A specific binding protein of CD3, bispecific antibody and preparation method and application thereof

Abstract

The application discloses a specific binding protein of CD3, a bispecific antibody and a preparation method and application thereof. The antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises HCDR1 shown as SEQ ID NO:1, HCDR2 shown as SEQ ID NO:2 and HCDR3 shown as SEQ ID NO:3; and the light chain variable region comprises LCDR1 shown as SEQ ID NO:4, LCDR2 shown as SEQ ID NO:5 and LCDR3 shown as SEQ ID NO:6. The antibody or antigen binding fragment thereof has good affinity and binding properties to CD3 protein, and can be used for preventing and / or treating CD3-mediated related diseases.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and in particular to a CD3-specific binding protein, a bispecific antibody, its preparation method, and its application. Background Technology

[0002] Anti-Müllerian hormone type II receptor (AMHR2) is a serine / threonine kinase receptor homologous to the type II receptor of the transforming growth factor-β (TGFβ) superfamily. The AMHR2 protein structure includes an N-terminal signal peptide, a 127-amino acid extracellular ligand-binding domain, a single transmembrane domain, and a 403-amino acid intracellular kinase domain. The extracellular domain contains two potential glycosylation sites, which are key regions for ligand binding and antibody recognition. Anti-Müllerian hormone (AMH) is a homologous ligand of AMHR2, and AMH binds to the extracellular domain of AMHR2. By activating intracellular SMAD, NF-κB, and Akt signaling, AMH alters gene transcription and apoptosis, leading to Müllerian duct degeneration during male development, regulation of oocyte development, and control of ovarian reserve and fertility in adult women.

[0003] Under physiological conditions, AMHR2 expression is highly tissue-specific, expressed at low levels only in ovarian granulosa cells and testicular Sertoli cells. It primarily participates in reproductive processes such as embryonic sex differentiation and follicle development regulation, with limited impact on the physiological functions of normal tissues. Pathologically, AMHR2 shows significantly high expression in various solid tumors, with a positive expression rate of approximately 70% in epithelial ovarian cancer (EOC) and approximately 53% in colorectal cancer. It also exhibits high expression levels in cervical cancer, endometrial cancer, breast cancer, and prostate cancer, making it a potential precision medicine target for gynecological tumors and some digestive system tumors. Ovarian cancer is one of the deadliest malignant tumors in gynecology. Current CSCO and NCCN guidelines for ovarian cancer treatment primarily rely on chemotherapy combined with bevacizumab as first-line therapy, with maintenance therapy depending on PARP inhibitors. Treatment options after recurrence are limited, necessitating the development of targeted immunotherapies with broad coverage and innovative mechanisms. The high expression of AMHR2 in approximately 70% of ovarian cancer patients offers a potential direction for addressing this clinical dilemma. Currently, the development of targeted drugs against AMHR2 is mainly based on monoclonal antibodies. A representative drug, Murlentamab (GM102), has entered Phase I / II clinical trials. It mediates tumor cell killing through ADCC / ADCP effects. Clinical data show that in Phase I monotherapy, only one patient with granular cell tumors achieved partial remission (PR); after combination therapy with chemotherapy, 4 out of 9 patients showed a treatment response (including 1 CR and 3 PR). In the Phase II colorectal cancer study, the disease control rate in the monotherapy group was low. Although the combination therapy group showed improvement, the objective response rate was still not ideal, and no clear ORR benefit was observed. Regarding safety, although no serious adverse events occurred, the anti-tumor activity of monotherapy was limited, with only some patients achieving disease stabilization. Furthermore, the efficacy was positively correlated with AMHR2 expression levels, resulting in insufficient overall clinical benefit.

[0004] Existing AMHR2 monoclonal antibodies have limited efficacy and cannot provide significant survival benefits for most patients with AMHR2-positive ovarian cancer. There is an urgent need to develop new AMHR2-targeted drugs to provide new solutions for enhancing the killing activity of AMHR2-positive tumor cells and more effectively treating AMHR2-positive cancers. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes an antibody that specifically binds to CD3 or an antigen-binding fragment thereof.

[0006] The present invention also provides a bispecific antibody.

[0007] The present invention also provides biological materials related to the above-described antibodies or their antigen-binding fragments or bispecific antibodies.

[0008] The present invention also provides a coupling agent.

[0009] The present invention also provides the application of the above-mentioned antibody or its antigen-binding fragment, bispecific antibody, the above-mentioned biological material or the above-mentioned conjugate.

