Bispecific antibody targeting CD3 and L1CAM and application thereof

Abstract

The invention discloses a bispecific antibody targeting CD3 and L1CAM and an application of the bispecific antibody. The bispecific antibody comprises a first functional area and a second functional area, the first functional area and the second functional area are connected through a connecting peptide, the first functional area is an anti-L1CAM antibody, and the second functional area is an anti-CD3 antibody. The invention also discloses nucleic acid for coding the bispecific antibody, a recombinant expression vector and a transformant containing the nucleic acid, a pharmaceutical composition containing the bispecific antibody, a kit and a kit kit. The invention also discloses application of the bispecific antibody in preparation of drugs for diagnosis, prevention and / or treatment of L1CAM mediated diseases or symptoms. The bispecific antibody disclosed by the invention shows killing activity superior to that of a parent monoclonal antibody on various tumor cell lines, and meanwhile, off-target toxicity is not generated; and the compound has relatively strong anti-tumor activity in vivo and has a great clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of tumor treatment, and specifically relates to a bispecific antibody targeting CD3 and L1CAM and an application thereof. Background Art

[0002] In the field of tumor treatment, bispecific antibodies are one of the most promising cancer immunotherapy drugs. Based on their mechanism of action, they can be roughly divided into immunomodulation, dual binding targeting of tumor cell receptors, and effector cell redirection (Loffler, Kufer, Lutterbüse et al., Blood, 2000, 2098-2103; Nie, Wang, Moscoso-Castro et al., Antibody therapeutics, 2020, 18-62). Among them, T cell-redirecting bispecific antibodies (TCEs) have two antigen arms, one that binds to CD3 on T cells and the other that binds to tumor-associated antigens (TAAs) on the surface of tumor cells, directing T cells to the vicinity of tumor cells. TCEs can initiate T cell activation without the need for additional auxiliary stimulation signals, directly guiding T cells to fight against tumor cells expressing TAAs in the tumor microenvironment (Sam, Colombetti, Fauti et al., Frontiers in Oncology, 2020, 575737). Therefore, TCEs are less affected by factors that limit the clinical efficacy of immunotherapy, such as antigen presentation, downregulation of MHC molecules, and loss of co-stimulatory signals (Sam, Colombetti, Fauti et al., Frontiers in Oncology, 2020, 575737), making them an attractive cancer immunotherapy strategy. Currently, many TCEs have been approved for marketing, but the targets of these antibodies are mostly located on B cells or bone marrow cells. Tebentafusp is the only antibody that targets the melanoma gp100 antigen (Lanyi Nora Chen and Carvajal, Expert Review of Anticancer Therapy, 2022, 1017-1027). The research progress of CD3-type TCEs in hematological malignancies is significantly ahead of solid tumors. It would be very desirable to develop more promising new TCEs targeting tumor-associated antigens to provide more possibilities for cancer immunotherapy.

[0003] L1 cell adhesion molecule (L1CAM) is a 200-200 kDa, heavily glycosylated, type I transmembrane protein belonging to the immunoglobulin superfamily. L1CAM is primarily involved in the development of neural cells. Because it is associated with cell migration and invasion (Maness and Schachner, Nature Neuroscience, 2007, 19-26), L1CAM also plays a crucial role in tumorigenesis and progression. L1CAM can promote tumor cell invasion, epithelial-to-mesenchymal transition (EMT), and the development of chemotherapy resistance (Kiefel, Bondong, Hazin et al., Celladhesion & Migration, 2012, 374-384). The signaling pathways involved in L1CAM-induced cancer development are very complex. Studies on gastric cancer (Ito, Yamada, Tanaka et al., Annals of surgical oncology, 2014, 560-568) and colon cancer (Fang, Zheng and Zhao, American Journal of Translational Research, 2020, 837) have shown that L1CAM may be involved in the occurrence and metastasis of cancer by activating the extracellular signal-regulated kinase (ERK) pathway. In patients with esophageal squamous cell carcinoma (ESCC) (Zhao, Liu, Chen et al., Cancer Biology & Medicine, 2021, 547), L1CAM promotes CCL22 expression by activating the PI3K / Akt / NF-κB signaling pathway, promoting the enrichment of Treg cells in the tumor microenvironment and exerting oncogenic activity.In recent years, an increasing number of studies have reported the effects of PD-1 on solid tumors such as melanoma, gastric cancer, breast cancer, ovarian cancer, endometrial cancer, and colorectal cancer (Fang, Zheng and Zhao, American Journal of Translational Research, 2020, 837; Fogel, Mechtersheimer, Huszar et al., Cancer letters, 2003, 237-247; Giordano, Decio, Battistini et al., Journal of Experimental & Clinical Cancer Research, 2021, 1-19; Havrilesky, Cragun, Calingaert et al., Gynecologic oncology, 2007, 401-405; Ito, Yamada, Tanaka et al., Annals of surgical oncology, 2014, 560-568; Moisini, Zhang, D'Aguiar et al., Applied Immunohistochemistry & Molecular Biology, 2014). Overexpression of L1CAM in human ovarian cancer cells has been reported (Morphology, 2021, 287-292). Clinical data show that high expression of L1CAM is a predictor of tumor metastasis and poor prognosis (Hua, Liu, Xin et al., Oncotarget, 2016, 85196; Wang, Wang, Wu et al., Clinics, 2022, 100040), indicating that L1CAM is an important target in cancer treatment.

[0004] Monoclonal antibodies (mAbs) or chimeric antigen receptor T cell therapies (CAR-T) targeting L1CAM have shown promising results in preclinical studies for the treatment of solid tumors (Arlt, Novak-Hofer, Gast et al., Cancer Research, 2006, 936-943; Künkele, Taraseviciute, Finn et al., Clinical Cancer Research, 2017, 466-477). However, clinical application has yet to progress, necessitating the development of novel immunotherapeutic agents targeting L1CAM. Currently, no TCEs targeting L1CAM have been reported. Currently, there are several TCE bispecific antibody platforms on the market, including IgG-(L)-scFv structures that can induce potent cytotoxic T lymphocyte responses against tumors by targeting TAAs and CD3. Studies have shown that IgG-(L)-scFv-based TCEs have higher potency than TCEs based on IgG-heterodimer or BiTE structures (Santich, Park, Tran et al., Science Translational Medicine, 2020, eaax1315). Two other tumor antigen-targeting TCE BsAbs by the inventors also verified the excellent antitumor activity of IgG-(L)-scFv-based TCEs (Jie Chen, Pan, Han et al., Biomedicines, 2021, 1059; Pan, Chen, Xiao et al., Acta Pharmaceutica Sinica B, 2022, 1928-1942). Summary of the Invention

[0005] To address the lack of an effective new immunotherapy drug targeting L1CAM in the prior art, the present invention provides a bispecific antibody targeting CD3 and L1CAM and its use. The inventors have proposed a novel bispecific antibody, CE7-TCE, targeting L1CAM and CD3, based on an IgG-(L)-scFv structure. This antibody is based on an L1CAM monoclonal antibody (chCE7 mAb), with an anti-CD3 scFv (derived from huOKT3) attached to the terminus of its light chain via a linker peptide. This novel bispecific antibody targeting L1CAM and CD3 exhibits excellent anti-tumor activity in animal models.

