Bispecific binding protein structure of T-cell connector, nucleic acid, carrier, cell, and preparation method and application of bispecific binding protein structure

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a bispecific binding protein structure of a T cell connector, nucleic acid, a carrier, a cell and a preparation method and application of the bispecific binding protein structure. The invention provides a bispecific binding protein of a T cell connector, the bispecific binding protein comprises a TCE double antibody structure of an anti-CD3 antibody sequence, a Fab / Fab-Fab Fc structure or a Fab / Fab-scFv Fc structure is used to construct a bispecific antibody of a 2 + 1 valence tumor-associated antigen and a CD3 protein, and the bispecific binding protein has good tumor cell killing activity after being combined with different TAAs.

Description

Technical Field

[0001] This application belongs to the field of biomedicine, specifically relating to a bispecific binding protein structure of a T-cell connector, nucleic acid, carrier, cell, its preparation method and application. Background Technology

[0002] Cancer, a major challenge in global public health, has become one of the leading causes of death worldwide, according to the latest statistics from the World Health Organization. While traditional surgical resection, radiotherapy, and chemotherapy have played a significant role in the history of cancer treatment, they have many limitations. Surgical treatment is only suitable for early-stage cancer and often leaves small lesions; radiotherapy and chemotherapy work by destroying cancer cell DNA or inhibiting their proliferation, but due to their lack of specificity, they inevitably damage normal tissue cells, leading to serious side effects such as bone marrow suppression and gastrointestinal reactions. Furthermore, tumor cells are prone to developing drug resistance through gene mutations, causing disease recurrence and deterioration.

[0003] In recent years, antibody-based immunotherapy has brought revolutionary breakthroughs to cancer treatment due to its high specificity and relatively few side effects. However, monoclonal antibody therapy faces insurmountable obstacles. The high heterogeneity of tumor cells is manifested in the significant differences in gene expression and antigen phenotype among cells within the same tumor tissue, making it difficult for monoclonal antibodies targeting a single point to cover all tumor cells. In the tumor microenvironment, immunosuppressive cells such as tumor-associated fibroblasts and myeloid-derived suppressor cells, as well as immunosuppressive cytokines such as transforming growth factor-β (TGF-β) and interleukin-10 (IL-10), jointly construct an immune escape barrier, severely weakening the ability of monoclonal antibodies to activate the immune system.

[0004] Against this backdrop, bispecific antibodies, as an innovative product in the field of antibody engineering, are endowed with the ability to simultaneously bind to two different antigens or epitopes through genetic engineering or chemical conjugation techniques. This unique structure enables them to precisely mediate the interaction between effector cells and tumor cells, achieving targeted killing of tumor cells and effectively circumventing the limitations of monoclonal antibody therapy.

[0005] Among the bispecific antibody family, CD3-targeting bispecific antibodies have become a focus of research in tumor immunotherapy. The CD3 molecule, composed of subunits such as γ, δ, ε, and ζ, together with the T cell receptor (TCR) αβ chain, forms the TCR-CD3 complex, playing a central role in T cell antigen recognition, signal transduction activation, and cell proliferation and differentiation. CD3 bispecific antibodies act as a molecular-level "bridge," specifically binding to the CD3 molecule on the surface of T cells at one end, activating the cytotoxic function of T cells; at the other end, they recognize tumor-associated antigens (such as EpCAM and HER2) or tumor-specific antigens (such as neoantigens) highly expressed on the surface of tumor cells, precisely guiding T cells to the vicinity of tumor cells. This prompts T cells to release cytotoxic substances such as perforin and granzymes, inducing tumor cell apoptosis, while simultaneously breaking the immunosuppressive state of the tumor microenvironment and reshaping the body's anti-tumor immune response.

[0006] Currently, the development of CD3 bispecific antibodies has achieved phased results, but it has also exposed many problems that urgently need to be solved. Catuximab, an early approved antibody, uses a complete IgG antibody structure. By binding its Fc fragment to the Fc receptor, it achieves a relatively long half-life in vivo. However, the immunogenicity caused by its murine component significantly reduces therapeutic efficacy, and some patients have experienced severe allergic reactions, limiting its clinical application. Amgen's belintumomab uses a single-chain variable fragment (scFv) fusion form. While it has advantages such as small molecular weight (approximately 55 kDa), strong tissue penetration, and low production cost, the lack of a stable Fc fragment structure leads to poor in vivo stability and a half-life of only about 2-3 hours. Continuous intravenous infusion is required, significantly increasing the treatment burden and potential side effects risk for patients.

[0007] Furthermore, existing CD3 bispecific antibodies still face key technological bottlenecks in structural design and manufacturing processes. At the binding activity level, precise control of affinity with CD3 and tumor antigens is crucial. Excessive affinity may lead to T cell overactivation, triggering serious adverse reactions such as cytokine release syndrome (CRS) and neurotoxicity; insufficient affinity, on the other hand, fails to effectively activate T cells, impacting therapeutic efficacy. Regarding manufacturing processes, some CD3 bispecific antibodies suffer from low expression levels (e.g., some recombinant proteins have expression levels below 1 g / L), high aggregation tendency, and high purification difficulty, resulting in high production costs and severely restricting their large-scale industrial production and widespread clinical application.

[0008] Therefore, developing novel CD3 bispecific antibody structures with excellent stability, high tumor targeting specificity, and good production performance has become a key breakthrough direction for promoting innovation in tumor immunotherapy technology and improving the quality of life of cancer patients. Summary of the Invention

[0009] Based on this, one embodiment of this application provides a bispecific binding protein of a T-cell connector, nucleic acid, carrier, cell, and its preparation method and application.

[0010] This application provides a dual-specific binding protein structure for a T-cell connector, comprising:

[0011] Bivalent binding units targeting the target protein and monovalent binding units targeting the CD3 protein;

[0012] The bivalent binding unit targeting the target protein comprises a Fab domain 1 and a Fab domain 2; the Fab domain 1 and Fab domain 2 are located at different ends;

[0013] The monovalent binding unit targeting the CD3 protein contains a Fab domain or an scFv domain.

[0014] The heavy chain C-terminus of the Fab domain on one side of the divalent binding unit of the target protein is connected to the N-terminus of the Fab domain or scFv domain of the target CD3 protein via a linker.