[0010] The present invention also provides a kit comprising the above-described antibody or its antigen-binding fragment, a bispecific antibody or the above-described conjugate.

[0011] The present invention also provides a pharmaceutical composition comprising the above-described antibody or its antigen-binding fragment, a bispecific antibody, or the above-described conjugate.

[0012] The present invention also provides a method for preparing the above-mentioned antibody or its antigen-binding fragment.

[0013] This invention also provides a method for preparing bispecific antibodies.

[0014] According to a first aspect of the present invention, an antibody or antigen-binding fragment thereof that specifically binds to CD3 comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, and LCDR3 as shown in SEQ ID NO:6.

[0015] The antibodies or antigen-binding fragments thereof according to embodiments of the present invention have at least the following beneficial effects: The antibodies or antigen-binding fragments of the embodiments described herein have good affinity and binding properties to CD3 protein and can be used for the prevention and / or treatment of CD3-mediated diseases.

[0016] According to some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment comprises: a1) The amino acid sequence shown in SEQ ID NO:7; or a2) A sequence of amino acids in SEQ ID NO:7 that has undergone substitution and / or deletion and / or addition of one or more amino acids, and has the same function as the protein shown in SEQ ID NO:7; or a3) has an amino acid sequence that is 80%, 85% or 90% or more homologous to SEQ ID NO:7 and has the same function as the protein shown in SEQ ID NO:7.

[0017] According to some embodiments of the present invention, the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment comprises: b1) The amino acid sequence as shown in SEQ ID NO:8; or b2) A sequence of amino acids in SEQ ID NO:8 that has undergone substitution and / or deletion and / or addition of one or more amino acids, and has the same function as the protein shown in SEQ ID NO:14; or b3) Having 80%, 85% or 90% or more homology with SEQ ID NO:8 and having the same function as the protein shown in SEQ ID NO:8.

[0018] According to some embodiments of the present invention, the amino acid sequence of the heavy chain of the antibody or its antigen-binding fragment is as shown in SEQ ID NO: 9 or as shown in SEQ ID NO: 13; The amino acid sequence of the light chain of the antibody or its antigen-binding fragment is shown in SEQ ID NO: 10 or SEQ ID NO: 14.

[0019] According to some embodiments of the present invention, the antibody or its antigen-binding fragment is selected from any one of Fab, Fab', F(ab')2, Fv, scFv, dimer, fusion antibody and intact antibody.

[0020] According to a second aspect of the present invention, a bispecific antibody is provided, comprising: a first antigen-binding domain that specifically binds to CD3 and a second antigen-binding domain that specifically binds to AMHR2 or GPC3; wherein the first antigen-binding domain that specifically binds to CD3 comprises the antibody or antigen-binding fragment of the first aspect described above.

[0021] According to some embodiments of the present invention, the second antigen-binding domain has a heavy chain variable region as shown in SEQ ID NO.11 and a light chain variable region as shown in SEQ ID NO.12.

[0022] The bispecific antibody according to embodiments of the present invention has at least the following beneficial effects: The CD3 antibody single-chain variable fragment in the first antigen-binding domain of the bispecific antibody provided by this invention can bind to T cells, while the second antigen-binding domain binds to antigens on the tumor surface. This binding mode can significantly activate T cell function. Compared with the CD3-terminal antibody in traditional T cell bispecific antibodies, it has a different binding epitope, which can maintain high tumor cell killing activity while having a low level of cytokine release, thereby balancing the efficacy and safety of T cell bispecific antibody therapy.

[0023] According to a third aspect of the present invention, a biomaterial is provided in relation to the antibody or its antigen-binding fragment described in the first aspect embodiment, or the bispecific antibody described in the second aspect embodiment, wherein the biomaterial is any one of c1) to c5); c1) A nucleic acid molecule encoding the antibody or its antigen-binding fragment, or a bispecific antibody; c2) An expression cassette containing the nucleic acid molecules described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1) or the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecule described in c1), the expression cassette described in c2), or the recombinant vector described in c3); c5) A transgenic cell line containing the nucleic acid molecule described in c1), the expression cassette described in c2), or the recombinant vector described in c3).

[0024] According to some embodiments of the present invention, the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0025] According to some embodiments of the present invention, the nucleic acid molecule is a DNA molecule encoding the antibody or its antigen-binding fragment.

