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

Abstract

The present application belongs to the field of biomedicine, and specifically relates to a CD3-specific binding protein, a bispecific antibody, and a preparation method and application thereof. The present application provides a bispecific antibody, which comprises a first antigen binding domain that specifically binds to CD3 and a second antigen binding domain that specifically binds to AMHR2 or GPC3. Among them, the CD3 antibody single-chain variable fragment (ScFV) in the first antigen binding domain can bind to T cells, and the second antigen binding domain binds to antigens on the surface of tumors. This binding mode can significantly activate T cell function. Compared with the CD3-terminal antibody in traditional T cell bispecific antibodies, it has different binding epitopes, which can maintain a high tumor cell killing activity while also having a lower cytokine release level, thereby balancing the effectiveness and safety of T cell bispecific antibody therapy.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine, and specifically relates to a CD3-specific binding protein, a bispecific antibody, and a preparation method and application thereof. Background Art

[0002] CD3 is a protein complex present on the surface of T cells. Its main function is to transduce the activation signal generated by TCR recognition of antigens into T cells, and participate in the differentiation of T cells, the differentiation and activation of helper T cells (Th), killer T cells (CTL) and regulatory T cells (Treg). In addition, CD3 is also involved in the secretion of cytokines and the regulation of apoptosis. It forms a complex with the T cell receptor (TCR). Among them, the CD3 protein complex is composed of four peptide chains, including γ chain, δ chain, ε chain and ζ chain, which are composed of δ-ε and γ-ε heterodimers containing extracellular and intracellular domains, and ζ-ζ homodimers with very short extracellular domains and very long intracellular domains.

[0003] During T cell activation, TCR binds to the cognate pMHC ligand. TCR needs to transmit the received signal to the cytoplasm, but because TCR α and β chains lack known intracellular signaling motifs, the transduction of activation signals to second messengers and intracellular transcription factors is completed by the CD3 protein complex. The number of immunoreceptor tyrosine-based activation motifs (ITAMs) contained in the γ chain, δ chain, ε chain and ζ chain is different. Among them, the γ, ε and δ chains each have one ITAM, while the ζ chain has three ITAMs. When TCR binds to pMHC ligands, the conformation of CD3 protein changes, which causes Lck to phosphorylate the Tyr residues in CD3 ITAMs, triggering phosphorylation signaling events involving ZAP70 and LAT in T cells, leading to the recruitment of multiple downstream adaptors and signaling molecules and the formation of LAT signalosomes. Downstream signaling events of these pathways include T cell activation, proliferation, cytokine production and effector function.

[0004] In summary, activating CD3 antibodies can be used directly for therapeutic purposes involving T cell activation. In addition, bispecific antibodies that can bind to CD3 and target tumor surface antigens can connect tumor cells and T cells, thereby directly activating T cells, releasing granzymes, perforins and cytokines to kill tumors, thereby achieving the therapeutic purpose of inhibiting tumors.

[0005] Although T cell bispecific antibodies have made some progress in the treatment of blood tumors, their efficacy in the treatment of solid tumors is poor. In clinical practice, T cell bispecific antibody treatment may produce strong side effects, such as cytokine release syndrome (CRS), neurotoxicity and other symptoms.

[0006] In addition, some anti-Glypican-3 (GPC3) T cell bispecific antibody molecules ERY974 currently in the clinical research stage have the disadvantages of low limiting toxic dose and large side effects of cytokine release syndrome (CRS), resulting in a toxic dose of only 0.81μg / kg, and no effective clinical treatment response. Another CD3 bispecific antibody AMG340 (PSMA×CD3) has a higher safety, but in the first phase of clinical trials, it was administered up to 800mg once every three weeks, but it failed to reach the toxic dose, and its efficacy was not obvious, with an objective response rate of 0 in 41 patients.

[0007] Therefore, effectiveness and safety in clinical treatment are key factors for the successful development of T cell bispecific antibodies. However, the selection of CD3 bispecific antibodies in traditional technologies is limited and cannot achieve both efficacy and safety. Summary of the invention

[0008] Based on this, an embodiment of the present application provides a CD3-specific binding protein, a bispecific antibody, and a preparation method and application thereof.

[0009] On the one hand, the present application provides a CD3-specific binding protein; the CD3-specific binding protein has a CD3 binding domain; the CD3 binding domain has a heavy chain CDR1 to a heavy chain CDR3 shown in SEQ ID NO.11 to SEQ ID NO.13, and a light chain CDR1 to a light chain CDR3 shown in SEQ ID NO.14 to SEQ ID NO.16.

[0010] In one embodiment, the heavy chain variable region of the CD3 binding domain is shown in SEQ ID.30.

[0011] In one embodiment, the light chain variable region of the CD3 binding domain is shown in SEQ ID.29.

[0012] In one embodiment, the heavy chain of the CD3 binding domain is shown as SEQ ID NO.3.

[0013] In one embodiment, the light chain of the CD3 binding domain is shown as SEQ ID NO.4.

[0014] On the other hand, the present application provides a bispecific antibody, comprising: a first antigen binding domain that specifically binds to CD3 and a second antigen binding domain that specifically binds to AMHR2 or GPC3.