[0015] In one embodiment, the amino acid sequence of the linker is (GGGGX)n, where X is G or S, and n is a natural number from 1 to 6.

[0016] In one embodiment, the monovalent binding unit targeting the CD3 protein is a Fab domain; the amino acid sequence of the heavy chain variable region of the monovalent binding unit targeting the CD3 protein is shown in SEQ ID NO.2; and the light chain variable region of the monovalent binding unit targeting the CD3 protein is shown in SEQ ID NO.1.

[0017] In one embodiment, the heavy chain of the monovalent binding unit targeting the CD3 protein is shown in SEQ ID NO.3; and the light chain of the monovalent binding unit targeting the CD3 protein is shown in SEQ ID NO.4.

[0018] In one embodiment, the monovalent binding unit targeting the CD3 protein is an scFv domain; the heavy chain variable region of the monovalent binding unit targeting the CD3 protein is shown in SEQ ID NO.5, and the light chain variable region of the monovalent binding unit targeting the CD3 protein is shown in SEQ ID NO.6.

[0019] In one embodiment, the target protein includes one or both of the GPC3 protein and the AMHR2 protein.

[0020] In one embodiment, the heavy chain of the Fab domain binding to the GPC3 protein is shown in SEQ ID NO.7; the light chain of the Fab domain binding to the GPC3 protein is shown in SEQ ID NO.8.

[0021] The heavy chain of the Fab domain binding to the AMHR2 protein is shown in SEQ ID NO.9; the light chain of the Fab domain binding to the AMHR2 protein is shown in SEQ ID NO.10.

[0022] This application also provides a nucleic acid molecule containing a bispecific binding protein structure for encoding a T-cell connector as described above.

[0023] This application also provides a carrier comprising the aforementioned nucleic acid molecules.

[0024] This application also provides a cell comprising the aforementioned nucleic acid molecules or the aforementioned vector.

[0025] This application also provides a method for preparing the above-mentioned cells, the method comprising the step of introducing the nucleic acid molecule or the vector into the target cell.

[0026] This application also provides a method for preparing the above-mentioned bispecific binding protein structure of the T-cell connector, the preparation method comprising the following steps:

[0027] The cells described above were cultured, and bispecific binding protein structures of T-cell connectors were isolated from the resulting cultures.

[0028] This application also provides a medicament comprising the above-described bispecific binding protein structure of the T-cell connector.

[0029] This application also provides the use of the above-mentioned bispecific binding protein structure of the T-cell connector, the above-mentioned nucleic acid molecule, the above-mentioned carrier, or the above-mentioned cell in the preparation of a drug for treating tumors.

[0030] This application provides a bispecific binding protein structure for a T-cell connector, comprising a TCE bispecific antibody structure containing an anti-CD3 antibody sequence. The bispecific antibody constructed using this structure, consisting of a 2+1 valence tumor-associated antigen (TAA) and CD3 protein, exhibits good tumor cell killing activity when combined with different TAAs. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 The structure of a bispecific binding protein of a T-cell connector provided in one embodiment of this application;

[0033] Figure 2 SDS-PAGE images of CR17-BA15N and CR04-BA12 provided in an embodiment of this application;

[0034] Figure 3 CR17-BA15N, provided in one embodiment of this application, can significantly bind to the CD3δε protein in humans and monkeys;

[0035] Figure 4 CR17-BA15N provided in one embodiment of this application can bind to human T cell line Jurkat cells and liver cancer cells HepG2;

[0036] Figure 5 Detection of the tumor cell killing activity of CR17-BA15N and CR04-BA12 provided in an embodiment of this application;

[0037] Figure 6 SDS-PAGE images of CR17-BA28 and CR04-BA7 provided in an embodiment of this application;

[0038] Figure 7 Detection of the binding activity of CR17-BA28 and CR04-BA7 with GPC3 or hAMHR2 positive tumor cells provided in an embodiment of this application;

[0039] Figure 8 This application provides an embodiment for the detection of the binding activity of CR17-BA28 and CR04-BA7 with human T cells;

[0040] Figure 9 The tumor cell killing activity of CR17-BA28 and CR04-BA7 provided in one embodiment of this application was detected. Detailed Implementation

[0041] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0042] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0043] the term

[0044] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0045] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0046] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0047] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0048] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0049] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0050] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0051] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0052] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0053] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0054] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0055] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0056] The term "CD3 (Cluster of Differentiation 3)" is an important cell surface protein complex, primarily found on T cells. It consists of multiple peptide chains, typically including γ, δ, ε, and ζ chains. The main function of CD3 is to work with the T cell receptor (TCR) in T cell activation and signal transduction.

[0057] The term "AMHR2" stands for Anti-Mullerian Hormone Receptor Type 2, a transmembrane protein belonging to the TGF-β (transforming growth factor-beta) superfamily of receptors. AMHR2 plays a role in various biological processes, including reproductive system development and tumorigenesis. AMHR2 expression levels can serve as biomarkers for certain tumors, aiding in diagnosis and prognostic assessment. Furthermore, AMHR2 is a potential target for developing novel cancer treatment strategies, particularly for tumors that express AMHR2.

[0058] The term "GPC3 (Glypican-3)" stands for phosphatidylinositol proteoglycan 3, a cell surface glycosylphosphatidylinositol (GPI) anchoring protein belonging to the heparin sulfate proteoglycan family. GPC3 plays a role in various biological processes, including cell proliferation, differentiation, and signal transduction. GPC3 expression levels can serve as biomarkers for hepatocellular carcinoma and other tumors, aiding in diagnosis and prognostic assessment. Furthermore, GPC3 is a potential target for developing novel cancer treatment strategies, especially for tumors that express GPC3.

[0059] The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target (such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination thereof) through at least one antigen recognition site located within the variable region of an immunoglobulin molecule. As used herein, the term "antibody" encompasses complete polyclonal antibodies, complete monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibody-containing fusion proteins, and any other modified immunoglobulin molecule, provided that the antibody exhibits the desired biological activity. Antibodies may be any of the five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), designated α, δ, ε, γ, and μ, respectively, based on the characteristic of their heavy chain constant structure. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional conformations. Antibodies may be naked or conjugated to other molecules (such as toxins, radioisotopes, etc.).