[0026] According to some embodiments of the present invention, the expression cassette refers to DNA capable of expressing the antibody or its antigen-binding fragment in a host cell. This DNA may include not only a promoter to initiate transcription of the DNA molecule encoding it, but also a terminator to terminate transcription of the DNA molecule encoding it. Furthermore, the expression cassette may also include a replication start site, a transcription start sequence, an enhancer sequence, a selection element, or a reporter gene.

[0027] According to some embodiments of the present invention, the recombinant vector may be a plasmid, bacteriophage, lentivirus, adenovirus, or adeno-associated virus. The recombinant vector is used to achieve the replication, integration, amplification, and / or expression of the exogenous target gene in recipient cells; it may be a cloning vector or an expression vector.

[0028] According to some embodiments of the present invention, the recombinant microorganism may be bacteria (such as Escherichia coli or Bacillus subtilis) or fungi (such as yeast or Aspergillus).

[0029] According to some embodiments of the present invention, the transgenic cell line may be insect cells (such as S2 Drosophila cells or Sf9 cells), animal cell lines (such as 293T cells, 293F cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells or HEK293 cells), or plant cells (such as Arabidopsis thaliana or tobacco). All transgenic cell lines may be non-reproductive materials.

[0030] A conjugate according to a fourth aspect of the present invention comprises: the antibody or its antigen-binding fragment described in the first aspect embodiment or the bispecific antibody described in the second aspect embodiment; And a coupling portion, the coupling portion comprising at least one of a detectable marker, a drug, a toxin, a radionuclide, and an enzyme.

[0031] According to some embodiments of the present invention, the detectable marker is selected from at least one of radioactive isotopes, fluorescent substances, chemiluminescent substances, and colored substances.

[0032] The use of the antibody or antigen-binding fragment thereof described in the first aspect embodiment of the fifth aspect embodiment of the present invention, the bispecific antibody described in the second aspect embodiment, the biological material described in the third aspect embodiment, or the conjugate described in the fourth aspect embodiment in any one of d1) to d3); d1) Preparation of drugs for the prevention and / or treatment of tumors; d2) Prepare products for CD3 detection; d3) Prepare products that bind to CD3.

[0033] According to some embodiments of the present invention, the tumor includes ovarian cancer.

[0034] According to some embodiments of the present invention, the product includes at least one of reagents, reagent kits, chips, and test strips.

[0035] A kit according to a sixth aspect of the present invention includes an antibody or antigen-binding fragment thereof as described in the first aspect embodiment, a bispecific antibody as described in the second aspect embodiment, a biological material as described in the third aspect embodiment, or a conjugate as described in the fourth aspect embodiment.

[0036] A pharmaceutical composition according to a seventh aspect of the present invention comprises, wherein the antibody or antigen-binding fragment thereof described in the first aspect embodiment, the bispecific antibody described in the second aspect embodiment, the biological material described in the third aspect embodiment, or the conjugate described in the fourth aspect embodiment.

[0037] According to some embodiments of the present invention, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0038] According to some embodiments of the present invention, the pharmaceutically acceptable excipients include, but are not limited to, diluents, carriers, solubilizers, emulsifiers, preservatives and / or adjuvants.

[0039] An eighth aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof from the first aspect of the present invention, obtained by culturing the transgenic cell line of the fourth aspect of the present invention.

[0040] According to a ninth aspect of the present invention, a method for preparing a bispecific antibody or ...

[0041] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0042] Figure 1 The graph shows the results of ELISA analysis of antibody binding activity with human and monkey CD3 protein. Figure 2 The image shows the results of the affinity test for bispecific antibodies against human CD3 protein. Figure 3 This is a graph showing the results of TDCC activity assay for the bispecific antibody; Figure 4 This is the result of the detection of bispecific antibody-induced cytokine release. Detailed Implementation

[0043] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0044] Unless otherwise specified, the amino acid sequences in this application are from the N-terminus to the C-terminus. "Amino acid" refers to the basic unit that constitutes a protein, giving it a specific molecular structure and enabling its biochemical activity. For example, the "amino acids" used in this invention include the following 20 natural amino acids: alanine (Ala or A), glycine (Gly or G), isoleucine (Ile or I), asparagine (Asn or N), arginine (Arg or R), lysine (Lys or K), lysine (Lys or K), cysteine ​​(Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), leucine (Leu or L), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), valine (Val or V), and tyrosine (Tyr or Y).

