include: LCDR1, LCDR2, and LCDR3, wherein, The 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 LCDR2 comprises an amino acid sequence as shown in SEQ ID NO:24 or having at least 80% homology with SEQ ID NO:

24. The LCDR3 comprises an amino acid sequence as shown in SEQ ID NO:25 or having at least 80% homology with SEQ ID NO:25; Preferably, the LCDR comprises: LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:23-25, respectively; Optionally, the anti-B7H7 antibody further comprises a heavy chain framework region (HFR), wherein the HFR comprises HFR1, HFR2, HFR3, and HFR4, respectively, as shown in the amino acid sequences of SEQ ID NO: 33–36; wherein, The HFR1 comprises an amino acid sequence as shown in SEQ ID NO:33 or having at least 80% homology with SEQ ID NO:

33. The HFR2 contains an amino acid sequence as shown in SEQ ID NO:34 or having at least 80% homology with SEQ ID NO:

34. The HFR3 contains an amino acid sequence as shown in SEQ ID NO:35 or having at least 80% homology with SEQ ID NO:

35. The HFR4 comprises an amino acid sequence as shown in SEQ ID NO:36 or having at least 80% homology with SEQ ID NO:36; Preferably, the HFR includes HFR1, HFR2, HFR3 and HFR4, respectively, as shown in the amino acid sequences of SEQ ID NO:33-36; Optionally, the anti-B7H7 antibody further comprises: a light chain framework region (LFR), wherein the LFR comprises: LFR1, LFR2, LFR3, and LFR4, respectively, as shown in SEQ ID NO: 29–32; wherein, The LFR1 contains an amino acid sequence as shown in SEQ ID NO:29 or having at least 80% homology with SEQ ID NO:

29. The LFR2 contains an amino acid sequence as shown in SEQ ID NO:30 or having at least 80% homology with SEQ ID NO:

30. The LFR3 contains an amino acid sequence as shown in SEQ ID NO:31 or having at least 80% homology with SEQ ID NO:

31. The LFR4 contains an amino acid sequence as shown in SEQ ID NO:32 or having at least 80% homology with SEQ ID NO:32; Preferably, the LFR includes LFR1, LFR2, LFR3 and LFR4, respectively, as shown in the amino acid sequences of SEQ ID NO:29-32; Preferably, the heavy chain variable region of the anti-B7H7 antibody comprises an amino acid sequence selected from or having 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 shown in SEQ ID NO:

42. Preferably, the light chain variable region of the anti-B7H7 antibody comprises an amino acid sequence as shown in SEQ ID NO:41 or having 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 shown in SEQ ID NO:

41.

4. The bispecific antibody according to claim 1, characterized in that, The first antigen-binding region includes a first anti-CD3 antibody scFv fragment, which includes an anti-CD3 antibody heavy chain variable region, a CD3 antibody light chain variable region, and a linker peptide 1. Optionally, the C-terminus of the variable region of the anti-CD3 antibody light chain is connected to the N-terminus of the linker peptide 1, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the variable region of the anti-CD3 antibody heavy chain. Optionally, the first antigen-binding region further includes a first Fc fragment; Optionally, the C-terminus of the variable region of the anti-CD3 antibody light chain is connected to the N-terminus of the linker peptide 1, the C-terminus of the linker peptide 1 is connected to the N-terminus of the variable region of the anti-CD3 antibody heavy chain, and the C-terminus of the variable region of the anti-CD3 antibody heavy chain is connected to the N-terminus of the first Fc fragment. Optionally, the second antigen-binding region includes a second anti-B7H7 antibody Fab fragment, the second anti-B7H7 antibody Fab fragment including an anti-B7H7 antibody heavy chain variable region, a B7H7 antibody light chain variable region and a linker peptide 1; Optionally, the C-terminus of the variable region of the light chain of the anti-B7H7 antibody is connected to the N-terminus of the linker peptide 1, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the variable region of the heavy chain of the anti-B7H7 antibody. Optionally, the second antigen-binding region further includes a second Fc fragment; Optionally, the C-terminus of the variable region of the light chain of the anti-B7H7 antibody is connected to the N-terminus of the linker peptide 1, the C-terminus of the linker peptide 1 is connected to the N-terminus of the variable region of the heavy chain of the anti-B7H7 antibody, and the C-terminus of the variable region of the heavy chain of the anti-B7H7 antibody is connected to the N-terminus of the second Fc fragment. Optionally, the linker peptide 1 has an amino acid sequence as shown in SEQ ID NO:

63.