[0015] The first antigen binding domain that specifically binds to CD3 includes the CD3-specific binding protein.

[0016] In one embodiment, the second antigen binding domain has heavy chain CDR1 to heavy chain CDR3 shown in SEQ ID NO.17 to SEQ ID NO.19, and light chain CDR1 to light chain CDR3 shown in SEQ ID NO.20 to SEQ ID NO.22.

[0017] In one embodiment, the second antigen binding domain has a heavy chain variable region as shown in SEQ ID NO.31 and a light chain variable region as shown in SEQ ID NO.32.

[0018] In one embodiment, the second antigen binding domain has heavy chain CDR1 to heavy chain CDR3 shown in SEQ ID NO.23 to SEQ ID NO.25, and light chain CDR1 to light chain CDR3 shown in SEQ ID NO.26 to SEQ ID NO.28.

[0019] In one embodiment, the second antigen binding domain has a heavy chain variable region shown in SEQ ID NO.33 and a light chain variable region shown in SEQ ID NO.34.

[0020] On the other hand, the present application provides a nucleic acid molecule containing a specific binding protein or bispecific antibody for encoding the CD3.

[0021] On the other hand, the present application provides a vector comprising the nucleic acid molecule.

[0022] On the other hand, the present application provides a cell comprising the nucleic acid molecule or the vector.

[0023] On the other hand, the present application provides a method for preparing the cells, which comprises the step of introducing the nucleic acid molecule or the vector into the target cell.

[0024] On the other hand, the present application provides a method for preparing the CD3 specific binding protein, which comprises the following steps: culturing the cells, and isolating antibodies from the obtained culture.

[0025] On the other hand, the present application provides a method for preparing the bispecific antibody or the like, which comprises the following steps: culturing the cells, and isolating antibodies from the obtained culture.

[0026] On the other hand, the present application provides a CD3 detection product, which includes the CD3 specific binding protein or bispecific antibody.

[0027] On the other hand, the present application provides a method for detecting CD3 in a sample to be tested, wherein the detection method uses the CD3-specific binding protein or bispecific antibody as a detection antibody, and determines whether the sample to be tested contains CD3 through an immune binding reaction.

[0028] On the other hand, the present application also provides a drug, which includes the bispecific antibody.

[0029] On the other hand, the present application provides the use of the CD3-specific binding protein, the bispecific antibody, the nucleic acid molecule, the vector, or the cell in the preparation of a drug for treating tumors.

[0030] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0032] Figure 1 The binding activity results of the bispecific antibody of the present application to CD3δε protein dimer;

[0033] Figure 2 The results of the binding activity of the bispecific antibody of the present application to the N-terminal peptide epitope of the CD3ε protein;

[0034] Figure 3 The results of the activity test of the bispecific antibody of the present application on T cell activation in the presence of target cells (ovarian cancer cells COV434-AMHR2);

[0035] Figure 4 The results of the activity test of the bispecific antibody of the present application on T cell activation in the presence of target cells (liver cancer cell HepG2);

[0036] Figure 5The test results of the bispecific antibody of the present application in specifically killing ovarian cancer cells COV434-AMHR2;

[0037] Figure 6 The detection result of the bispecific antibody of the present application specifically killing GPC3-positive liver cancer cell Huh7;

[0038] Figure 7 The detection results of T cell-mediated cytokine release during the process of the bispecific antibody of the present application killing ovarian cancer cells COV434-AMHR2;

[0039] Figure 8 These are the results of T cell-mediated cytokine release detection during the process of the bispecific antibody of the present application specifically killing GPC3-positive liver cancer cell Huh7. DETAILED DESCRIPTION

[0040] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0042] the term

[0043] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0044] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the 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 by 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 that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of 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, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0045] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0046] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0047] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0048] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0049] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0050] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0051] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges included therein.

[0052] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0053] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0054] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0055] The term "CD3 (Cluster of Differentiation 3)" refers to an important cell surface protein complex that is mainly present on T cells. It is composed of multiple peptide chains, usually including γ chain, δ chain, ε chain and ζ chain. The main function of CD3 is to participate in T cell activation and signal transduction together with the T cell receptor (TCR).

[0056] The term "AMHR2" stands for Anti-Mullerian Hormone Receptor Type 2, which is a transmembrane protein that belongs to the TGF-β (transforming growth factor-beta) superfamily of receptors. AMHR2 plays a role in a variety of biological processes, including the development of the reproductive system and the occurrence of tumors. The expression level of AMHR2 can be used as a biomarker for certain tumors to help with diagnosis and prognosis assessment. In addition, AMHR2 is a potential target for the development of new cancer treatment strategies, especially for those tumors that express AMHR2.

[0057] The term "GPC3 (Glypican-3)" stands for hepatocellular carcinoma-associated antigen 3, which is a cell surface glycosylphosphatidylinositol (GPI) anchored protein belonging to the heparin sulfate proteoglycan family. GPC3 plays a role in a variety of biological processes, including cell proliferation, differentiation, and signal transduction. The expression level of GPC3 can be used as a biomarker for hepatocellular carcinoma and other tumors to help with diagnosis and prognosis assessment. In addition, GPC3 is a potential target for the development of new cancer treatment strategies, especially for those tumors that express GPC3.