[0045] Unless otherwise specified in the following examples, the conditions should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0046] Example 1: Design and screening of CD3 antibodies 1. Animal immunization CD3δε protein dimers were purchased from Peptasys (catalog number: CDD-H52W1). A peptide fragment containing the N-terminal 27 amino acid sequence of the human CD3ε protein (sequence: QDGNEEMGGITQTPYKVSISGTTVILT (SEQ ID NO:15)) was synthesized. Its C-terminus was conjugated to the widely used carrier proteins keyfora hemocyanin (KLH) or bovine serum albumin (BSA) by adding a cysteine ​​residue to obtain CD3ε-N27-KLH or CD3ε-N27-BSA to induce a significant immune response.

[0047] Select 4-6 week old Balb / c (albino lab rats) and administer subcutaneous injections of a mixture of CD3δε protein dimer or CD3ε-KLH and an adjuvant (TiterMax or Sigma adjuvant system) at multiple sites every 2-3 weeks for a total of 3-4 immunizations. Based on serum titer, select high-titer animals 3 days before spleen collection for a pulse immunization via intraperitoneal injection of the antigen solution.

[0048] Mouse spleen cells were collected and antibody light and heavy chain variable region sequences were obtained using a single B-cell screening platform. Expression vectors constructed using PCR were co-transfected into 293T cells, and cell culture supernatants were collected.

[0049] 2. ELISA and FCM screening Human CD3δε protein dimer, CD3ε-N27-BSA, or cynomolgus CD3δε protein dimer (purchased from Pypsis, catalog number: CDD-C52W4) were coated into 96-well microplates at 100 μL / well and incubated overnight at 4°C. The next day, after washing four times with PBST, 150 μL of 5% skim milk blocking buffer was added, and the plate was incubated at 37°C for 1 hour; after washing four times with PBST, 100 μL of transfection supernatant was added, and the plate was incubated at 37°C for 1 hour; after washing four times with PBST, 100 μL of Anti-Human IgG HRP (1:10000 dilution) was added, and the plate was incubated at 37°C for 0.5 hours; after washing four times with PBST, 50 μL of TMB chromogenic buffer was added, and the plate was incubated at 37°C for 8 minutes. The reaction was terminated by adding 50 μL of LELISA stop solution to each well, and the absorbance was measured at 450 nm using a microplate reader.

[0050] Jurkat-NFAT-Luc Effector Cells were harvested and treated with 2×10⁻⁶ cells. 6 Cells were resuspended at 1 / mL in FACS buffer (PBS containing 4% FBS). 50 μL of the resuspended solution was added to each well of a 96-well U-plate, centrifuged at 300g for 5 minutes, the supernatant was discarded, and 100 μL of transfection supernatant was added. The cells were incubated at 4°C for 1 hour. Cells were washed three times with FACS buffer, and after centrifugation, 100 μL of APC-labeled anti-human IgG antibody was added to each well. Cells were incubated at 4°C for 30 minutes, washed three times, the supernatant was discarded, and the cells were resuspended in 150 μL of FACS buffer for flow cytometry analysis.

[0051] Based on the ELISA and flow cytometry binding activity of the transfection supernatant (containing the corresponding antibody protein), this application screened clones that bind to human CD3δε protein dimer or CD3ε-N27-BSA peptide and have Jurkat cell binding activity, while also satisfying the requirement of cross-binding activity with cynomolgus CD3δε protein dimer. The detection results are shown in Table 1.

[0052] Table 1

[0053] The amino acid sequence of the complementarity-determining region of the M167 antibody is shown below: Heavy chain complementarity determinant region: HCDR1:AYGLS (SEQ ID NO:1); HCDR2:EIYPRSGNTYFNEKFRG (SEQ ID NO:2); HCDR3: LKFITTVVSSYWYFDV (SEQ ID NO: 3).

[0054] Light chain complementarity determinant region: LCDR1: KASQSVSFAGTSLMH (SEQ ID NO:4); LCDR2: RASNLEA (SEQ ID NO:5); LCDR3: QQSREYPRT (SEQ ID NO:6).

[0055] M167 antibody heavy chain variable region amino acid sequence: EVQVVESGAEVARPGASVKLSCKASGYTFTAYGLSWVKQMTGQGLEWIGEIYPRSGNTYFNEKFRGKATLTADKSSSTAYMELRSLTSEDSAVYFCARLKFITTVVSSYWYFDVWGTGTTVTVSS (SEQ ID NO: 7).