[0060] The term "antibody fragment" refers to a portion of a complete antibody. "Antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to a portion of a complete antibody that specifically binds to an antigen. An antigen-binding fragment may contain an antigen recognition site of the complete antibody (e.g., a CDR region (complementarity-determining region) sufficient to specifically bind to an antigen). Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab'.F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. Antigen-binding fragments of antibodies may be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or may be artificially generated.

[0061] The terms “anti-CD3 antibody,” “CD antibody,” and “antibody that binds to CD3” refer to antibodies that specifically bind to CD3 with sufficient affinity, making them suitable for use as diagnostic and / or therapeutic agents targeting CD3. As used herein, the terms “specifically bind,” “immunely specifically bind,” “immunely specifically recognize,” and “specifically recognize” are similar terms in the context of antibodies or their antigen-binding fragments. These terms indicate that the antibody or its antigen-binding fragment binds to the epitope via its antigen-binding domain, and that binding requires a degree of complementarity between the antigen-binding domain and the epitope.

[0062] As used herein, the terms “variable region” or “variable domain” are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a portion of the light or heavy chain, typically about 110 to 120 or 110 to 125 amino acids from the amino terminus of the mature heavy chain and about 90 to 115 amino acids from the mature light chain, which differ in sequence between antibodies and are responsible for the binding and specificity of a particular antibody to its specific antigen. Sequence variability is concentrated in those regions called CDRs, while more conserved regions within a variable domain are called FR regions (Framework regions). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for antibody-antigen interactions and specificity. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region is a rodent or mouse variable region.

[0063] The term "heavy chain variable region" consists of 4 FR regions and 3 CDR regions. According to the IMGT database, the genes of the heavy chain variable region are defined as the V gene, D gene, and J gene.

[0064] The term "light chain variable region" consists of 4 FR regions and 3 CDR regions. According to the IMGT database, the genes of the light chain variable region are defined as the V gene and the J gene.

[0065] The term "heavy chain" generally includes one variable region and three constant regions (CH1 / CH2 / CH3).

[0066] The term "light chain" generally includes one variable region and one constant region (CL).

[0067] As used herein, the terms “constant region” and “constant domain” are interchangeable and have their common meanings in the art. A constant region is an antibody portion that does not directly participate in antibody-antigen binding but can exhibit a variety of effector functions, such as an antibody portion interacting with an Fc receptor, or, for example, the carboxyl-terminal portion of the light chain and / or heavy chain. Compared to the variable domains of immunoglobulins, the constant regions of immunoglobulin molecules typically have a more conserved amino acid sequence. In some respects, antibody or antigen-binding fragments contain a constant region or a portion thereof sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0068] As used herein, based on the amino acid sequence of constant structural domains, the term "heavy chain" in relation to antibody use can refer to any different type, for example, α, δ, ε, γ, and μ that produce antibodies of the IgA, IgD, IgE, IgG, and IgM classes, including subclasses of IgG, such as IgG1, IgG2, IgG3, and IgG4. The amino acid sequence of the heavy chain is well known in the art. In a specific embodiment, the heavy chain is a human heavy chain. In a specific embodiment, the heavy chain is a rodent or mouse heavy chain.

[0069] As used herein, based on the amino acid sequence of a constant domain, the term "light chain" can refer to any different type, such as κ or λ, in relation to antibody use. Light chain amino acid sequences are well known in the art. In a specific embodiment, the light chain is a human light chain. In a specific embodiment, the light chain is a rodent or mouse light chain.

[0070] Or an antigen-binding fragment thereof, wherein such antibody or antigen-binding fragment thereof is prepared using any technique known in the art. This definition of human antibody or antigen-binding fragment thereof includes complete or full-length antibodies and fragments thereof.

[0071] "Binding affinity" generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or its antigen-binding fragment) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody or its antigen-binding fragment and an antigen). The affinity of molecule X for its partner Y can generally be represented by a dissociation constant (K). Affinity can be measured and / or represented in a variety of ways known in the art, including but not limited to the equilibrium dissociation constant.

[0072] As used herein, "epitope" is a term in the art and refers to a localized region of an antigen to which an antibody or its antigen-binding fragment can specifically bind. An epitope can be, for example, a continuous amino acid sequence of a polypeptide (linear or continuous epitope), or an epitope can be, for example, derived from two or more discontinuous regions of one or more polypeptides (conformal, nonlinear, discontinuous, or non-continuous epitopes). In some embodiments, the epitope to which the antibody or its antigen-binding fragment specifically binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assays, hydrogen / atmosphere exchange in combination with mass spectrometry (e.g., liquid chromatography-electrospray ionization mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any method known in the art (e.g., Giegé R et al., (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303). Antibody / its antigen-binding fragment: Antigen crystals can be studied using well-known X-ray diffraction techniques and can be improved using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, for example, Meth Enzymo1 (1985) Vols. 114 and 115, edited by Wyckoff HW et al.; US2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt1): 37-60; Bricogne G (1997) Meth Enzymo 276A: 361-423, edited by Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt10): 1316-1323). Mutagenesis mapping studies can be performed using any method known to those skilled in the art. For descriptions of mutagenesis techniques, including alanine scanning mutagenesis, see, for example, Champe M et al., (1995) JBiol Chem 270: 1388-1394 and Cunningham BC and Wells JA (1989) Science 244: 1081-108.

[0073] If an antibody binds to a given epitope or overlapping epitope of a reference antibody such that it partially blocks the binding of the reference antibody to the epitope, then it can be said that the antibody "competitively inhibits" the binding of the reference antibody to the epitope. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. It can be said that the antibody competitively inhibits the binding of the reference antibody to the given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0074] The “sample” used in this application has a biological origin. In a preferred embodiment, the sample is a human sample, but animal samples may also be used in the practice of this application. Non-limiting sources of samples used in this application include, for example, solid tissues, biopsies, ascites, aspirates, fluid leachates, blood (including circulating tumor cells), plasma, serum, cerebrospinal fluid, lymph, external sections of skin, respiratory tract, intestinal and genitourinary tract, tears, saliva, breast milk, tumors, organs, cell cultures and / or cell culture components.