[0058] The term "antibody" means 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 in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses complete polyclonal antibodies, complete monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising antibodies, and any other modified immunoglobulin molecules, as long as the antibody exhibits the desired biological activity. The antibody may have any of the following five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or their subclasses (isotypes) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), which are referred to as α, δ, ε, γ, and μ, respectively, based on the properties of their heavy chain constant structures. Different types of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies may be naked or conjugated to other molecules (such as toxins, radioisotopes, etc.).

[0059] The term "antibody fragment" refers to a portion of an intact antibody. "Antigen-binding fragment," "antigen-binding domain," or "antigen-binding region" refers to a portion of an intact antibody that specifically binds to an antigen. An antigen-binding fragment may contain the antigen recognition site of an intact 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 produced.

[0060] The terms "anti-CD3 antibody", "CD antibody" and "antibody that binds to CD3" refer to antibodies that are capable of specifically binding to CD3 with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent in targeting CD3. As used herein, the terms "specifically binds", "immunospecifically binds", "immunospecifically recognizes" and "specifically recognizes" are similar terms in the context of an antibody or antigen-binding fragment thereof. These terms indicate that the antibody or antigen-binding fragment thereof binds to the epitope via its antigen-binding domain, and that binding requires a certain complementarity between the antigen-binding domain and the epitope.

[0061] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. The variable region generally refers to a portion of an antibody, generally a portion of a light chain or a heavy chain, typically about the amino-terminal 110 to 120 amino acids or 110 to 125 amino acids in a mature heavy chain and about 90 to 115 amino acids in a mature light chain, which differ in sequence between antibodies and are used for the binding and specificity of a particular antibody to its specific antigen. The variability in the sequence is concentrated in those regions called CDRs, while the more conserved regions in the 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 the interaction and specificity of the antibody with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region is a rodent or murine variable region.

[0062] The term "heavy chain variable region" is composed of 4 FR regions and 3 CDR regions. According to the IMGT database, the genes of the heavy chain variable region are defined as V gene, D gene and J gene.

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

[0064] The term "heavy chain" generally comprises one variable region and three constant regions (CH1 / CH2 / CH3).

[0065] The term "light chain" generally comprises one variable region and one constant region (CL).

[0066] The term "Kabat numbering" and similar terms are well-known in the art and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody or its antigen-binding fragment. In certain aspects, CDRs may be determined according to the Kabat numbering system (see, e.g., Kabat EA and Wu TT (1971) Ann NY Acad Sci 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35 (which may optionally include one or two additional amino acids after 35 (referred to as 35A and 35B in the Kabat numbering scheme)) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule typically occur at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In a specific embodiment, the CDRs of the antibodies described herein have been identified according to the Kabat numbering scheme.

[0067] As used herein, the terms "constant region" and "constant domain" are interchangeable and have their common meanings in the art. The constant region is an antibody portion that is not directly involved in the binding of the antibody to the antigen, but can exhibit a variety of effector functions, such as interactions with Fc receptors, such as the carboxyl terminal portion of the light chain and / or heavy chain. Relative to the immunoglobulin variable domain, the constant region of the immunoglobulin molecule generally has a more conserved amino acid sequence. In certain aspects, the antibody or antigen-binding fragment comprises a constant region or portion thereof sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0068] As used herein, the term "heavy chain" when used with respect to an antibody can refer to any of the different types, e.g., α, δ, ε, γ, and μ, based on the amino acid sequence of the constant domain, to produce antibodies of the IgA, IgD, IgE, IgG, and IgM categories, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3, and IgG4. Heavy chain amino acid sequences are 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 murine heavy chain.

[0069] As used herein, the term "light chain" when used with respect to an antibody can refer to any of the different types, such as kappa or lambda, based on the amino acid sequence of the constant domain. 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 murine light chain.

[0070] The term "chimeric" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof in which the amino acid sequence is derived from two or more species. Typically, the variable regions of the light and heavy chains correspond to the variable regions of an antibody or antigen-binding fragment thereof derived from one mammalian species (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capacity, while the constant regions are homologous to sequences in an antibody or antigen-binding fragment thereof derived from another species (usually human) to avoid eliciting an immune response in said species.

[0071] The term "human" antibody or antigen-binding fragment thereof refers to an antibody or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin locus, wherein such antibody or antigen-binding fragment thereof is prepared using any technique known in the art. This definition of human antibodies or antigen-binding fragments thereof includes complete or full-length antibodies and fragments thereof.

[0072] "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 antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless otherwise indicated, 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 antigen-binding fragment thereof and an antigen). The affinity of a 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, an equilibrium dissociation constant.