[0056] M167 antibody light chain variable region amino acid sequence: DILMTQSPASLAVSLGQRAIISCKASQSVSFAGTSLMWYRQKPGQQPKLLIYRASNLEAGVPTRFSGSGSRTDFTLNIHPVEEDDAAIYYCQQSREYPRTFGGGTKLEIK (SEQ ID NO: 8).

[0057] M167 antibody heavy chain amino acid sequence: EVQVVESGAEVARPGASVKLSCKASGYTFTAYGLSWVKQMTGQGLEWIGEIYPRSGNTYFNEKFRGKATLTADKSSSTAYMELRSLTSEDSAVYFCARLKFITTVVSSYWYFDV WGTGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:9).

[0058] M167 antibody light chain amino acid sequence: DILMTQSPASLAVSLGQRAIISCKASQSVSFAGTSLMWYRQKPGQQPKLLIYRASNLEAGVPTRFSGSGSRTDFTLNIHPVEEDDAAIYYCQQSREYPRTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:10).

[0059] Example 2 Construction and preparation of bispecific antibodies The CD3 antibody-positive clone (M167) selected above was combined with the humanized anti-AMHR2 antibody 7E1 to construct a bispecific antibody (CR04-7E1-M167) with a natural IgG conformation targeting AMHR2 / CD3 in a "1+1" form. The antibody sequence is as follows: AMHR2 terminal heavy chain amino acid sequence: DIVMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPNLLIYAASTLQSGVPSRFSGSGSGTEFTLTIISLQPEDFATYYCQQLNSYPLTFGGGTKVDIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:11).

[0060] AMHR2-side light chain amino acid sequence: QVQLVQSGPGLVKPSETLSLTCTVSGDSISSYYWSWIRQPPGKGLEWIGYISYSGSTDYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCARHAYREKGQFVPYYFYYGMDVWGQGTTVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:12).

[0061] CD3-side heavy chain amino acid sequence: EVQVVESGAEVARPGASVKLSCKASGYTFTAYGLSWVKQMTGQGLEWIGEIYPRSGNTYFNEKFRGKATLTADKSSSTAYMELRSLTSEDSAVYFCARLKFITTVVSSYWYFDVWGTGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 13).

[0062] Amino acid sequence of the CD3 terminal light chain: DILMTQSPASLAVSLGQRAIISCKASQSVSFAGTSLMHWYRQKPGQQPKLLIYRASNLEAGVPTRFSGSGSRTDFTLNIHPVEEDDAAIYYCQQSREYPRTFGGGTKLEIKRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 14).

[0063] The verified sequence was expanded and cultured, and plasmids were extracted using an endotoxin-free plasmid extraction kit (ZYMO RESEARCH, D4201-A), following the manufacturer's instructions. HEK-293T cells were cultured in DMEM basic medium (Gbico, catalog number: C11995500BT) at 37°C in a 5% CO2 cell culture incubator, according to the manufacturer's instructions. After cell preparation, the plasmid was transfected into HEK293-T cells using a transfection kit (Polysciences, catalog number: 24765-1) to express the anti-AMHR2 / CD3 bispecific antibody. After 9 days of culture, the expression supernatant was harvested by centrifugation. The expression supernatant was filtered through a 0.22 μM filter and the antibody was purified using a Protein A affinity chromatography column.

[0064] Example 3: ELISA analysis of human-monkey cross-binding activity of bispecific antibody protein levels Human CD3δ&ε (manufacturer: Acro, catalog number: CDD-H52W1) and monkey CD3δ&ε (purchased from Bisprosys, catalog number: CDD-C52W4) were aliquoted into 96-well ELISA plates, 50 μL / well, and incubated overnight at 4°C. After washing three times with PBST, 100 μL of 3% skim milk blocking buffer was added, and the plates were incubated at 37°C for 0.5 hours. After washing three times with PBST, 50 μL of serially diluted bispecific antibody CR04-7E1-M167 sample (starting concentration 100 nM, 3-fold serial dilution, total 8 concentration points) was added, and the plates were incubated at 37°C for 0.5 hours. After washing three times with PBST, Anti-Human IgG was added. Incubate 50 μL of HRP (1:10000 dilution, manufacturer: Jackson, catalog number: 109-035-170) at 37°C for 0.5 hours; wash 4 times with PBST, add 50 μL of TMB chromogenic solution, incubate at 37°C for 5 minutes, add 50 μL of ELISA stop solution per well to stop the reaction, and measure the absorbance at 450 nm using a microplate reader.