[0006] To solve the above technical problems, one of the technical solutions provided by the present invention is: a bispecific antibody, comprising a first functional region and a second functional region, wherein the first functional region and the second functional region are connected by a connecting peptide, the first functional region is an anti-L1CAM antibody, and the second functional region is an anti-CD3 antibody; the amino acid sequence of the light chain variable region of the anti-L1CAM antibody is as shown in SEQ ID NO: 2, and / or, the amino acid sequence of the heavy chain variable region of the anti-L1CAM antibody is as shown in SEQ ID NO: 9; and / or, the amino acid sequence of the light chain variable region of the anti-CD3 antibody is as shown in SEQ ID NO: 7, and / or, the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody is as shown in SEQ ID NO: 5.

[0007] In the present invention, the "first functional region" and the "second functional region" are merely two different functional regions that distinguish the bispecific antibody, and do not impose any additional restrictions on these two functional regions.

[0008] In a specific embodiment of the present invention, the anti-L1CAM antibody is an IgG antibody, and the anti-CD3 antibody is a scFv antibody; and / or, the N-terminus of the scFv antibody is connected to the C-terminus of the light chain of the IgG antibody via a connecting peptide.

[0009] In the present invention, the bispecific antibody has the structure of "IgG-(L)-scFv" as described herein ("L" refers to the connecting peptide), with reference to Figure 1 Schematic diagram, that is, when the IgG antibody has a quaternary structure of a tetramer, the N-termini of the two scFv antibodies are symmetrically connected to the C-termini of the two light chains of an IgG antibody through connecting peptides.

[0010] In a specific embodiment of the present invention, the heavy chain constant region of the anti-L1CAM antibody is derived from the heavy chain of a human antibody, and / or the light chain constant region of the anti-L1CAM antibody is derived from the κ chain of a human antibody.

[0011] In a specific embodiment of the present invention, the Fc segment of the heavy chain constant region of the anti-L1CAM antibody comprises a PGLALA mutation; and / or the CH1 segment of the heavy chain constant region of the anti-L1CAM antibody comprises an F126C mutation and the light chain constant region of the anti-L1CAM antibody comprises an S121C mutation.

[0012] It is known in the art that the PGLALA mutation (L234A, L235A and P329G) in the Fc segment of an antibody can reduce the biological activities of the antibody, such as ADCC and complement fixation; the F126C / S121C mutation (heavy chain CH1F126C and light chain S121C mutation) occurs on CH1 and CL, mainly introducing cysteine by amino acid mutation at the antibody Fab interface, and ultimately forming a new disulfide bond by the introduced cysteine to improve the stability of the bispecific antibody.

[0013] In a specific embodiment of the present invention, the amino acid sequence of the heavy chain constant region of the anti-L1CAM antibody is shown in SEQ ID NO: 10 or SEQ ID NO: 13; and / or the amino acid sequence of the light chain constant region of the anti-L1CAM antibody is shown in SEQ ID NO: 3.

[0014] In a specific embodiment of the present invention, the amino acid sequence of the connecting peptide connecting the IgG antibody and the scFv antibody is shown in SEQ ID NO: 4, and / or the amino acid sequence of the connecting peptide connecting the light chain variable region and the heavy chain variable region of the scFv antibody is shown in SEQ ID NO: 6.

[0015] In a specific embodiment of the present invention, the amino acid sequence of the heavy chain of the bispecific antibody is shown as SEQ ID NO: 8, and / or the amino acid sequence of the light chain of the bispecific antibody is shown as SEQ ID NO: 1.

[0016] To solve the above technical problems, the present invention provides a second technical solution: an isolated nucleic acid encoding the bispecific antibody as described in one of the technical solutions of the present invention.

[0017] To solve the above technical problems, the third technical solution provided by the present invention is: a recombinant expression vector, which comprises the isolated nucleic acid as described in the second technical solution of the present invention.

[0018] In a specific embodiment of the present invention, the backbone of the recombinant expression vector is pcDNA3.4.

[0019] To solve the above technical problems, the fourth technical solution provided by the present invention is: a transformant, which comprises the isolated nucleic acid as described in the second technical solution of the present invention or the recombinant expression vector as described in the third technical solution of the present invention.

[0020] In a specific embodiment of the present invention, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

[0021] In a specific embodiment of the present invention, the prokaryotic cell is Escherichia coli, such as Escherichia coli DH5α.

[0022] To solve the above technical problems, the present invention provides a fifth technical solution: a method for preparing a bispecific antibody as described in one of the technical solutions of the present invention, the preparation method comprising: culturing the transformant as described in the fourth technical solution of the present invention, and obtaining the bispecific antibody from the culture.

[0023] To solve the above technical problems, the sixth technical solution provided by the present invention is: a pharmaceutical composition, comprising the bispecific antibody as described in one of the technical solutions of the present invention, and a pharmaceutically acceptable carrier.

[0024] In a specific embodiment of the present invention, the pharmaceutical composition further contains an inhibitor that blocks the interaction between PD1 and PD-L1.

[0025] In a specific embodiment of the present invention, the inhibitor is an anti-PD1 antibody, such as Pembrolizumab.

[0026] To solve the above technical problems, the present invention provides a seventh technical solution: use of the bispecific antibody described in one of the technical solutions of the present invention and / or the pharmaceutical composition described in the sixth technical solution of the present invention in the preparation of a drug for diagnosing, preventing and / or treating L1CAM-mediated diseases or conditions.

[0027] In a specific embodiment of the present invention, the disease or condition is a tumor, preferably gastric cancer, esophageal squamous cell carcinoma, melanoma, breast cancer, ovarian cancer, endometrial cancer or colorectal cancer.

[0028] To solve the above technical problems, the eighth technical solution provided by the present invention is: a kit comprising the bispecific antibody according to one of the technical solutions of the present invention and / or the pharmaceutical composition according to the sixth technical solution of the present invention.

[0029] In a specific embodiment of the present invention, the kit further comprises (i) a device for administering the bispecific antibody or pharmaceutical composition; and / or (ii) instructions for use.

[0030] To solve the above technical problems, the ninth technical solution provided by the present invention is: a kit of parts, comprising kit A and kit B, wherein kit A contains the bispecific antibody as described in one of the technical solutions of the present invention; kit B contains an inhibitor that blocks the interaction between PD1 and PD-L1, preferably an anti-PD1 antibody such as Pembrolizumab; or, kit A contains the pharmaceutical composition as described in the sixth technical solution of the present invention; kit B contains other anti-tumor antibodies or a pharmaceutical composition containing the other anti-tumor antibodies.