[0075] Terms such as “treatment,” “curative,” “treatment,” “relief,” and “relief” refer to therapeutic measures that can cure, slow down, alleviate, and stop the progression of a pathological disease or condition. Therefore, those requiring treatment include those already diagnosed with or suspected of having the stated condition. In some embodiments, a subject’s cancer is successfully “treated” according to the method of this application if the patient exhibits one or more of the following: a reduction in the number of cancer cells or their complete absence; a reduction in tumor size; inhibition or absence of cancer cell infiltration into surrounding organs, including, for example, cancer spread to soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; relief of one or more symptoms associated with the specific cancer; a reduction in morbidity and mortality; an improvement in quality of life; a reduction in tumorigenicity, tumorigenic frequency, or tumorigenic capacity of the tumor; a reduction in the number or frequency of cancer stem cells in the tumor; differentiation of tumorigenic cells into a non-tumorigenic state; an increase in progression-free survival (PFS), disease-free survival (DFS), overall survival (OS), complete response (CR), partial response (PR), stable disease (SD), progressive disease (PD), time to progression (TTP), or any combination thereof.

[0076] The terms “cancer” and “cancerous” refer to or describe a physiological disorder in mammals in which a population of cells is characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, gynecological cancers (e.g., breast cancer (including triple-negative breast cancer, ductal carcinoma, ovarian cancer, and endometrial cancer), non-small cell lung cancer, pancreatic cancer, thyroid cancer, kidney cancer (e.g., renal cell carcinoma), and bladder cancer (e.g., urothelial carcinoma). Cancer can also be “cancer expressing AMHR2 or GPC3.” Such terms refer to cancers containing cells that express AMHR2 or GPC3. Cancer can be a solid tumor expressing AMHR2 or GPC3. Cancer can be a primary tumor or can be an advanced or metastatic cancer.

[0077] The term "T cell connector" or T cell engager (TCE) is an alternative therapy to cytokines, checkpoint inhibitors, and T cell therapies, which are typically based on antibodies or antibody fragments and can connect any T cell in the body to a cell expressing selected surface markers.

[0078] The term "bivalent binding unit" refers to a molecule containing two identical binding units / binding sites that can bind to the same target, meaning that the molecule can bind to two identical target molecules simultaneously.

[0079] The term "monovalent binding unit" refers to a domain or region in a molecule that can independently recognize and bind to a specific target (such as an antigen or receptor). In other words, a binding unit typically contains one binding site.

[0080] This application provides a bispecific binding protein for a T-cell connector, comprising:

[0081] Bivalent binding units targeting the target protein and monovalent binding units targeting the CD3 protein;

[0082] The bivalent binding unit targeting the target protein comprises a Fab domain 1 and a Fab domain 2; the Fab domain 1 and Fab domain 2 are located at different ends;

[0083] The monovalent binding unit targeting the CD3 protein contains a Fab domain or an scFv domain.

[0084] The heavy chain C-terminus of the Fab domain on one side of the divalent binding unit of the target protein is connected to the N-terminus of the Fab domain or scFv domain of the target CD3 protein via a linker.

[0085] In one embodiment, the amino acid sequence of the linker is (GGGGX)n, where X is Gly or Ser, and n is a natural number from 1 to 6. For example, a linker linker is (G4S)×3.

[0086] In one embodiment, the bispecific binding protein of the T-cell connector satisfies one or both of the following conditions:

[0087] (1) The monovalent binding unit targeting the CD3 protein is the Fab domain;

[0088] The amino acid sequence of the light chain variable region of the Fab domain is shown in SEQ ID NO.1; the heavy chain variable region of the Fab domain is shown in SEQ ID NO.2.

[0089] SEQ ID NO.1:

[0090] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYTLRTFGGGTKVEIK.

[0091] SEQ ID NO.2:

[0092] QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDYYMHWMRQAPGQGLEWMGWIVPEQGDTRYDPKF QGRATITADTSNTAYMELSSLRSEDTAVYYCARDQYGNYFFAHWGQGTLVTVSS.

[0093] In one embodiment, the heavy chain of the Fab domain is shown in SEQ ID NO.3; the light chain of the Fab domain is shown in SEQ ID NO.4.

[0094] SEQ ID NO.3:

[0095] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYTLRTFGGG TKVEIKSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV.

[0096] SEQ ID NO.4:

[0097] QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDYYMHWMRQAPGQGLEWMGWIVPEQGDTRYDPKFQGRATITADTSNTAYMELSSLRSEDTAVYYCARDQYGNYFFAHWGQGT LVTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0098] (2) The monovalent binding unit targeting the CD3 protein is the scFv domain; the heavy chain variable region of the scFv domain is shown in SEQ ID NO.5, and the light chain variable region of the scFv domain is shown in SEQ ID NO.6.

[0099] SEQ ID NO.5:

[0100] QVQLVQSGAEVKKPGSSVKVSCKASGFNIKDYYMHWMRQAPGQCLEWMGWIVPEQGDTRY DPKFQGRATITADTSNTAYMELSSLRSEDTAVYYCARDQYGNYFFAHWGQGTLVTVSS.

[0101] SEQ ID NO.6:

[0102] DIVMTQSPDSLAVSLGERATINCKSSQSLLNSRTRKNYLAWYQQKPGQPPKLLIYWASTRESGV PDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYTLRTFGCGTKVEIK.

[0103] To further reduce heavy chain mismatches during antibody expression and purification, Knob-into-Hole mutations were introduced: S354C and T366W mutations were introduced at CH3 of the heavy chain containing the CD3scFv domain, and Y349C, T366S, L368A, and Y407V mutations were introduced at CH3 of the other heavy chain. To enhance the structural stability of scFv, cysteine ​​residues were introduced at specific positions in VH and VL (e.g., position 44 of VH and position 100 of VL) to enable the formation of additional disulfide bonds. N297G was introduced into the CH2 region to remove the ADCC-like effector function from the Fc terminus of the antibody.

[0104] The two bispecific binding protein structures of the T-cell connectors in this application share the following common features:

[0105] 1. All are in a 2+1 valence state, meaning each antibody molecule has two Fab domains targeting the TAA and one domain targeting the CD3 antigen; 2. The sequence of the anti-CD3 domain was specifically designed and optimized for this application. The anti-CD3 domain can be either Fab or scFv (a pair of disulfide bond mutations were added to increase the structural stability of the scFv); 3. To prevent heavy chain mismatch, KiH mutations were incorporated; 4. To prevent heavy and light chain mismatch, a Crossmab structure was added.