[0065] The results are as follows Figure 1 As shown in the figure. The results indicate that both bispecific antibodies can bind to the CD3δ&ε proteins of humans and cynomolgus monkeys, exhibiting good human-monkey cross-binding activity.

[0066] Example 4: Bispecific antibody affinity assay Using surface plasmon resonance (SPR) technology, the bispecific antibody CR04-7E1-M167 was captured on a Protein A probe. The binding affinity to human CD3δε protein dimers at multiple concentrations (200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 0 nM) was detected using a Biacore 8k instrument. The affinity constant KD was obtained based on the binding and dissociation rates. Affinity data are shown below. Figure 2 And Table 2.

[0067] Table 2

[0068] The results show that the bispecific antibody provided by the present invention has diverse affinity activities (KD values) and binding / dissociation kinetics for human CD3δε protein dimers.

[0069] Example 5: Assay of the antitumor activity of bispecific antibodies This embodiment uses a T-cell-dependent cytotoxicity (TDCC) system based on lactate dehydrogenase (LDH) release to quantitatively evaluate the in vitro killing activity of the CD3-targeting bispecific antibody CR04-7E1-M167 against AMHR2-positive tumor cells.

[0070] The specific experimental procedure is as follows: Human PBMC cells (purchased from Miaoshun Biotechnology) were revived in advance and cultured overnight to ensure cell stability. COV434 tumor cells (purchased from Fuheng Biotechnology, product number FH0724) in the logarithmic proliferation phase and highly expressing hAMHR2 were selected. Cell digestion was performed using Accutase cell digestion solution. After terminating the digestion reaction with complete culture medium containing fetal bovine serum, the cell pellet was collected by centrifugation at 1300 rpm for 5 min. The cell pellet was washed once with an analytical buffer prepared from 1640 basal medium (Gibco, catalog number 11835030), 10% fetal bovine serum (FBS), and 1% penicillin-dextrose antibody (PS) to thoroughly remove residual digestion solution and metabolic impurities. After cell counting, the target cell suspension density was adjusted to 8 × 10⁶ cells / mL. 5 Cells / mL. After gentle pipetting and mixing, seed 50 μL into each well of a 96-well U-shaped culture plate. Then add 50 μL of serially diluted candidate bispecific antibody working solution to each well; the initial antibody concentration was set at 50 nM, with 6 detection points using a 10-fold serial dilution. Centrifuge overnight revived PBMC effector cells at 400 × g for 10 min at room temperature, discard the supernatant, resuspend the cells in the same analytical buffer as described above, and adjust the cell suspension concentration to 2 × 10⁻⁶. 6100 μL of PBMC cell suspension was added to each well to construct a complete antibody-effect cell-target cell co-culture system. The 96-well U-shaped plates were incubated continuously at 37℃ and 5% CO2 for 24 h. Following the method of the LDH detection kit (Promega, G1780), 50 μL of supernatant was aspirated from each well for OD490 detection. Results are as follows... Figure 3 As shown in Table 3, the statistical results are presented. Detailed activity detection results obtained in this experiment are attached. Figure 3 The corresponding statistical analysis data are detailed in Table 3.

[0071] Table 3

[0072] The results showed that the bispecific antibody against CR04-7E1-M167 of the present invention has high killing activity against tumor target cells.

[0073] Example 6: Cytokine Release Assay of Bispecific Antibody The level of cytokine release induced by the CD3 bispecific anti-drug of this invention was detected using CBA flow cytometry microsphere technology. The specific procedures were as follows: Human peripheral blood mononuclear cells (PBMCs) purchased from Miaoshun Biotechnology were resuscitated and incubated overnight at 37°C and 5% CO2 to restore viability; COV434-AMHR2 tumor cells in logarithmic growth phase with a viability ≥95% were selected, and the cell density was adjusted to 8 × 10⁶ cells / mL using sterile RPMI 1640 medium. 5 The sample was seeded at a density of 50 μL / well in a 96-well U-shaped plate, followed by 100 μL / well at a density of 2 × 10⁻⁶. 6 PBMCs were resuscitated at a concentration of 100 μL / well to construct a co-culture system of tumor cells and immune cells. Six concentration points of the CD3 penicillin antibody to be tested were set up using a 10-fold serial dilution, with the highest concentration being 100 nM. Each concentration of the drug was added to the co-culture system at 50 μL / well, for a final volume of 200 μL / well. The cells were incubated at 37°C with 5% CO2 for 24 h. After incubation, the cell supernatant was collected by centrifugation. The Th1 / Th2 cytokine release levels in each group were quantitatively analyzed using the BD Human Th1 / Th2 Cytokine Kit II (catalog number: 551809) strictly following the instructions. The results are shown below. Figure 4 As shown, the dose-related characteristics of the drug-induced cytokine release of the present invention can be clearly understood.