[0031] To solve the above technical problems, the present invention provides a tenth technical solution: a method for immunodetection or determination of L1CAM and / or CD3, which comprises using the bispecific antibody described in one of the technical solutions of the present invention and / or the pharmaceutical composition described in the sixth technical solution of the present invention.

[0032] In a specific embodiment of the present invention, the detection is a detection for non-diagnostic and / or therapeutic purposes.

[0033] The application scenarios of "non-diagnostic and / or therapeutic purposes" described in the present invention include, but are not limited to: for example, detecting the presence of the antigen L1CAM and / or CD3 in the laboratory; or using it as a positive antibody to screen other anti-L1CAM and / or CD3 antibodies; or competing with other anti-L1CAM and / or CD3 antibodies for binding to detect whether there is competition between antibodies, that is, whether the antigen epitopes are the same or similar, and other application scenarios.

[0034] To solve the above technical problems, the present invention provides an eleventh technical solution: a method for diagnosing, treating and / or preventing L1CAM-mediated diseases or conditions, the method comprising administering a therapeutically effective amount of the bispecific antibody according to one of the technical solutions of the present invention and / or the pharmaceutical composition according to the sixth technical solution of the present invention to a patient in need, or using the kit according to the ninth technical solution of the present invention to treat a patient in need.

[0035] In a specific embodiment of the present invention, the disease or condition is a tumor, preferably gastric cancer, esophageal squamous cell carcinoma, melanoma, breast cancer, ovarian cancer, endometrial cancer or colorectal cancer.

[0036] To solve the above technical problems, the present invention provides a twelfth technical solution: a combination therapy, comprising administering a bispecific antibody as described in one of the technical solutions of the present invention and a second therapeutic agent to a patient in need thereof; the second therapeutic agent preferably comprises other anti-tumor antibodies or a pharmaceutical composition comprising the other anti-tumor antibodies.

[0037] In a specific embodiment of the invention, the second therapeutic agent is an inhibitor that blocks the interaction between PD1 and PD-L1.

[0038] In a specific embodiment of the present invention, the inhibitor is an anti-PD1 antibody, such as Pembrolizumab.

[0039] To solve the above technical problems, the present invention provides a thirteenth technical solution: the bispecific antibody according to one of the technical solutions of the present invention, the pharmaceutical composition according to the sixth technical solution of the present invention, and / or the kit according to the ninth technical solution of the present invention, which are used for diagnosing, treating and / or preventing L1CAM-mediated diseases or conditions.

[0040] In a specific embodiment of the present invention, the disease or condition is a tumor, preferably gastric cancer, esophageal squamous cell carcinoma, melanoma, breast cancer, ovarian cancer, endometrial cancer or colorectal cancer.

[0041] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0042] The reagents and raw materials used in the present invention are commercially available.

[0043] The positive progress effect of the present invention is:

[0044] 1) Based on the chCE7 sequence, the L1CAM-targeting T cell-engaging bispecific antibody CE7-TCE with an IgG-(L)-scFv structure was constructed for the first time. This antibody is close to the natural antibody structure and has a good in vivo half-life.

[0045] 2) Compared with its parental monoclonal antibody chCE7, CE7-TCE exhibits superior anti-tumor activity both in vivo and in vitro, which can further meet clinical needs.

[0046] 3) Currently, the development of L1CAM targets is concentrated in the fields of monoclonal antibodies and chimeric antigen receptor T cell therapy. This invention demonstrates the anti-tumor activity of TCE targeting L1CAM in vivo and in vitro through a series of experiments, further verifying the development potential of this target and enriching L1CAM targeted immunotherapy.

[0047] In this invention, the inventors for the first time employed an IgG-(L)-scFv structure to construct a novel T cell engager, CE7-TCE, targeting L1CAM and CD3. This antibody possesses a full-length IgG conformation and a symmetrical structure, avoiding the light-heavy chain mispairing issues of traditional asymmetric bispecific antibodies, simplifying purification steps and improving antibody production efficiency. Furthermore, the full-length IgG conformation allows for a half-life close to that of native antibodies, theoretically resulting in lower immunogenicity.

[0048] In in vitro functional studies, the inventors determined through cell binding experiments that CE7-TCE has similar binding activity to its parental monoclonal antibody, CE7 mAb. Subsequently, the inventors investigated the mechanism of T cell killing of tumor cells mediated by this antibody. The results demonstrated that the antibody can effectively bridge CD3-positive cells and L1CAM-positive tumor cells, promoting T cell activation and proliferation and the release of multiple cytokines in an L1CAM antigen-dependent manner, with a high safety profile. The inventors also evaluated the cytotoxicity of CE7-TCE on different L1CAM-positive tumor cells. The results showed that CE7-TCE exhibited superior cytotoxicity to the parental monoclonal antibody, CE7 mAb, across multiple tumor cell lines without producing off-target toxicity.

[0049] The evaluation results of the two animal models also demonstrated that the CE7-TCE in this patent has strong anti-tumor activity in mice, and the tumor-suppressing effect of CE7-TCE can be further enhanced when used in combination with Pembrolizumab. These results indicate that CE7-TCE has significant clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is a schematic diagram of the CE7-TCE structure.

[0051] Figure 2 CE7 mAb was analyzed by SDS-PAGE.

[0052] Figure 3 Analyze CE7-TCE for SDS-PAGE.

[0053] Figure 4 SEC-HPLC was used to detect CE7 mAb and CE7-TCE.

[0054] Figure 5 Detection of binding to cell surface antigens.

[0055] Figure 6 CE7-TCE-induced cross-linking between CD3-positive cells and L1CAM-positive cells.

[0056] Figure 7 The cytokine release of PBMC cells co-cultured with NCI-N87 cells and CE7-TCE.

[0057] Figure 8 CE7-TCE-induced T cell proliferation.

[0058] Figure 9This represents the lytic activity of CE7-TCE against L1CAM-positive tumor cells. After adding the test protein and PBMCs, SK-OV-3, NCI-H292, and NCI-N87 cells were co-cultured for 48 hours, and A2780, Caov-3, OVCAR-3, PANC-1, and JIMT-1 cells were co-cultured for 36 hours. Duplicate wells were prepared for each group, and data are presented as mean ± SEM.

[0059] Figure 10 It is the drug-time curve and PK parameters of the antibody.