[0106] In one embodiment, the target protein includes one or both of the GPC3 protein and the AMHR2 protein.

[0107] In one embodiment, the heavy chain of the Fab domain binding GPC3 protein is shown in SEQ ID NO.7; the light chain of the Fab domain binding GPC3 protein is shown in SEQ ID NO.8.

[0108] SEQ ID NO.7:

[0109] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGAIEPETGGTAYNREFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCTRYYSFAYWGQ GTLVTVSSASTKGPSVFPLAPSSKSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV.

[0110] SEQ ID NO.8:

[0111] DVVMTQTPLSLSVTPGQPASISCRSGQSLVHSNRNTYLQWYLLKPGQSPQLLIFKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQTSHVPLTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0112] The heavy chain of the Fab domain binding AMHR2 protein is shown in SEQ ID NO.9; the light chain of the Fab domain binding AMHR2 protein is shown in SEQ ID NO.10.

[0113] SEQ ID NO.9:

[0114] QVQLVQSGPGLVKPSETLSLTCTVSGDSISSYYWSWIRQPPGKGLEWIGYISYSGSTDYNPSLKSRVTISSVDTSKNQFSLKLNSVTAADTAVYYCARHAYREKGQFVPYYFYY GMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV.

[0115] SEQ ID NO.10:

[0116] DIVMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPNLLIYAASTLQSGVPSRFSGSGSGTEFTLTIISLQPEDFATYYCQQLNSYPLTFGGGTKVDIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0117] This application, in another respect, provides a nucleic acid molecule containing a bispecific binding protein for encoding a T-cell connector as described above.

[0118] This application also provides a nucleic acid molecule containing a component for encoding the aforementioned bispecific antibody.

[0119] This application also provides a vector comprising the aforementioned nucleic acid molecule. This application does not specifically limit the type of recombinant expression vector, which may include, but is not limited to, antibody expression vectors. This application does not specifically limit the antibody expression vector, which may include, but is not limited to, bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or combinations thereof. For example, the PCDNA3.1 expression vector.

[0120] This application also provides a cell comprising the aforementioned nucleic acid molecule or the aforementioned vector. This application does not specifically limit the type of host cell, including but not limited to CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0121] This application also provides a method for preparing the cells, the method comprising the step of introducing the nucleic acid molecule or the vector into the target cell.

[0122] In one example, the import method uses transfection.

[0123] The term “transfection” refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques used for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ibid.; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.

[0124] This application also provides a method for preparing the above-mentioned bispecific binding protein of the T-cell connector, the preparation method comprising the following steps:

[0125] The cells described above are cultured, and antibodies are isolated from the resulting cultures.

[0126] This application also provides a medicament comprising the aforementioned bispecific binding protein of the T-cell connector.

[0127] This application also provides the use of the above-mentioned bispecific binding protein of the T-cell connector, the above-mentioned nucleic acid molecule, the above-mentioned carrier, or the above-mentioned cell in the preparation of a drug for treating tumors.

[0128] This application also provides the use of the antibody or the recombinant protein in the preparation of a medicament for treating tumors.

[0129] It is understood that this application does not specifically limit the type of tumor; it can be any type of tumor that is positive for AMHR2 or GPC3, or a T-cell connector prepared by binding CD3 to the target antigen using the structure of this application. For example, it could be ovarian cancer that is positive for AMHR2, or liver cancer and lung cancer that are positive for GPC3.

[0130] This application, in another aspect, provides a treatment method comprising administering to a subject an effective dose of the bispecific binding protein of the T-cell connector or the drug.

[0131] In this context, "application," "giving," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Application," "giving," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Application," "giving," and "treatment" also mean the treatment of, for example, cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. "Treatment," when applied to humans, veterinary, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0132] "Treatment" means administering an oral or topical therapeutic agent, such as a composition comprising any antibody or antigen-binding fragment of this disclosure, to a patient who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more symptoms of the disease, inducing the regression of such symptoms or inhibiting their progression to any clinically measured extent. The amount of a therapeutic agent that effectively relieves any specific disease symptom (also referred to as a "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0133] "Effective amount" or "effective dose" means the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic outcome. For prophylactic use, beneficial or desired outcomes include eliminating or reducing risk, mitigating severity, or delaying the onset of a condition, including the condition itself, its complications, and the biochemical, histological, and / or behavioral symptoms of intermediate pathological phenotypes presented during the development of the condition. For therapeutic use, beneficial or desired outcomes include clinical outcomes such as reducing the incidence of various target antigen-related conditions of this application or improving one or more symptoms of said conditions, reducing the dosage of other agents required to treat the condition, enhancing the efficacy of another agent, and / or delaying the progression of the target antigen-related condition of this disclosure in patients.

[0134] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0135] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0136] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0137] Example 1

[0138] This embodiment provides a bispecific binding protein structure for a T-cell connector with a CD3 Fab domain.

[0139] 1. Structural design of bispecific antibodies

[0140] A 2+1 valent TAA×CD3 bispecific antibody was constructed using a Fab / Fab-Fab Fc structure. The TAA terminus is divalent (Fab type), and the CD3 terminus is monovalent (Fab type). The TAA heavy chain Fab region and the CD3 light chain are linked by a (G4S)×2 linker. The antibody structure is shown below. Figure 1 As shown in A in the diagram.

[0141] To further reduce heavy chain mismatches during antibody expression and purification, Knob-into-Hole mutations were introduced: S354C and T366W mutations were introduced at CH3 of the heavy chain containing the CD3 domain, and Y349C, T366S, L368A, and Y407V mutations were introduced at CH3 of the other heavy chain. To reduce mismatches between the heavy and light chains, a CrossMab structure was introduced, which interchanges the variable regions of the heavy and light chains at the CD3 terminus. Furthermore, N297G was introduced into the CH2 region to reduce the effector function of the antibody's Fc terminus, such as ADCC.

[0142] 2. Preparation of bispecific antibodies based on the 2+1 valence state of GPC3×CD3 and AMHR2×CD3

[0143] Based on the bispecific antibody structure in step 1, two different structures with 2+1 valence states, GPC3×CD3 and AMHR2×CD3 bispecific antibodies, were constructed, and their sequences are shown in Table 1.