[0073] As used herein, "epitope" is a term in the art and refers to a local region of an antigen to which an antibody or antigen-binding fragment thereof can specifically bind. An epitope can be, for example, a continuous amino acid of a polypeptide (a linear or continuous epitope), or an epitope can be, for example, from two or more non-continuous regions of one or more polypeptides (a conformational, non-linear, discontinuous or non-continuous epitope). In certain embodiments, the epitope to which an antibody or antigen-binding fragment thereof specifically binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / atmosphere exchange in combination with mass spectrometry (e.g., liquid chromatography electrospray 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., Giege 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). Antibodies / antigen-binding fragments thereof: 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, e.g., Meth Enzymol (1985) Vols. 114 and 115, edited by Wyckoff HW et al.; US2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, edited by Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10): 1316-1323). Mutagenesis mapping studies can be performed using any method known to those skilled in the art. For a description of mutagenesis techniques, including alanine scanning mutagenesis, see, for example, Champe M et al. (1995) J Biol Chem 270: 1388-1394 and Cunningham BC and Wells JA (1989) Science 244: 1081-108.

[0074] If an antibody binds to a given epitope or an overlapping epitope of a reference antibody such that it blocks the binding of the reference antibody to the epitope to some extent, then the antibody is said to "competitively inhibit" the binding of the reference antibody to the epitope. Competitive inhibition can be determined by any method known in the art, for example, a competitive ELISA assay. An antibody can be said to competitively inhibit the binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0075] In the preferred embodiment, the "sample" of the present application is a human sample, but animal samples can also be used in the practice of the present application. Non-limiting sources of samples used in the present application include, for example, solid tissues, biopsies, ascites, aspirates, fluid leaches, blood (including circulating tumor cells), plasma, serum, cerebrospinal fluid, lymph fluid, external sections of the skin, respiratory tract, intestinal and urogenital tracts, tears, saliva, milk, tumors, organs, cell cultures and / or cell culture components.

[0076] The word "label" when used herein refers to a detectable compound or composition that is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody. The label can itself be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzyme label, can catalyze chemical alteration of a substrate compound or composition that is detectable.

[0077] Terms such as "treat", "treatment", "treatment", "relieve" and "relieve" refer to therapeutic measures that can cure, slow down, alleviate the symptoms of a pathological disorder or condition and / or prevent its progression. Therefore, those who need treatment include those who have been diagnosed with or suspected of having the condition. In certain embodiments, a subject's cancer is successfully "treated" according to the methods of the present application if the patient shows one or more of the following: a decrease in the number of cancer cells or the complete absence of cancer cells; a decrease 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; alleviation of one or more symptoms associated with a particular cancer; reduced morbidity and mortality; improved quality of life; reduced tumorigenicity, tumorigenic frequency, or tumorigenic capacity of a tumor; reduced number or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells into a non-tumorigenic state; increased progression-free survival (PFS), disease-free survival (DFS), overall survival (OS), complete response (CR), partial response (PR), stable disease (SD), reduced progressive disease (PD), reduced time to progression (TTP), or any combination thereof.

[0078] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells is characterized by unregulated 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 cell carcinoma). A cancer may be a "cancer expressing AMHR2 or GPC3." Such terms refer to a cancer comprising cells expressing AMHR2 or GPC3. A cancer may be a solid tumor expressing AMHR2 or GPC3. A cancer may be a primary tumor, or may be an advanced or metastatic cancer.

[0079] Among them, the properties of antibodies targeting the CD3 end have a significant impact on the activity of T cell bispecific antibodies. In addition to the binding activity of the antibody, the epitope recognized by the antibody is also a very important influencing factor. Among the T cell bispecific antibodies that have been approved for marketing and are in the research stage, most of the antibodies recognize the same site, that is, the 27 amino acid-based peptide epitope at the N-terminus of the CD3ε subunit. A large part of the CD3-end antibodies are sequenced for activity optimization or humanization based on SP34 as the parent, while others such as Mosunetuzumab, Cevostamab, Elranatamab, Tarlatamab, ERY974, etc., all recognize the peptide epitope at the N-terminus of the CD3ε subunit.

[0080] Based on this, the present application provides a CD3-specific binding protein on one hand;

[0081] The CD3-specific binding protein has a CD3 binding domain; the CD3 binding domain has heavy chain CDR1 to heavy chain CDR3 shown in SEQ ID NO.11 to SEQ ID NO.13, and light chain CDR1 to light chain CDR3 shown in SEQ ID NO.14 to SEQ ID NO.16.

[0082] In a specific example, the heavy chain variable region of the CD3-specific binding protein is shown in SEQ ID NO.30.

[0083] In a specific example, the light chain variable region of the CD3-specific binding protein is shown in SEQ ID NO.29.

[0084] In a specific example, the heavy chain of the CD3-specific binding protein is shown in SEQ ID NO.3.

[0085] In a specific example, the light chain of the CD3-specific binding protein is shown in SEQ ID NO.4.

[0086] The specific sequence is shown in Table 1 below:

[0087] Table 1

[0088]

[0089] The CD3-specific binding protein of the present application may have the above-mentioned CDRs, or may be a derivative fragment of the above-mentioned CDRs. The derivative fragment is formed by amino acid substitution ("conservative modification" or "conservative substitution or replacement") at no more than 6 sites relative to its corresponding CDR, and retains the biological activity consistent with its corresponding complementary determining region. For example, the derivative fragment is substituted at 1, 2, 3, 4, 5 or 6 sites relative to its corresponding complementary determining region, and may be one amino acid replaced by another amino acid, or one amino acid replaced by multiple (for example, 2) amino acids.