[0060] Figure 11 Pharmacodynamic analysis of CE7-TCE in the NCI-N87 / PBMC mixed model. To evaluate the in vivo efficacy of CE7-TCE, female NOD / SCID mice (n=5) were inoculated with NCI-N87 (5×10 6 ) and PBMC (1.66×10 6 A mixture of 100 (1000) cells was then treated with PBS, CE7 mAb (5 mg / kg), or CE7-TCE (5 mg / kg) twice weekly for 3 weeks. (a) Tumor growth curves of mice during the study; (b) Mouse body weight curves; (c) Tumor mass weights. Statistical analysis was based on a one-way ANOVA multiple comparison test. Data are expressed as mean ± SD, n = 5. ns: not significantly different; ** P<0.01.

[0061] Figure 12 Figure 3. Effects of CE7-TCE on the expression of immune checkpoint-related molecules in a PBMC / tumor cell co-culture system. (a) Changes in PD-L1 expression on NCI-N87 cells and PD1 expression on PBMC cells in the presence or absence of CE7-TCE; (b) Changes in PD-L1 expression on NCI-H292 cells and PD1 expression on PBMC cells in the presence or absence of CE7-TCE.

[0062] Figure 13 The cytotoxicity of CE7-TCE combined with pembrolizumab against NCI-N87 and NCI-H292 cells was evaluated. Two replicate wells were set up in each group, and data are presented as mean ± SEM.

[0063] Figure 14 To evaluate the in vivo antitumor activity of CE7-TCE combined with Pembrolizumab, female NOD / SCID mice (n=5) were given 5×10 6 NCI-H292 cells and 1.66×10 6PBMCs were then treated with different drugs via tail vein injection twice weekly for 3 weeks. (a) Tumor growth curves of mice during the study. (b) Histogram of tumor mass weights in each group. Statistical analysis was performed using a one-way ANOVA multiple comparison test. Data are presented as mean ± SD, n = 6. *, ***, and **** indicate significant differences compared to the PBS group. * P<0.05, *** P<0.001, **** P<0.0001. # and ## represent significant differences compared with the CE7-TCE group. # P<0.05, ## P<0.01. DETAILED DESCRIPTION

[0064] High expression of L1CAM has been shown to be a predictor of tumor metastasis and poor prognosis, indicating that L1CAM is an important target in cancer treatment. This invention is the first to construct a novel bispecific antibody targeting L1CAM and CD3, which exhibits excellent anti-tumor activity in animal models.

[0065] The IgG-(L)-scFv structure used in the bispecific antibody of the present invention has been used in the study of bispecific antibodies against CD3 and other tumor-associated antigens. In addition, Brian H. Santich et al. (Santich, Park, Tran et al., Science translational medicine, 2020, eaax1315) have demonstrated that due to the appropriate intrachain distance and spatial conformation, the TCE based on the IgG-(L)-scFv structure has stronger tumor inhibitory activity than the bispecific antibodies of the IgG and BiTE structures. Facts have also proved that the bispecific antibody targeting L1CAM and CD3 with the novel IgG-(L)-scFv structure designed by the inventor can not only specifically activate huPBMCs in vitro and mediate target cell killing, but also can exert strong anti-tumor activity in animals.

[0066] In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the procedures in molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA used herein are conventional procedures widely used in the corresponding fields. In addition, for a better understanding of the present invention, the following definitions and explanations of relevant terms are provided:

[0067] In the present invention, the letters in the amino acid sequence represent the single-letter abbreviations of amino acids known in the art, such as those described in J.Biol.Chem, 243, p3558 (1968): alanine: Ala-A, arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T.

[0068] In the present invention, the scFv refers to a single chain antibody fragment, which includes a heavy chain variable region, a light chain variable region and a connecting peptide of 15 to 20 amino acids. The VL and VH domains are paired to form a monovalent molecule by a connecting peptide that enables them to be produced as a single polypeptide chain [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules can have a general structure: NH2-VL-connecting peptide-VH-COOH or NH2-VH-connecting peptide-VL-COOH.

[0069] In the present invention, the IgG antibody refers to immunoglobulin G, whose basic monomeric structure consists of two identical heavy chains and two identical light chains. Disulfide bonds connect the two heavy chains to each other and to a light chain, forming a Y-shaped structure. The heavy chain of an IgG antibody is composed of multiple components, including CH1, CH2, CH3, hinge, and VH (heavy chain variable region). The light chain is composed of VL (light chain variable region) and a kappa chain or a lambda chain.

[0070] As known in the art, "nucleic acid" in the present invention refers to a nucleotide chain of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.

[0071] The recombinant expression vector of the present invention can be any suitable recombinant expression vector that can be used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably express the genes or sequences in the host cell. Suitable vectors include those designed for expansion and amplification or for expression or both, examples of vectors include but are not limited to viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0072] As used herein, the term "host cell" refers to any type of cell that can contain a nucleic acid or vector described herein. In exemplary aspects, the host cell is a eukaryotic cell, such as a plant, animal, fungus, or algae; or it can be a prokaryotic cell, such as a bacterium or protozoa.

[0073] In the present invention, the pharmaceutical composition may include suitable pharmaceutically acceptable carriers such as pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients as known in the art, including buffers."Pharmaceutically acceptable carriers" include any and all solvents, dispersion media, isotonic agents and absorption delay agents that are physiologically compatible. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. The pharmaceutical composition comprising the present invention can be prepared by mixing the bispecific antibodies of the present invention with the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of a lyophilized formulation or an aqueous solution.

[0074] The pharmaceutical composition of the present invention can also include more than one active ingredient, which is required for the specific indication being treated, preferably those active ingredients with complementary activities that do not adversely affect each other. For example, it is desirable to also provide other active ingredients, such as other antibodies, antiviral agents, small molecule drugs or immunomodulators, etc. The active ingredients are suitably combined in an amount effective for the intended use. Sustained release formulations can be prepared, and suitable examples thereof include a semi-permeable matrix of a solid hydrophobic polymer containing the bispecific antibody of the present invention, the matrix being a shaped article, such as a film or microcapsule form.

[0075] As used herein, the term "effective amount" refers to an amount of a drug or pharmaceutical agent that elicits the biological or pharmaceutical response of a tissue, system, animal, or human that is being sought, for example, by a researcher or clinician. Additionally, the term "therapeutically effective amount" refers to an amount that results in improved treatment, cure, prevention, or alleviation of a disease, condition, or side effect, or that reduces the rate of progression of a disease or condition, compared to a corresponding subject that has not received that amount. The term also includes within its scope amounts that are effective to enhance normal physiological function.

[0076] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0077] Example 1 Sources of plasmids, strains and cell lines

[0078] The pcDNA3.4 plasmid was purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0079] Escherichia coli DH5α competent cells were purchased from Suzhou Xinsaimei Company.

[0080] HEK293 F, Jurkat, SK-OV-3, Caov-3, OVCAR-3, A2780, NCI-N87, NCI-H292, PANC-1, and JIMT-1 cell lines were previously maintained in the laboratory. All cell lines were cultured under standard conditions specified by the supplier and used within 2 months of thawing. PBMCs were purchased from Shanghai Saili Biotechnology Co., Ltd.