[0144] Table 1

[0145]

[0146]

[0147] Genes for each chain of the bispecific antibody were cloned into the pCDNA3.4 expression vector, and antibody expression plasmids were prepared. CHO-S cells were co-transfected with the heavy chain and light chain expression plasmids, and cultured for 5-10 days. The culture supernatant was collected and purified by Protein A affinity chromatography, molecular sieve chromatography, and ultrafiltration to 1×PBS to obtain bispecific antibodies with a purity >95%. SDS-PAGE gel images of CR17-BA15N and CR04-BA12 are shown below. Figure 2 A and Figure 2 As shown in B in the diagram.

[0148] 3. Binding activity of GPC3×CD3 bispecific antibody to CD3 protein

[0149] Human CD3δ&ε (manufacturer: Acro, catalog number: CDD-H52W1) or monkey CD3δ&ε (manufacturer: Acro, catalog number: CDD-C52W4) were aliquoted into 96-well microplates, 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 antibody sample (starting concentration 100 nM, 3-fold serial dilution, totaling 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.

[0150] The results are as follows Figure 3 As shown, the results indicate that CR17-BA15N can significantly bind to human CD3δε protein and has human-monkey cross-binding activity.

[0151] 4. Binding activity of GPC3×CD3 bispecific antibody to T cells

[0152] Collect suspended Jurkat-NFAT-Luc Effector Cells (expressing CD3 protein) by centrifugation, or digest adherent HepG2 cells with Accutase, wash once with PBS, and then centrifuge at 2×10⁻⁶. 6 Cells were suspended in FACS buffer (PBS containing 3% FBS) at a rate of 50 μL / well in a 96-well U-plate. 50 μL of serially diluted antibody sample (200 nM starting concentration, 3-fold serial dilutions, for a total of 8 concentration points) was added to each well, and the plates were incubated at 4°C for 45 minutes. Cells were washed once with FACS buffer, centrifuged, and then 100 μL of APC-labeled anti-human IgG antibody was added to each well. Cells were incubated at 4°C for 30 minutes, followed by a second wash and removal of the supernatant. Cells were resuspended in 200 μL of FACS buffer and analyzed by flow cytometry.

[0153] The results are as follows Figure 4 As shown, the results indicate that CR17-BA15N can bind to human T cell line Jurkat cells and hepatocellular carcinoma HepG2 cells.

[0154] 5. GPC3×CD3 Bispecific Antibody Affinity Assay

[0155] Using surface plasmon resonance (SPR) technology, the binding affinity of amino-conjugated antigen human CD3δ&ε protein dimers on a CM5 chip with different antibody concentrations (400 nM, 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, and 0 nM) was measured using a Biacore 8k instrument. The affinity constant KD was obtained based on the binding and dissociation rates, and the affinity data are shown in Table 2.

[0156] Table 2

[0157] Antibody Antigen ka (1 / Ms) kdis (1 / s) KD (M) CR17-BA15N CD3 6.35e+04 6.38e-03 1.01e-07

[0158] 6. TDCC activity assay of GPC3×CD3 bispecific antibody

[0159] PBMCs were used as effector cells. Antibodies induced TDCC (transient-target cell lysis) to lyse target cells, and the TDCC effect of candidate antibodies was reflected by detecting the release of LDH from the cells. Specific procedures included: resuscitating PBMCs (derived from Miaoshun Biotechnology; PBMC donor 1 and PBMC donor 2 were catalog numbers P123031202C and P123090504C, respectively); the next day, Huh-7 cells (derived from Procell, CL-0120) or HepG2 cells (derived from CCTCC) grown to the logarithmic growth phase were adjusted to a cell density of 4 × 10⁶ cells using 1640 medium (manufacturer: Gibco, catalog number: 11835030). 5 Add 50 μL / well to a 96-well U-bottom plate; the cell density is 2 × 10⁶ cells / mL. 6 PBMC cells (number of cells / mL), 100 μL / well; antibody (100 nM, 10-fold serial dilution, 6 concentration points), 50 μL / well; incubate at 37℃ and 5% CO2 for 24 hours. Following the method of the LDH detection kit (manufacturer: Promega, catalog number: G1780), 50 μL / well of the supernatant was aspirated from the test wells for OD490 detection. Results are as follows. Figure 5 As shown in A and Table 3.

[0160] Table 3

[0161] CR17-BA15N EC50 (donor 1) 53.07 pM EC50 (donor 2) 17.91 pM

[0162] The results showed that the antibody CR17-BA15N had significant TDCC activity and could specifically kill tumor cells.

[0163] 7. TDCC activity assay of AMHR2×CD3 bispecific antibody

[0164] Using PBMCs as effector cells, antibodies induce T-cell-mediated cellular cytotoxicity (TDCC) to lyse target cells. The TDCC effect of candidate antibodies is reflected by detecting the release of cellular lactate dehydrogenase (LDH).

[0165] Specific procedures: Resuscitate PBMC cells (purchased from Miaoshun Biotechnology, catalog number P123120918C); the next day, adjust the cell density of COV434-AMHR2 cells that have grown to the logarithmic growth phase to 4 × 10⁻⁶ cells using 1640 medium (Gibco, 11835030). 5 Add 50 μL / well to a 96-well U-bottom plate; the cell density is 2 × 10⁶ cells / mL. 6 PBMC cells (number of cells / mL), 100 μL / well; antibody (initial concentration 100 nM, serially diluted 10-fold), 50 μL / well; incubate at 37°C and 5% CO2 for 24 hours. Following the method of the LDH detection kit (Promega, G1780), 50 μL / well of the supernatant was aspirated from the test wells for OD490 detection. Results are as follows. Figure 5 As shown in B and Table 4.

[0166] Table 4

[0167] EC50 PBMC donor 1 1.66 pM

[0168] The results showed that the candidate antibody CR04-BA12 had significant TDCC activity and could specifically kill tumor cells.

[0169] Example 2

[0170] This embodiment provides a bispecific binding protein structure for a T-cell connector with CD3 as the scFv domain.