[0090] In the CDRs provided in the present application, the derivative fragment (conservative variant) refers to a polypeptide formed by replacing 1, 2, or 3 amino acids with amino acids of similar or similar properties compared to the amino acid sequence of the antibody of the present application. These conservative variant polypeptides are preferably generated by amino acid replacement according to Table 2.

[0091] Table 2

[0092]

[0093] "Conservative modification" or "conservative substitution or replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein. Those skilled in the art know that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). In addition, replacement of amino acids with similar structure or function is unlikely to destroy biological activity.

[0094] The specific binding protein of CD3 provided in the present application may be an antibody, an antigen-binding fragment of an antibody, or a small modular immune drug. Further, the binding protein is a monoclonal antibody, a F(ab')2 fragment, a Fab' fragment, a Fab fragment, a Fv fragment, a ScFv fragment, a biclonal antibody, a multispecific antibody, a mini antibody, a chelated recombinant antibody, an intrabody, a nanobody, a binding domain immunoglobulin fusion protein, or a small modular immune drug.

[0095] Among them, the properties of antibodies targeting the CD3 end have a significant impact on the activity of T cell bispecific antibodies. In addition to the binding activity of the antibody, the epitope recognized by the antibody is also a very important influencing factor. Among the T cell bispecific antibodies that have been approved for marketing and are in the research stage, most of the antibodies recognize the same site, that is, the 27 amino acid-based peptide epitope at the N-terminus of the CD3ε subunit. A large part of the CD3-end antibodies are sequenced for activity optimization or humanization based on SP34 as the parent, while others such as Mosunetuzumab, Cevostamab, Elranatamab, Tarlatamab, ERY974, etc., all recognize the peptide epitope at the N-terminus of the CD3ε subunit.

[0096] On the one hand, the present application provides a bispecific antibody, comprising:

[0097] A first antigen binding domain that specifically binds CD3 and a second antigen binding domain that specifically binds AMHR2 or GPC3.

[0098] In a specific example, the heavy chain CDR1 of the second antigen binding domain is shown as SEQ ID NO.17, the heavy chain CDR2 is shown as SEQ ID NO.18, and the heavy chain CDR3 is shown as SEQ ID NO.19.

[0099] In a specific example, the light chain CDR1 of the second antigen binding domain is shown as SEQ ID NO.20, the light chain CDR2 is shown as SEQ ID NO.21, and the light chain CDR3 is shown as SEQ ID NO.22.

[0100] In a specific example, the heavy chain CDR1 of the second antigen binding domain is shown as SEQ ID NO.23, the heavy chain CDR2 is shown as SEQ ID NO.24, and the heavy chain CDR3 is shown as SEQ ID NO.25.

[0101] In a specific example, the light chain CDR1 of the second antigen binding domain is shown as SEQ ID NO.26, the light chain CDR2 is shown as SEQ ID NO.27, and the light chain CDR3 is shown as SEQ ID NO.28.

[0102] The details are shown in Table 3 below:

[0103] Table 3

[0104]

[0105] In a specific example, the light chain variable region of the first antigen binding domain of the antibody is shown in SEQ ID NO.29: DILMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLEIK (SEQ ID NO.29)

[0106] In a specific example, the heavy chain of the first antigen binding domain of the antibody is shown as SEQ ID NO.3.

[0107] DILMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLEIKSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ IDNO.3)

[0108] In a specific example, the heavy chain variable region of the first antigen binding domain of the antibody is shown in SEQ ID NO.30:

[0109] QVQLQQSGTELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSSNYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVFYCARNCYGCSFDFDYWGQGTTLTVSS (SEQ ID NO.30)

[0110] In a specific example, the light chain of the first antigen binding domain of the antibody is shown as SEQ ID NO.4.

[0111] QVQLQQSGTELMKPGASVKISCKATGYTFSRYWIEWVKQRPGHGLEWIGEILPGSGSSNYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVFYCARNCYGCSFDFDYWGQGTT LTVSSASVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO.4)

[0112] In a specific example, the heavy chain variable region of the second antigen binding domain of the antibody is shown as SEQ ID NO.31.

[0113] QVQLVQSGPGLVKPSETLSLTCTVSGDSISSYYWSWIRQPPGKGLEWIGYISYSGSTDYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCARHAYREKGQFVPYYFYYGMDVWGQGTTVTVSS (SEQ ID NO.31)

[0114] In a specific example, the heavy chain of the second antigen binding domain of the antibody is shown as SEQ ID NO.1.

[0115] QVQLVQSGPGLVKPSETLSLTCTVSGDSISSYYWSWIRQPPGKGLEWIGYISYSGSTDYNPSLKSRVTISSVDTSKNQFSLKLNSVTAADTAVYYCARHAYREKGQFVPYYFYYGM DVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO.1)

[0116] In a specific example, the light chain variable region of the second antigen binding domain of the antibody is shown as SEQ ID NO.32.

[0117] DIVMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPNLLIYAASTLQSGVPSRFSGSGSGTEFTLTIISLQPEDFATYYCQQLNSYPLTFGGGTKVDIK (SEQ ID NO.32)

[0118] In a specific example, the light chain of the second antigen binding domain of the antibody is shown as SEQ ID NO.2.