[0081] Example 2 Design and Construction of Antibodies

[0082] The heavy chain variable region (VH) and light chain variable region (VL) sequences of CE7 monoclonal antibody (CE7 mAb) and CE7-TCE bispecific antibody are derived from the chimeric monoclonal antibody chCE7 (Amstutz, Rytz, Novak-Hofer et al., International journal of cancer, 1993, 147-152). Anti-CE7 mAb was named CE7 mAb by cloning the VH and VL sequences into the wild-type human IgG1κ antibody framework (CE7 mAb light chain is shown in SEQ ID NO: 11, and CE7 mAb heavy chain is shown in SEQ ID NO: 12). Anti-CE7-TCE adopts an IgG-(L)-scFv structure, mainly based on the IgG1 framework, and introduces PGLALA mutations (L234A, L235A and P329G) into the Fc segment. CE7-TCE ( Figure 1, the light chain is shown in SEQ ID NO: 1, and the heavy chain is shown in SEQ ID NO: 8) has a symmetrical molecular structure and normal antibody heavy and light chains, but is fused to the C-terminus of the light chain via a (G4S)3 connecting peptide (SEQ ID NO: 4). The CD3 scFv is formed by connecting the VH and VL of humanized OKT3 (huOKT3 monoclonal antibody, from Adair, Athwal, Bodmer et al., Human Antibodies, 1994, 41-47) with a (G4S)6 connecting peptide (SEQ ID NO: 6).

[0083] The antibody expression sequence was codon-optimized and synthesized by Anhui General Biotechnology Co., Ltd. and constructed into the pcDNA3.4 expression vector. After sequencing verification, it was transformed into Escherichia coli DH5α, and an endotoxin-free sterile expression plasmid was prepared using a plasmid extraction kit.

[0084] The isotype control CD22-TCE has a similar IgG-(L)-scFv configuration, and its sequence is adapted from M971 (U.S. Patent No. 9,598,492; the CD22-TCE light chain sequence is shown in SEQ ID NO: 14, and the CD22-TCE heavy chain sequence is shown in SEQ ID NO: 15). The sequence of the control antibody CD3 mAb is also derived from humanized OKT3 (i.e., huOKT3 monoclonal antibody, from Adair, Athwal, Bodmer et al., Human Antibodies, 1994, 41-47). The CD22-TCE and CD3 mAb proteins were previously constructed and expressed in the laboratory.

[0085] Example 3 Expression and purification of antibodies

[0086] Expression of CE7 mAb and CE7-TCE was performed by transient transfection of HEK293F cells mediated by polyethylenimine (PEI). On the day of transfection, the cell viability was ensured to be above 95%, and the density of HEK293 F cells was adjusted to 1×10 6 cells / mL. The plasmid light chain to heavy chain mass ratio was 2:1. The required amount of plasmid was calculated by adding 0.8 μg per mL of cells. The plasmid was diluted with Opti-MEM medium to a final concentration of 40 ng / μL. PEI was added to the plasmid dilution at a PEI:DNA mass ratio of 2:1 to form a PEI / DNA complex. After incubation at room temperature for 20 minutes, the PEI / DNA complex was slowly added dropwise to the cell culture and mixed thoroughly. Five to six days after transfection, cell viability dropped to approximately 60%. The cell supernatant was collected for purification.

[0087] A MabSelect SuRe affinity chromatography column (Cytiva) was used for affinity purification of the antibody. The column was first equilibrated with a pH 7.2 phosphate equilibration buffer (20 mM NaH2PO4, 150 mM NaCl) and then loaded with the sample. After removing impurities with a pH 5.0 100 mM citric acid buffer, the target protein was eluted with a pH 3.0 100 mM citric acid buffer to collect the target protein, and an appropriate amount of pH 9.0 1 M Tris-HCl buffer was immediately added to neutralize the protein sample.

[0088] The obtained samples were subjected to SDS-PAGE analysis. A small amount of protein sample was added to reducing and non-reducing loading buffer, heated at 95°C for 5 minutes, and the electrophoresis sample was prepared. Figure 2 This is a gel image of CE7 mAb purification. The non-reduced sample shows a single band, while the reduced sample shows two bands, namely a 50 kDa heavy chain and a 25 kDa light chain. Figure 3 This is a CE7-TCE purification gel image. The non-reduced sample shows clear and distinct bands, while the reduced sample shows very close proximity between the light and heavy chain bands. This is due to the increased molecular weight of the light chain after attachment to the scFv, bringing it closer to that of the heavy chain (approximately 50 kDa). The antibody purity, calculated by grayscale value, is above 95%, indicating successful purification of the target protein. The sample was then dialyzed into PBS buffer.

[0089] Example 4 Antibody Purity Detection

[0090] The obtained sample was analyzed for purity by size exclusion chromatography (SEC-HPLC), and the specific method was as follows:

[0091] The protein to be tested was diluted to a concentration of 0.5 mg / mL with PBS and analyzed using a Tosoh TSKgelG3000SWxl column (7.8 mm × 300 mm, 5 μm) and an Agilent 1260 high-performance liquid chromatograph. The mobile phase consisted of 150 mM sodium phosphate, 100 mM NaCl, pH 7.2. 30 μg of sample was injected each time, and elution was performed at a flow rate of 1 mL / min for 20 min. The eluted protein was detected by UV absorption at a wavelength of 280 nm. The results are shown in Figure 2. Figure 4 As shown in Table 1, the main peak of CE7mAb accounted for 98.06%, and the main peak of CE7-TCE accounted for 96.94%, proving that affinity purification was successful and protein samples with a purity of more than 95% could be obtained.

[0092] Table 1 SEC-HPLC detection parameters

[0093]

[0094] Example 5 Binding of CE7-TCE to Cell Surface CD3 and L1CAM Antigens

[0095] The CD3-positive Jurkat cell line and the L1CAM-positive tumor cell lines Caov-3, OVCAR-3 and SK-OV-3 were selected for cell binding activity detection.

[0096] Cells were added to 96-well plates (2 × 10 5 cells), the test proteins CD3 mAb, CE7 mAb, CE7-TCE and isotype control CD22-TCE (Isotype Control) were diluted 6 times in a concentration range of 0.001nM to 500nM, added to the cells and incubated at 4°C for 1 hour. After incubation, the cells were washed three times with wash buffer (2% fetal bovine serum + PBS buffer) and stained with FITC-labeled anti-human IgG antibody (ThermoFisher, 31529). After incubation at 4°C in the dark for 0.5 hours, the cells were washed three times with wash buffer and the proteins bound to the target cells were detected using a flow cytometer (Beckman Coulter). The mean fluorescence signal intensity (MFI) of each sample was recorded and the data were analyzed and processed using GraphPad Prism 8 software to draw the binding curve and calculate the binding EC 50 .