[0171] 1. Preparation of AMHR2×CD3 bispecific antibodies with a 2+1 valence state based on Fab / Fab-scFv Fc structure

[0172] A 2+1 valent TAA×CD3 bispecific antibody was constructed using a Fab / Fab-scFv Fc structure. The TAA terminus is bivalent and entirely Fab-type; the CD3 terminus is monovalent and scFv-type. The heavy chain Fab region of the TAA terminus and the scFv region of CD3 are linked by a (G4S)×2 linker. The heavy and light chains within the scFv are linked by a (G4S)×3 linker. The antibody structure is shown below. Figure 1 As shown in B in the diagram.

[0173] To further reduce heavy chain mismatches during antibody expression and purification, Knob-into-Hole mutations were introduced: S354C and T366W mutations were introduced on CH3 of the heavy chain containing the CD3scFv domain, and Y349C, T366S, L368A, and Y407V mutations were introduced on CH3 of the other heavy chain. An N297G mutation was introduced in the CH2 region to remove the ADCC effector function at the Fc terminus of the antibody, constructing the AMHR2×CD3 bispecific antibody, the sequence of which is shown in Table 5.

[0174] Table 5

[0175]

[0176]

[0177] The correct sequence was amplified 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 (Gibco, catalog number: C11995500BT) at 37°C in a 5% CO2 cell culture incubator, according to the manufacturer's instructions (Polysciences, catalog number: 24765-1). After cell preparation, the plasmids were transfected into HEK293-T cells using a transfection kit to express the bispecific antibody against AMHR2×CD3, according to the manufacturer's instructions. 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. SDS-PAGE gel images of CR17-BA28 and CR04-BA7 are shown below. Figure 6 A and Figure 6 As shown in B in the diagram.

[0178] 2. Affinity assay of bispecific antibody AMHR2×CD3

[0179] Surface plasmon resonance (SPR) technology was used to measure the binding affinity of the amino-coupled antigen hAMHR2 (approximately 30 RU) on a CM5 chip with different concentrations of antibody using a Biacore 8k instrument. The concentration ranges were 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, and 0 nM. Binding took 180 s, dissociation 1200 s, and regeneration with Glycine 1.7 for 20 s. The affinity constant KD was obtained based on the binding and dissociation rates. The affinity data are shown in Table 6.

[0180] Table 6

[0181]

[0182] Using surface plasmon resonance (SPR) technology, the binding affinity of amino-conjugated antigen human CD3δ&ε protein dimers on a CM5 chip with different concentrations of antibody was determined using a Biacore 8k instrument. The concentration ranges were 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.56 nM, and 0 nM, with binding time of 100 s, dissociation time of 180 s, and regeneration with Glycine 1.5 for 30 s. The affinity constant KD was obtained based on the binding and dissociation rates, and the affinity data are shown in Table 7.

[0183] Table 7

[0184]

[0185] 3. Binding activity of bispecific antibodies to cell lines expressing AMHR2 or GPC3

[0186] Logarithmically growing COV434-hAMHR2 cells (COV434 cells purchased from Fuheng Biotechnology, CAT#FH0724) were harvested. After digestion, the harvested cells were centrifuged at 300×g for 5 minutes at room temperature. The supernatant was discarded, and the cells were washed once with analysis buffer (PBS + 2% FBS) and the cell density was adjusted to 1×10⁻⁶. 6 Cells / mL. Add 100 μL of the prepared cell suspension to each well of a 96-well U-plate, followed by 100 μL of diluted antibody solution (starting concentration 320 nM, serially diluted 5-fold, 10 concentration points). Mix well and incubate at 4°C for 1 h. Centrifuge at 300 × g for 5 min at 4°C, discard the supernatant, wash cells twice with analysis buffer, add 100 μL of the appropriate secondary antibody solution (manufacturer: Sigma, catalog number: F9512) to each well, and incubate at 4°C for 0.5 h. Centrifuge at 300 × g for 5 min at 4°C, discard the supernatant, wash cells twice with analysis buffer, and finally resuspend cells in analysis buffer. Detect the mean fluorescence signal (MFI) using a BD flow cytometer.

[0187] See results Figure 7 As shown in Figure A. The results indicate that the CR04-BA7 bispecific antibody has good binding activity with hAMHR2-positive tumor cells.

[0188] HepG2 adherent cells (endogenously expressing GPC3 protein) were digested with Accutase, washed once with PBS, and then subjected to 2×10⁻⁶ ppm digestion. 6Cells were suspended in FACS buffer (PBS containing 3% FBS) at a concentration of 50 μL / well in a 96-well U-plate. 50 μL of serially diluted antibody sample (starting concentration 30 μg / mL, 3-fold serial dilutions, for a total of 8 concentration points) was added to each well, and the plates were incubated at 4°C for 45 min. Cells were washed once with FACS buffer, centrifuged, and 100 μL of APC-labeled anti-human IgG antibody was added to each well. Cells were incubated at 4°C for 30 min, followed by a second wash and removal of supernatant. Cells were resuspended in 200 μL of FACS buffer and analyzed by flow cytometry.

[0189] The results are as follows Figure 7 As shown in B in the figure. The results indicate that the candidate antibody CR17-BA28 can bind to HepG2 liver cancer cells.

[0190] 4. Binding activity of bispecific antibodies to T cells

[0191] One day in advance, thaw frozen PBMC cells and adjust the density to 2 × 10⁶ cells / day using culture medium (1640 + 10% FBS + 1% PS). 6 Cells were cultured overnight at 2E6 cells / mL. The next day, cells were collected, resuspended in analytical buffer (DPBS containing 2% FBS), counted, and the cell density was adjusted to 2E6 cells / mL for later use. The prepared cell suspension was blocked with a human FC block. 50 μL of the blocked cell suspension was added to each well of a 96-well U-plate, followed by 50 μL of diluted antibody solution (starting concentration 320 nM, 3-fold serial dilution, 11 concentration points). The mixture was incubated at 4°C for 1 hour. The cells were then centrifuged at 300 × g for 5 minutes at 4°C, the supernatant was discarded, and the cells were washed twice with analytical buffer. 100 μL of a mixed antibody solution of PE anti-human CD4, APC anti-human CD8, and goat anti-human IgG Fc specific-FITC was added to each well, and the cells were incubated at 4°C for 0.5 hours. Centrifuge at 300×g for 5 minutes at 4℃, discard the supernatant, wash the cells twice with analysis buffer, and finally resuspend the cells with analysis buffer. Detect the mean fluorescence signal (MFI) on a BD flow cytometer.