[0119] DIVMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPNLLIYAASTLQSGVPSRFSGSGSGTEFTLTIISLQPEDFATYYCQQLNSYPLTFGGGTKVDIKRT VAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO.2)

[0120] In a specific example, the heavy chain variable region of the second antigen binding domain of the antibody is shown as SEQ ID NO.33.

[0121] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGAIEPETGGTAYNREFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCTRYYSFAYWGQGTLVTVSS (SEQ ID NO.33)

[0122] In a specific example, the heavy chain of the second antigen-binding domain of the antibody is shown in SEQ ID NO.8.

[0123] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGAIEPETGGTAYNREFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCTRYYSFAYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDEKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQID NO.8)

[0124] In a specific example, the light chain variable region of the second antigen binding domain of the antibody is shown in SEQ ID NO.34.

[0125] DVVMTQTPLSLSVTPGQPASISCRSGQSLVHSNRNTYLQWYLLKPGQSPQLLIFKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQTSHVPLTFGQGTKLEIK (SEQ ID NO. 34)

[0126] In a specific example, the light chain of the second antigen binding domain of the antibody is shown in SEQ ID NO.9.

[0127] DVVMTQTPLSLSVTPGQPASISCRSGQSLVHSNRNTYLQWYLLKPGQSPQLLIFKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQTSHVPLTFGQGTKLEI KRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO.9)

[0128] It is understandable that when preparing bispecific antibodies, the present application uses antibody engineering to exchange the variable regions of the heavy and light chains at the CD3 end, which can further prevent mispairing between antibody chains.

[0129] Optionally, the antibody also includes a heavy chain constant region and a light chain constant region.

[0130] Further optionally, the present application does not specifically limit the species of origin of the heavy chain constant region and the light chain constant region, and can be any species including but not limited to human, mouse, rabbit, sheep, cow, horse, pig, dog, cat, camel, donkey, deer, mink, chicken, duck and goose.

[0131] The present application does not particularly limit the types of the heavy chain constant region and the light chain constant region of the antibody, which may be, but are not limited to, IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

[0132] On the other hand, the present application provides a nucleic acid molecule containing a nucleic acid encoding the bispecific antibody.

[0133] On the other hand, the present application provides a vector comprising the nucleic acid molecule. The present application does not specifically limit the type of recombinant expression vector, which may be, but is not limited to, an antibody expression vector. The present application does not specifically limit the antibody expression vector, which may be, but is not limited to, a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus or a combination thereof. For example, a PCDNA3.1 expression vector.

[0134] The present application also provides a cell comprising the nucleic acid molecule or the vector. The present 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.

[0135] On the other hand, the present application provides a method for preparing the cells, which comprises the step of introducing the nucleic acid molecule or the vector into the target cell.

[0136] In one example, the method of introduction is transfection.

[0137] The term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques 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 disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al, 1981, Gene 13:197.

[0138] On the other hand, the present application provides a method for preparing the bispecific antibody, comprising the following steps: culturing the host cells, and isolating the antibody or recombinant protein from the obtained culture.

[0139] In some examples, the CD3 specific binding protein of the present application is obtained by biosynthesis, such as culturing the cells. It can be understood by those skilled in the art that, when the amino acid sequence of the CD3 specific binding protein is known, it can also be obtained by other methods such as solid phase synthesis.

[0140] On the other hand, the present application provides a CD3 detection product, which includes the CD3 specific binding protein.

[0141] The embodiments of the present application do not specifically limit the type of the detection product, such as a diagnostic reagent, a test strip, a detection plate or a test kit. In the detection product, the CD3 specific binding protein competes for binding to CD3 in the test product.

[0142] On the other hand, the present application provides a method for detecting CD3 in a sample to be tested, wherein the detection method uses the CD3-specific binding protein as a detection antibody to determine whether the sample to be tested contains CD3 through an immune binding reaction.

[0143] The term "immunobinding" refers to a specific binding reaction that occurs between an antibody molecule and an antigen for which the antibody is specific. The strength or affinity of the immunobinding interaction can be expressed as the equilibrium dissociation constant (KD) of the interaction, where a smaller KD value indicates a higher affinity. The immunobinding properties between two molecules can be quantified using methods known in the art. One method involves measuring the rate at which the antigen binding site / antigen complex forms and dissociates.

[0144] The present application relates to a method for immunodetection or determination of a target antigen (e.g., CD3), a reagent for immunodetection or determination of a target antigen (e.g., CD3), a method for immunodetection or determination of cells expressing a target antigen (e.g., CD3), and a diagnostic agent for diagnosing a disease associated with target antigen (e.g., CD3)-positive cells, which comprises an antibody or antibody fragment of the present application that specifically recognizes the target antigen (e.g., CD3) and binds to the amino acid sequence of the extracellular region or its three-dimensional structure as an active ingredient.