[0097] like Figure 5 As shown in the figure, for CD3-positive Jurkat cell lines, CE7-TCE, CD22-TCE (Isotype control) and CD3 mAb can effectively bind to the cell surface and bind to EC 50 The EC values of CE7-TCE binding to CD3 antigen were 7.959 nM, 6.354 nM and 0.7044 nM, respectively, compared with CD3 mAb. 50The binding capacity increased approximately 10-fold, while the maximum binding rate decreased. This reduction in CD3 antigen binding is likely due to the influence of interchain distance, but it does not necessarily imply reduced activity (Mandikian, Takahashi, Lo et al., Molecular Cancer Therapeutics, 2018, 776-785; Staflin, deZafra, Schutt et al., JCI Insight, 2020). CE7-TCE and CE7 mAb effectively bound to the tumor cell surface of three L1CAM-positive tumor cell lines, with similar fluorescence intensity and binding curves. The isotype control, CD22-TCE, showed no binding to L1CAM-positive cells. CE7-TCE has a symmetrical antibody structure and shares the same antigen-recognition fragment as the CE7 mAb, enabling bivalent binding to the target antigen. This demonstrates that the IgG-(L)-scFv bispecific antibody retains the high affinity of the parent mAb despite the introduction of the scFv fragment at the C-terminus of the light chain. The overall data suggest that CE7-TCE has the activity of binding to two target antigens on the cell surface.

[0098] Example 6 CE7-TCE Bridging T Cells and Tumor Cells

[0099] The ability of CE7-TCE to mediate T cell adhesion to tumor cells was tested. Since Jurkat cells express CD3, Jurkat cells were used instead of T cells to evaluate antibody-mediated cell adhesion.

[0100] Following the instructions in the reagent manual, CD3-positive Jurkat cells were labeled with PKH26 (Sigma Aldrich, PKH26GL), and L1CAM-positive NCI-N87 cells were labeled with CellTrace CFSE (ThermoFisher, C34570). The two labeled cells were then mixed in a 1:1 ratio using washing buffer (2% FBS + PBS) at a cell density of 1×10 6 cells / mL. 1 μg / mL of test protein (CE7 mAb, CE7 mAb + CD3 mAb, and CE7-TCE) was then added, and the cell mixture was incubated in the dark for 30 min. The cells were gently washed three times with wash buffer and then analyzed by flow cytometry. Cross-linking of Jurkat and NCI-N87 cells showed PKH26 + / CFSE + (PE / FITC) double-positive cell population, so the proportion of cell cross-linking was obtained by counting the proportion of double-positive cells in the upper right corner of the flow cytometry histogram.

[0101] like Figure 6 As shown, compared with the blank control group, CE7-TCE effectively induced cross-linking of Jurkat and L1CAM-positive NCI-N87 cells (13%). Addition of CE7 mAb alone or in combination with CD3 mAb did not increase the proportion of FITC / PE double-positive cells. This experiment confirms the cell redirecting effect of CE7-TCE, which can specifically recruit T cells to the vicinity of L1CAM-positive tumor cells. Therefore, CE7-TCE can act as a cell adaptor to connect T cells and tumor target cells, thereby guiding the tumor immune response.

[0102] Example 7 CE7-TCE antigen specifically activates T cells to release cytokines

[0103] In the cytokine release assay, NCI-N87 cells (1.2×10 4 ) were seeded into 96-well plates and cultured overnight. Then, effector PBMCs (1.2×10 per well) were added. 5 The effector cell / target cell ratio was 10:1. After co-culture with cells for 27 hours, CD22-TCE (isotype control) and CE7-TCE were added, and the cell supernatant was collected. The cytokines IL-2 and IFN-γ released by PBMCs were detected using the Human IL-2 DuoSet ELISA Kit (R&D Systems, DY202) and the Human IFN-gamma DuoSet ELISA Kit (R&D Systems, DY285B) according to the manufacturer's instructions.

[0104] The results showed that CE7-TCE induced T cell activation in a dose-dependent manner, and the levels of IL-2 and IFN-γ in the cell supernatant were significantly increased ( Figure 7 ), while the isotype control CD22-TCE (Isotype control) did not increase cytokine secretion. This indicates that the novel CE7-TCE bispecific antibody constructed in the present invention has strong T cell activation ability and can promote T cells to secrete cytokines to fight tumors.

[0105] Example 8 CE7-TCE-mediated T cell proliferation and activation

[0106] NCI-H292 cells were added to 96-well plates (2 × 10 4 The next day, fresh PBMC cells were stained with CellTrace CFSE (ThermoFisher, C34570) at a final concentration of 1 μM and 2×10 5Cells were cultured at a density of 1000 cells / mL into NCI-H292 cell cultures, and 1 μg / mL of CE7-TCE or CD22-TCE (isotype control) was added. After incubation at 37°C in the dark for 96 hours, PBMCs were harvested and cell proliferation was analyzed by flow cytometry.

[0107] The results showed that compared with the isotype control CD22-TCE (Isotype control) Figure 8 d), after adding CE7-TCE, significant T cell proliferation was observed, and CFSE weakly positive cells accounted for 35.12% ( Figure 8 Furthermore, in conjunction with the results in Example 7, in the absence of L1CAM-positive tumor cells, the addition of CE7-TCE alone to PBMCs did not induce T cell proliferation ( Figure 8 b) and cytokine release ( Figure 7 ), which proves that the activation of T cells by CE7-TCE is target-dependent, thereby minimizing the risk of nonspecific activation and preliminarily confirming the safety of CE7-TCE.

[0108] Example 9 CE7-TCE mediates PBMC killing of tumor cells in vitro

[0109] To evaluate the in vitro cytotoxicity of CE7 mAb, CE7-TCE, and CD22-TCE (Isotype control), the inventors selected seven L1CAM-positive tumor cells and one L1CAM-negative tumor cell as target cells, and used huPBMC as effector cells for the experiment. Different tumor cells (target cells) were seeded into 96-well plates and cultured overnight, and then serially diluted antibodies (from 5×10 -6 nM to 50nM). PBMCs were added to the well plates at an effector cell / target cell ratio of 12.5:1. After incubation at 37°C for 36 or 48 hours, the cells were centrifuged at 1000 rpm for 10 minutes, and the culture supernatant was collected. Tumor cell killing efficiency was determined by measuring LDH released in the culture supernatant after tumor cell lysis. LDH levels were measured using the CytoTox96 Non-Radioactive Cytotoxicity Assay Kit (Promega, G1780). The target cell lysis efficiency was calculated as follows:

[0110] Cytotoxicity (%) = (experimental cell lysis rate - PBMC cell spontaneous lysis rate - target cell spontaneous lysis rate) / (target cell maximum lysis rate - target cell spontaneous lysis rate) × 100%.