5. The bispecific antibody according to claim 1, characterized in that, The first antigen-binding region includes a first anti-B7H7 antibody Fab fragment and a first anti-CD3 antibody scFv fragment, which are linked by a linker peptide 2. The first anti-B7H7 antibody Fab fragment includes 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 includes an anti-CD3 antibody heavy chain variable region, a CD3 antibody light chain variable region, and a linker peptide 1. Optionally, the C-terminus of the variable region of the heavy chain of the first anti-B7H7 antibody is connected to the N-terminus of the first CH1 fragment, the C-terminus of the variable region of the light chain of the first anti-B7H7 antibody 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 linker peptide 2, the C-terminus of the linker peptide 2 is connected to the N-terminus of the variable region of the light chain of the first anti-CD3 antibody, the C-terminus of the variable region of the light chain of the first anti-CD3 antibody is connected to the N-terminus of the linker peptide 1, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the variable region of the heavy chain of the first anti-CD3 antibody. Optionally, the C-terminus of the variable region of the heavy chain of the first anti-B7H7 antibody is connected to the N-terminus of the first CH1 fragment, the C-terminus of the variable region of the light chain of the first anti-B7H7 antibody 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 linker peptide 2, the C-terminus of the linker peptide 2 is connected to the N-terminus of the variable region of the heavy chain of the first anti-CD3 antibody, the C-terminus of the variable region of the heavy chain of the first anti-CD3 antibody is connected to the N-terminus of the linker peptide 1, and the C-terminus of the linker peptide 1 is connected to the N-terminus of the variable region of the light chain of the first anti-CD3 antibody. Optionally, the first antigen-binding region further includes a first Fc fragment; Optionally, the first anti-CD3 antibody scFv fragment is linked to the first Fc fragment; Optionally, the linker peptide 2 has an amino acid sequence as shown in SEQ ID NO:

64.

6. The bispecific antibody according to claim 5, characterized in that, The second antigen-binding region includes a second anti-B7H7 antibody Fab fragment, which includes an anti-B7H7 antibody heavy chain variable region, a B7H7 antibody light chain variable region, a second CL fragment, and a second CH1 fragment. Optionally, the C-terminus of the variable region of the heavy chain of the second anti-B7H7 antibody is connected to the N-terminus of the second CH1 fragment, the C-terminus of the variable region of the light chain of the second anti-B7H7 antibody is connected to the N-terminus of the second CL fragment, and the second CL fragment and the second CH1 fragment are connected by disulfide bonds. Optionally, the second antigen-binding region further includes a second Fc fragment; Optionally, the second anti-B7H7 antibody Fab fragment is linked to the second Fc fragment; Optionally, the linker peptide 2 has an amino acid sequence as shown 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 each independently selected from human antibody IgG1; Preferably, the human antibody IgG1 has an L234A / L235A mutation; Optionally, the first Fc segment and the second Fc segment are connected via a Knob-in-hole structure.

8. The bispecific antibody according to claim 1, characterized in that, The first antigen-binding region of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO:47, and the second antigen-binding region of the bispecific antibody has an amino acid sequence as shown in SEQ ID NO:

48. Optionally, the first antigen-binding region of the bispecific antibody has the amino acid sequences shown in SEQ ID NO:51 and SEQ ID NO:53, and the second antigen-binding region of the bispecific antibody has the amino acid sequences shown in SEQ ID NO:52 and SEQ ID NO:

53. Optionally, the first antigen-binding region of the bispecific antibody has the amino acid sequences shown in SEQ ID NO:68 and SEQ ID NO:53, and the second antigen-binding region of the bispecific antibody has the amino acid sequences shown in SEQ ID NO:52 and SEQ ID NO:

53. Optionally, the first antigen-binding region of the bispecific antibody has the amino acid sequences shown in SEQ ID NO:57 and SEQ ID NO:59, and the second antigen-binding region of the bispecific antibody has the amino acid sequences shown 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 according to any one of claims 1 to 8; Optionally, the nucleic acid molecule is selected from DNA.

10. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 9.

11. A method for preparing the bispecific antibody according to any one of claims 1 to 8, characterized in that, include: The expression vector of claim 10 is introduced into cells; The cells are cultured under conditions suitable for protein expression and secretion in order to obtain the bispecific antibody; Optionally, the cells are eukaryotic cells.

12. A recombinant cell, characterized in that, The recombinant cells carry the bispecific antibody as described in any one of claims 1 to 8, the nucleic acid molecule as described in claim 9, or the expression vector as described in claim 10.

13. A pharmaceutical composition, characterized in that, include: The bispecific antibody according to any one of claims 1 to 8, the nucleic acid molecule according to claim 9, the expression vector according to claim 10, or the recombinant cell according to claim 12; Optionally, it may further include pharmaceutically acceptable excipients; Optionally, the excipients include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers; Optionally, the pharmaceutical composition is an injection.

14. Use of the bispecific antibody according to any one of claims 1 to 8, the nucleic acid molecule according to claim 9, the expression vector according to claim 10, the recombinant cell according to claim 12, or the pharmaceutical composition according to claim 13 in the preparation of a medicament for the treatment or prevention of tumors.

15. The use according to claim 14, characterized in that, The tumor includes at least one of the following: lung cancer and colorectal cancer.

16. Use of the bispecific antibody of any one of claims 1 to 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 preparation of a kit for detecting B7H7 and / or CD3.

17. A reagent kit, characterized in that, The kit contains the bispecific antibody as described in any one of claims 1 to 8.

18. The kit according to claim 17, characterized in that, The kit is used to detect at least one of B7H7 and CD3.

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