[0145] In the present application, the method for detecting or measuring the amount of the target antigen (e.g., CD3) can be any known method. For example, it includes an immunoassay or assay method. An immunoassay or assay method is a method for detecting or measuring the amount of an antibody or an antigen using a labeled antigen or antibody. Examples of immunoassay or assay methods include radioactive material labeled immunoantibody methods (RIA), enzyme immunoassays (EIA or ELISA), fluorescent immunoassays (FIA), luminescent immunoassays, protein immunoblotting, physicochemical methods, etc.

[0146] To detect cells expressing the polypeptide, known immunodetection methods can be used, and preferably immunoprecipitation, fluorescent cell staining, immunohistostaining, etc. In addition, fluorescent antibody staining using FMAT8100 HTS system (Applied Biosystem) can be used.

[0147] In the application, there is no particular limitation on the sample to be tested for detecting or measuring the target antigen (e.g., CD3), as long as it has the possibility of containing cells expressing the target antigen (e.g., CD3), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture fluid.

[0148] Depending on the desired detection method, the test product may also contain reagents for performing antigen-antibody reactions or reagents for detecting reactions. Reagents for performing antigen-antibody reactions include buffers, salts, etc. Reagents for detection include reagents commonly used in immunoassays or assays, such as a labeled second antibody that recognizes the monoclonal antibody, its antibody fragment, or its conjugate, and a substrate corresponding to the label, etc.

[0149] On the other hand, the present application provides the use of the antibody or the recombinant protein in the preparation of a drug for treating tumors.

[0150] It is understandable that the present application does not specifically limit the type of tumor, and it can be any type of tumor that is positive for AMHR2 or GPC3 expression, such as lung cancer or liver cancer that is positive for AMHR2 expression, ovarian cancer, lung adenocarcinoma or neuroblastoma that is positive for GPC3 expression.

[0151] On the other hand, the present application provides a treatment method, which comprises administering an effective dose of the CD3-specific binding protein or the drug to a subject.

[0152] Wherein, "administering," "giving," and "treating" as applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administering," "giving," and "treating" may refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administering," "giving," and "treating" also means in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnosis, a combination composition, or by another cell. "Treatment," when applied to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0153] "Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments disclosed herein, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measurable extent. The amount of the therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the disease symptom has been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptom. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating every symptom of the target disease, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0154] "Effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results. For preventive uses, beneficial or desired results include eliminating or reducing the risk, reducing the severity, or delaying the onset of a condition, including the biochemical, histological, and / or behavioral symptoms of the condition, its complications, and intermediate pathological phenotypes presented during the development of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as reducing the incidence of various target antigen-related conditions of the present application or improving one or more symptoms of the condition, reducing the dose 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 the present disclosure in a patient.

[0155] The CD3-specific binding protein provided in the embodiments of the present application has specific CDRs, can bind to human CD3 epsilon & delta dimer protein with high affinity and has Jurkat cell binding activity. Compared with the CD3-terminal antibody in the traditional T cell bispecific antibody, the bispecific antibody prepared by using the CD3-specific binding protein of the present application has a different binding epitope, and can maintain a high tumor cell killing activity while also having a low cytokine release level, thereby balancing the effectiveness and safety of T cell bispecific antibody therapy.

[0156] On the other hand, the present application provides a bispecific antibody, which comprises a first antigen binding domain that specifically binds to CD3 and a second antigen binding domain that specifically binds to AMHR2 or GPC3. Among them, the CD3 antibody single-chain variable fragment (ScFV) in the first antigen binding domain can bind to T cells, and the second antigen binding domain binds to antigens on the surface of tumors. This binding mode can significantly activate T cell function. It can maintain a high tumor cell killing activity while also having a low cytokine release level, thereby balancing the effectiveness and safety of T cell bispecific antibody therapy.

[0157] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0158] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0159] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean 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 the present application.

[0160] Example 1

[0161] This embodiment provides a method for preparing a CD3-specific binding protein:

[0162] 1. Animal immunization and CD3 antibody screening

[0163] 1. Animal immunization

[0164] CD3δε protein dimer was purchased from Biopsy (Cat. No. CDD-H52W1). A peptide fragment containing the N-terminal 27 amino acid sequence of human CD3ε protein was synthesized: QDGNEEMGGITQTPYKVSISGTTVILT (SEQ ID NO.10). Its C-terminus was coupled with the widely used carrier protein keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) by adding a cysteine ​​to obtain CD3ε-N27-KLH or CD3ε-N27-BSA, in which the purpose of coupling with KLH is to produce a significant immune response.

[0165] 4-6 week old Balb / c (albino laboratory mice) or SJL mice were selected, and the animals were injected subcutaneously at multiple points with a mixture of CD3δε protein dimer and immune adjuvant (TiterMax or Sigma adjuvant system), once every 2-3 weeks, for a total of 3-4 immunizations. According to the serum titer, the high titer animals selected 3 days before the spleen was collected were immunized by intraperitoneal injection of antigen solution. The collected mouse spleen cells were used to obtain the antibody light chain and heavy chain variable region sequences through the single B cell screening technology platform, and the expression vector constructed by PCR was co-transfected into 293T cells to collect the cell culture supernatant.