[0111] The results are as follows Figure 9As shown in Figure 2, CE7-TCE showed stronger cytotoxicity than CE7 mAb for L1CAM-positive cells, as evidenced by a lower EC 50 The killing platform of NCI-N87 cells and SK-OV-3 cells was higher ( Figure 9 , Table 2). This indicates that CE7-TCE has a better in vitro target cell killing effect than the parental monoclonal antibody CE7 mAb.

[0112] Table 2 EC values of the test proteins for lysis of 8 tumor cell lines 50 value

[0113]

[0114] ND, no cell lytic activity was observed.

[0115] For A2780 cells (L1CAM expression negative, Figure 9 ), no target cell lysis was detected in either the CE7 mAb or CE7-TCE groups, indicating that the tumor killing mediated by them is antigen-dependent. This suggests that CE7-TCE has limited toxicity against L1CAM-negative normal tissues in vivo. CD22 is an adhesion cytokine expressed restrictively on B cells. The inventors observed that CD22-TCE (isotype control) had no killing effect on L1CAM-positive tumor cells, indicating that cell killing mediated by the IgG-(L)-scFv-structured T cell-redirecting bispecific antibody is not guided by the CD3 arm but is instead dependent on the antigen-targeting arm.

[0116] Example 10 In vivo pharmacokinetic testing

[0117] Male BALB / c mice aged 6-8 weeks were grouped by weight and received a single tail vein injection of 5 mg / kg of CE7 mAb and CE7-TCE. Blood samples were collected before dosing and at 0.25 hours, 8 hours, and 1, 2, 3, 6, 10, 15, 21, and 28 days after dosing. Plasma samples were centrifuged at 3000 rpm and 4°C for 15 minutes, and the supernatant was aspirated and stored at -80°C for determination of plasma antibody concentrations. Plasma antibody concentrations were determined by ELISA using an anti-human IgGκ chain antibody (Merck, AP502) as the primary antibody and an HRP-anti-human IgG Fc antibody (ThermoFisher, A18829) as the secondary antibody. PK parameters for CE7 mAb and CE7-TCE were calculated using a non-compartmental model using the PKsolver program.

[0118] From the perspective of the antibody plasma concentration-time curve, the drug-time curves of CE7-TCE and CE7 mAb are similar ( Figure 10 ), indicating that the metabolic activity of CE7-TCE after the scFv was linked to the C-terminus was not significantly affected compared with the parental monoclonal antibody CE7 mAb. The maximum plasma concentration of CE7-TCE was higher than that of CE7 mAb, but the clearance rate was also faster. The two had similar elimination half-lives, 14.98 days and 18.74 days, respectively. A long half-life means stable blood drug levels, fewer doses are required to maintain effective blood drug concentrations, and improved drug administration convenience has advantages in clinical applications. The longer half-life of CE7-TCE may be due to the IgG1 skeleton, in which the presence of Fc has been shown to effectively prolong the half-life (Peters, Blood, 2013, SCI-8; Strohl, BioDrugs, 2015, 215-239).

[0119] Example 11 Efficacy of CE7-TCE in the NCI-N87 / PBMC hybrid model

[0120] 5×10 6 NCI-N87 cells and 1.66×10 6 PBMC cells were mixed with 100 μL of RPMI-1640 medium (ThermoFisher, 11875119) and injected subcutaneously into the right armpit of female NOD / SCID mice (6-8 weeks old). The mice were randomly divided into three groups (5 mice in each group) according to their body weight. The control group PBS, CE7 mAb (5 mg / kg) and CE7-TCE (5 mg / kg) were injected into the tail vein twice a week for a total of six times. The body weight and tumor size (length and width) of the mice were measured twice a week, and a line graph of the tumor volume was drawn. The formula for calculating the tumor volume is: tumor volume = tumor length × tumor width × tumor width / 2. When the tumor volume of the mouse exceeds 1000 mm 3 The experiment was terminated at 4 hr, and the tumor was removed and weighed.

[0121] The results show that ( Figure 11 ) CE7-TCE showed superior tumor inhibitory effects compared to the parental mAb in the NCI-N87 / PBMC model. Compared with the PBS group, at a dose of 5 mg / kg, CE7-TCE effectively inhibited NCI-N87 tumor growth. There was no significant difference between the CE7 mAb and PBS groups. Figure 11 After the experiment, the tumor was peeled off and tested. The tumor mass in the CE7-TCE group was significantly lower than that in the PBS group ( Figure 11 c). Preliminary in vivo efficacy studies demonstrated that CE7-TCE could effectively inhibit the growth of L1CAM-positive NCI-N87 xenograft tumors compared to the parental CE7 monoclonal antibody. The body weight of mice did not decrease significantly during the entire treatment process due to drug administration ( Figure 11b), indicating that the treatment with CE7 mAb and CE7-TCE did not induce severe systemic toxicity.

[0122] Example 12 Detection of Upregulated Expression of Immunosuppressive Molecules During Tumor Killing by CE7-TCE

[0123] To examine the effect of CE7-TCE on PD1 expression on PBMCs and PD-L1 expression on tumor cells, PBMCs and target cells (NCI-N87 or NCI-H292) were mixed at a 10:1 ratio and co-cultured with CE7-TCE (1 μg / mL) for 24 hours. PBMCs were then harvested and stained with a PE-labeled anti-PD1 antibody (Sino Biological, 10377-M140-P). NCI-N87 or NCI-H292 tumor cells were harvested and stained with an APC-labeled anti-PD-L1 antibody (Sino Biological, 10084-MM36-A). PD1 and PD-L1 expression were determined by flow cytometry after staining according to the manufacturer's instructions.

[0124] The results showed that co-culture with PBMCs alone could induce NCI-N87 cells ( Figure 12 a) and NCI-H292 cells ( Figure 12 b) PD-L1 expression on the surface is upregulated, and the addition of CE7-TCE further increases PD-L1 expression. For PBMC cells, there is no significant change in PD1 expression after co-culture with tumor cells alone, but upregulation of PD1 expression is observed after the addition of CE7-TCE ( Figure 12 These results suggest that CE7-TCE-induced T cell-mediated tumor killing is accompanied by PD1-PD-L1 interaction, resulting in immunosuppression. Blocking PD1 inhibition may restore T cell anti-tumor activity and enhance the efficacy of CE7-TCE.

[0125] Example 13: Evaluation of the efficacy of the combined use of CE7-TCE and PD1 mAb

[0126] To test the synergistic cytotoxicity of the combination of CE7-TCE and anti-PD1 mAb (Pembrolizumab) against tumor cells in vitro, NCI-H292 cells (1×10 4 ) and NCI-N87 cells (1×10 per well) 4PBMCs were seeded into 96-well plates and cultured overnight. CE7-TCE was then diluted in series and a fixed concentration of pembrolizumab (7 nM) was added. PBMCs were added to the culture medium of NCI-N87 and NCI-H292 cells at effector / target cell ratios of 10:1 and 15:1, respectively. After 36 hours of culture, LDH levels in the culture supernatant were measured.