[0192] See results Figure 8 The results showed that the CR04-BA7 bispecific antibody had weak binding activity with human primary T cells.

[0193] Collect suspended Jurkat-NFAT-Luc Effector Cells (expressing CD3 protein) by centrifugation, wash once with PBS, and then centrifuge at 2×10⁻⁶. 6Cells were suspended in FACS buffer (PBS containing 3% FBS) at a concentration of 50 μL / well in a 96-well U-plate. 50 μL of serially diluted antibody sample (200 nM starting concentration, 3-fold serial dilutions, for a total of 8 concentration points) was added to each well, and the plates were incubated at 4°C for 45 min. Cells were washed once with FACS buffer, centrifuged, and then 100 μL of APC-labeled anti-human IgG antibody was added to each well. Cells were incubated at 4°C for 30 min, followed by a second wash and removal of supernatant. Cells were resuspended in 200 μL of FACS buffer and analyzed by flow cytometry.

[0194] The results are as follows Figure 8 As shown in B, the candidate antibody CR17-BA28 can bind to the human T cell line Jurkat cells.

[0195] 5. Assay for the antitumor activity of bispecific antibodies

[0196] PBMCs were used as effector cells. Antibodies induced T-cell-mediated cellular cytotoxicity (TDCC), causing target cell lysis. The TDCC effect of the candidate antibodies was reflected by detecting the release of cellular lactate dehydrogenase (LDH). Specific procedures included: resuscitating PBMCs (purchased from Miaoshun Biotechnology, catalog number P123120918C); the following day, COV434-AMHR2 or Huh7 cells (endogenously expressing GPC3 protein) grown to the logarithmic growth phase were adjusted to a cell density of 4 × 10⁶ cells using 1640 medium (Gibco, 11835030). 5 Add 50 μL / well to a 96-well U-bottom plate; the cell density is 2 × 10⁶ cells / mL. 6 PBMC cells (number of cells / mL), 100 μL / well; antibody (initial concentration 100 nM, serially diluted 10-fold), 50 μL / well; incubated at 37℃ and 5% CO2 for 24 hours. Following the method of the LDH detection kit (Promega, G1780), 50 μL / well of the supernatant was aspirated from the test wells for OD490 detection. The cytotoxic activity results of CR17-BA28 and CR04-BA7 are as follows: Figure 9 As shown in A in 9 and B in 9, the statistical results are shown in Table 8 and Table 9, respectively.

[0197] Table 8

[0198] EC50 PBMC donor 1 19.39 pM PBMC donor 2 12.36 pM

[0199] Table 9

[0200] EC50 PBMC donor 1 1.047 pM PBMC donor 2 0.373 pM

[0201] The results showed that the CR17-BA28 and CR04-BA7 antibodies of this application had high killing activity against tumor target cells.

[0202] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A bispecific binding protein structure for a T-cell connector, characterized in that, include: Bivalent binding units targeting the target protein and monovalent binding units targeting the CD3 protein; The bivalent binding unit targeting the target protein comprises a Fab domain 1 and a Fab domain 2; the Fab domain 1 and Fab domain 2 are located at different ends; The monovalent binding unit targeting the CD3 protein contains a Fab domain or an scFv domain. The heavy chain C-terminus of the Fab domain on one side of the divalent binding unit of the target protein is connected to the N-terminus of the Fab domain or scFv domain of the target CD3 protein via a linker.

2. The bispecific binding protein structure of the T-cell connector according to claim 1, characterized in that, The amino acid sequence of the linker is (GGGGX)n, where X is G or S, and n is a natural number from 1 to 6; Optionally, the bispecific binding protein of the T-cell connector satisfies one or both of the following conditions: (1) The monovalent binding unit targeting CD3 protein is a Fab domain; the amino acid sequence of the heavy chain variable region of the monovalent binding unit targeting CD3 protein is shown in SEQ ID NO.2; the light chain variable region of the monovalent binding unit targeting CD3 protein is shown in SEQ ID NO.1; Optionally, the heavy chain of the monovalent binding unit targeting the CD3 protein is as shown in SEQ ID NO.3; the light chain of the monovalent binding unit targeting the CD3 protein is as shown in SEQ ID NO.4; and, (2) The monovalent binding unit targeting CD3 protein is an scFv domain; the heavy chain variable region of the monovalent binding unit targeting CD3 protein is shown in SEQ ID NO.5, and the light chain variable region of the monovalent binding unit targeting CD3 protein is shown in SEQ ID NO.

6.

3. The bispecific binding protein structure of the T-cell connector according to any one of claims 1 to 2, characterized in that, The target protein includes one or both of GPC3 protein and AMHR2 protein; Optionally, the heavy chain binding the Fab domain of the GPC3 protein is shown in SEQ ID NO.7; the light chain binding the Fab domain of the GPC3 protein is shown in SEQ ID NO.

8. The heavy chain of the Fab domain binding to the AMHR2 protein is shown in SEQ ID NO.9; the light chain of the Fab domain binding to the AMHR2 protein is shown in SEQ ID NO.

10.

4. A nucleic acid molecule, characterized in that, It contains a bispecific binding protein structure for encoding the T-cell connector as described in any one of claims 1 to 3.

5. A carrier, characterized in that, It includes the nucleic acid molecule as described in claim 4.

6. A cell, characterized in that, It comprises the nucleic acid molecule of claim 4 or the vector of claim 5.

7. A method for preparing cells according to claim 6, characterized in that, The preparation method includes the step of introducing the nucleic acid molecule of claim 4 or the vector of claim 5 into target cells.

8. A method for preparing the bispecific binding protein structure of the T-cell connector according to any one of claims 1 to 3, characterized in that, The preparation method includes the following steps: The cells of claim 6 are cultured, and the bispecific binding protein structure of the T-cell connector is isolated from the resulting culture.

9. A drug, characterized in that, The drug comprises a bispecific binding protein structure of the T-cell connector according to any one of claims 1 to 3.

10. The use of the bispecific binding protein structure of the T-cell connector according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4, the carrier according to claim 5, or the cell according to claim 6 in the preparation of a medicament for treating tumors.

Images