[0074] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to CD3, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:3; and the light chain variable region comprises LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, and LCDR3 as shown in SEQ ID NO:

6.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment includes: a1) The amino acid sequence shown in SEQ ID NO:7; or a2) A sequence of amino acids in SEQ ID NO:7 that has undergone substitution and / or deletion and / or addition of one or more amino acids, and has the same function as the protein shown in SEQ ID NO:7; or a3) has an amino acid sequence that is 80%, 85% or 90% or more homologous to SEQ ID NO:7 and has the same function as the protein shown in SEQ ID NO:7; And / or, The amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment includes: b1) The amino acid sequence as shown in SEQ ID NO:8; or b2) A sequence of amino acids in SEQ ID NO:8 that has undergone substitution and / or deletion and / or addition of one or more amino acids, and has the same function as the protein shown in SEQ ID NO:14; or b3) The amino acid sequence that has 80%, 85% or 90% or more homology with SEQ ID NO:8 and has the same function as the protein shown in SEQ ID NO:8; And / or, the antibody or its antigen-binding fragment is selected from any one of Fab, Fab', F(ab')2, Fv, scFv, dimer, fusion antibody and intact antibody.

3. A bispecific antibody, characterized in that, It comprises: a first antigen-binding domain that specifically binds to CD3 and a second antigen-binding domain that specifically binds to AMHR2 or GPC3; wherein the first antigen-binding domain that specifically binds to CD3 comprises an antibody or an antigen-binding fragment thereof as described in any one of claims 1-2; Preferably, the second antigen-binding domain has a heavy chain variable region as shown in SEQ ID NO.11 and a light chain variable region as shown in SEQ ID NO.

12.

4. A biomaterial relating to the antibody or antigen-binding fragment thereof as described in any one of claims 1-2 or the bispecific antibody as described in claim 3, characterized in that, The biomaterial is any one of c1) to c5); c1) A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in any one of claims 1-2 or the bispecific antibody as described in claim 3; c2) An expression cassette containing the nucleic acid molecules described in c1); c3) A recombinant vector containing the nucleic acid molecule described in c1) or the expression cassette described in c2); c4) Recombinant microorganisms containing the nucleic acid molecule described in c1), the expression cassette described in c2), or the recombinant vector described in c3); c5) A transgenic cell line containing the nucleic acid molecule described in c1), the expression cassette described in c2), or the recombinant vector described in c3).

5. A coupling agent, characterized in that, Includes the antibody or antigen-binding fragment thereof as described in any one of claims 1-2 or the bispecific antibody as described in claim 3; And a coupling portion, the coupling portion comprising at least one of a detectable marker, a drug, a toxin, a radionuclide, and an enzyme.

6. The use of the antibody or antigen-binding fragment thereof according to any one of claims 1-2, the bispecific antibody according to claim 3, the biomaterial according to claim 4, or the conjugate according to claim 5 in any one of d1) to d3); d1) Preparation of drugs for the prevention and / or treatment of tumors; d2) Prepare products for CD3 detection; d3) Preparation of products that bind to CD3; Preferably, the tumor includes ovarian cancer.

7. A reagent kit, characterized in that, It includes the antibody or antigen-binding fragment thereof as described in any one of claims 1-2, the bispecific antibody as described in claim 3, or the conjugate as described in claim 5.

8. A pharmaceutical composition, characterized in that, It includes the antibody or antigen-binding fragment thereof as described in any one of claims 1-2, the bispecific antibody as described in claim 3, or the conjugate as described in claim 5.

9. A method for preparing the antibody or its antigen-binding fragment according to any one of claims 1-2, characterized in that, The preparation method includes the following steps: preparing the transgenic cell line as described in claim 4 by culturing it.

10. The method for preparing the bispecific antibody according to claim 3, characterized in that, The preparation method includes the following steps: preparing the transgenic cell line as described in claim 4 by culturing it.

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