[0127] By calculating the target cell lysis rate, the inventors found that the addition of Pembrolizumab did not change the killing EC of CE7-TCE. 50 The maximum lysis rate of the NCI-N87 group increased from 21.86% to 33.82% ( Figure 13 a), the maximum lysis rate of the NCI-H292 group increased from 64.81% to 85.07% ( Figure 13 b), demonstrating that combined pembrolizumab blockade of PD1 can indeed enhance the anti-tumor activity of CE7-TCE.

[0128] For the efficacy of CE7-TCE and Pembrolizumab combination in NCI-H292 / PBMC mixed model, 5×10 6 NCI-H292 cells and 1.66×10 6 PBMCs were pre-mixed in 100 μL of RPMI-1640 medium and subcutaneously inoculated into the right axilla of female NOD / SCID mice. Mice were randomly divided into four groups (n=6) based on body weight and treated the following day with PBS, pembrolizumab (5 mg / kg), CE7-TCE (5 mg / kg), or a combination of CE7-TCE and pembrolizumab (5 mg / kg).

[0129] CE7-TCE was injected into the tail vein twice a week, and Pembrolizumab was injected intraperitoneally once a week for three weeks. Tumor size was measured twice a week. 3 After the experiment, the mice were euthanized with carbon dioxide, and the tumor tissues were removed and weighed.

[0130] From the tumor growth Figure 14 According to a), 5mg / kg of Pembrolizumab inhibited tumor growth by 30.1%, CE7-TCE significantly inhibited tumor growth by 61.5%, and the combination of CE7-TCE and Pembrolizumab produced a synergistic effect, with a tumor inhibition rate of 84.8%. The experiment was terminated on day 25, and tumors were collected and weighed. The tumor weight of the combination group was the smallest ( Figure 14 b), further proved that the combination of CE7-TCE and PD1 monoclonal antibody can inhibit tumor growth to the greatest extent. Amino acid sequence:

[0131]

[0132]

Claims

1. A bispecific antibody comprising a first functional region and a second functional region, wherein the first functional region and the second functional region are connected by a connecting peptide, characterized in that: The first functional region is an anti-L1CAM antibody, and the second functional region is an anti-CD3 antibody; The amino acid sequence of the light chain variable region of the anti-L1CAM antibody is shown in SEQ ID NO: 2, and / or the amino acid sequence of the heavy chain variable region of the anti-L1CAM antibody is shown in SEQ ID NO: 9; and / or the amino acid sequence of the light chain variable region of the anti-CD3 antibody is shown in SEQ ID NO: 7, and / or the amino acid sequence of the heavy chain variable region of the anti-CD3 antibody is shown in SEQ ID NO:

5.

2. The bispecific antibody according to claim 1, wherein The anti-L1CAM antibody is an IgG antibody, and the anti-CD3 antibody is a scFv antibody; and / or, the N-terminus of the scFv antibody is connected to the C-terminus of the light chain of the IgG antibody via a connecting peptide; Preferably, the heavy chain constant region of the anti-L1CAM antibody is derived from the heavy chain of a human antibody, and / or; the light chain constant region of the anti-L1CAM antibody is derived from the κ chain of a human antibody; More preferably, the Fc segment of the heavy chain constant region of the anti-L1CAM antibody comprises a PGLALA mutation; and / or the CH1 segment of the heavy chain constant region of the anti-L1CAM antibody comprises an F126C mutation and the light chain constant region of the anti-L1CAM antibody comprises an S121C mutation; More preferably, the amino acid sequence of the heavy chain constant region of the anti-L1CAM antibody is shown as SEQ ID NO: 10 or SEQ ID NO: 13; and / or the amino acid sequence of the light chain constant region of the anti-L1CAM antibody is shown as SEQ ID NO:

3.

3. The bispecific antibody according to claim 1 or 2, wherein The amino acid sequence of the connecting peptide connecting the IgG antibody and the scFv antibody is shown in SEQ ID NO: 4, and / or the amino acid sequence of the connecting peptide connecting the light chain variable region and the heavy chain variable region of the scFv antibody is shown in SEQ ID NO: 6; Preferably, the amino acid sequence of the heavy chain of the bispecific antibody is shown as SEQ ID NO: 8, and / or the amino acid sequence of the light chain of the bispecific antibody is shown as SEQ ID NO:

1.

4. An isolated nucleic acid, characterized in that The nucleic acid encodes the bispecific antibody according to any one of claims 1 to 3.

5. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the isolated nucleic acid according to claim 4; preferably, the backbone of the recombinant expression vector is pcDNA3.

4.

6. A transformant, characterized in that The transformant comprises the isolated nucleic acid according to claim 4 or the recombinant expression vector according to claim 5; preferably, the host cell of the transformant is a prokaryotic cell or a eukaryotic cell; more preferably, the prokaryotic cell is Escherichia coli, such as Escherichia coli DH5α.

7. A method for preparing the bispecific antibody according to any one of claims 1 to 3, characterized in that: The preparation method comprises: culturing the transformant according to claim 6, and obtaining the bispecific antibody from the culture.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the bispecific antibody according to any one of claims 1 to 3, and a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition further contains an inhibitor that blocks the interaction between PD1 and PD-L1; More preferably, the inhibitor is an anti-PD1 antibody, such as Pembrolizumab.

9. Use of the bispecific antibody according to any one of claims 1 to 3 and / or the pharmaceutical composition according to claim 8 in the preparation of a medicament for diagnosing, preventing and / or treating a disease or condition mediated by L1CAM; Preferably, the disease or condition is a tumor, preferably gastric cancer, esophageal squamous cell carcinoma, melanoma, breast cancer, ovarian cancer, endometrial cancer or colorectal cancer.

10. A kit, characterized in that The kit comprises the bispecific antibody according to any one of claims 1 to 3 and / or the pharmaceutical composition according to claim 8; Preferably, the kit further comprises (i) a device for administering the bispecific antibody or pharmaceutical composition; and / or (ii) instructions for use.

11. A medicine kit comprising medicine box A and medicine box B, characterized in that: The drug kit A contains the bispecific antibody according to any one of claims 1 to 3; the drug kit B contains an inhibitor that blocks the interaction between PD1 and PD-L1, preferably an anti-PD1 antibody such as Pembrolizumab; Alternatively, the medicine kit A contains the pharmaceutical composition according to claim 8; and the medicine kit B contains other anti-tumor antibodies or a pharmaceutical composition containing the other anti-tumor antibodies.

12. A method for immunodetection or determination of L1CAM and / or CD3, characterized in that: The method comprises using the bispecific antibody according to any one of claims 1 to 3 and / or the pharmaceutical composition according to claim 8; preferably, the detection is for non-diagnostic and / or therapeutic purposes.

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