[0166] 2. ELISA and flow cytometry screening

[0167] Human CD3δε protein dimer was coated in a 96-well ELISA plate at a concentration of 1μg / mL, 100μL / well, and incubated overnight at 4℃. The next day, after washing 4 times with PBST, 150μL of 5% skim milk powder blocking solution was added and incubated at 37℃ for 1 hour; after washing 4 times with PBST, 100μL of transfection supernatant was added and incubated at 37℃ for 1 hour; after washing 4 times with PBST, 100μL of Anti-Human IgGHRP (1:10000 dilution) was added and incubated at 37℃ for 0.5 hour; after washing 4 times with PBST, 50μL of TMB colorimetric solution was added and color developed at 37℃ for 8 minutes, 50μL of ELISA stop solution was added to each well to terminate the reaction, and the absorbance value was measured at a wavelength of 450nm by an ELISA reader.

[0168] Jurkat-NFAT-Luc Effector Cells were taken and 2×10 6 Cells / mL were resuspended in cell staining buffer (PBS containing 4% FBS). The resuspension was added to a 96-well U-bottom plate at 50 μL / well, centrifuged at 300g for 5 minutes, supernatant was discarded, 100 μL of transfection supernatant was added, and incubated at 4°C for 1 hour. The cells were washed three times with cell staining buffer, centrifuged, 100 μL APC-labeled anti-human IgG antibody was added to each well, incubated at 4°C for 30 minutes, and then the cells were washed three times, the supernatant was removed, and the cells were resuspended with 100 μL~200 μL cell staining buffer and analyzed by flow cytometry.

[0169] Based on the ELISA and flow cytometry binding activity of the transfection supernatant (containing the corresponding antibody protein), clones (M132 clones) that bind to human CD3δε protein dimers and have binding activity to Jurkat cells were further screened. The ELISA and flow cytometry test results of the M132 clone are shown in Table 4:

[0170] Table 4: Antibody binding activity to human CD3δε protein and human T cell line Jurkat in transfection supernatant

[0171]

[0172] 2. Construction and preparation of CD3 bispecific antibodies targeting AMHR2 or GPC3

[0173] The CD3 antibody-positive clone (M132 clone) obtained from the above screening was combined with the humanized anti-Mullerian Hormone Receptor Type 2 (AMHR2) antibody 7E1 or the anti-GPC3 antibody Hu285 to construct a "1+1" bispecific antibody with a recombinant IgG configuration.

[0174] The T cell bispecific antibody ERY974 from Chugai Pharmaceutical was used as the positive control antibody for GPC3×CD3. All candidate and reference antibody sequences are shown in Tables 5 to 7.

[0175] Table 5: AMHR2 × CD3 bispecific antibodies

[0176]

[0177] Table 6: List of GPC3 × CD3 bispecific antibodies

[0178]

[0179] Table 7: ERY974, a T cell bispecific antibody produced by Chinese and foreign pharmaceutical companies

[0180]

[0181] ERY974 antibody GPC3 end heavy chain:

[0182] QVQLVQSGAEVKKPGASVTVSCKASGYTFTDYEMHWIRQPPGEGLEWIGAIDGPTPDTAYSEKFKGRVTLTADKSTSTAYMELSSLTSEDTAVYYCTRFYSYTYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFRGGPKVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQESLSLSP (SEQ ID NO.5)

[0183] ERY974 antibody GPC3-terminal - light chain and CD3-terminal light chain (SEQ ID NO.6)

[0184] DIVMTQSPLSLPVTPGEPASISCRSSQPLVHSNRNTYLHWYQQKPGQAPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCGQGTQVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO.6)

[0185] ERY974 antibody CD3-terminal heavy chain:

[0186] QVQLVESGGGVVQPGGSLRLSCAASGFTFSNAWMHWVRQAPGKGLEWVAQIKDKSQNYATYVAESVKGRFTISRADSKNSIYLQMNSLKTEDTAVYYCRYVHYAAGYGVDIW GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFRGGPKVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTI SKAKGQPREPQVYTLPPSQKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSP (SEQ ID NO.7)

[0187] The correct sequence was expanded and the plasmid was extracted using an endotoxin-free extraction kit (ZYMO RESEARCH, D4201-A), and the specific operation was carried out according to the instructions provided by the manufacturer. HEK-293T cells were cultured in DMEM basic medium (Gbico, Cat. No.: C11995500BT) at 37°C and 5% CO2 in a cell culture incubator according to the instructions provided by the manufacturer. After the cells were prepared, the plasmid was transfected into HEK293-T cells using a transfection kit according to the manufacturer's instructions (Polysciences, Cat. No.: 24765-1) to express the anti-GPC3×CD3 bispecific antibody. After 9 days of culture, the expression supernatant was harvested by centrifugation. The expression supernatant was filtered with a 0.22μM filter membrane and the antibody was purified using a Protein A affinity chromatography column.

[0188] Verification results:

[0189] 1. Affinity determination of M132-7E1 antibody and M132-Hu285 antibody

[0190] The bispecific antibody was captured on the Protein A probe using surface plasmon resonance (SPR) technology, and the binding ability to human CD3δε protein dimer at multiple concentrations (200, 100, 50, 25, 12.5, 6.25, 3.125, 0 nM) was detected by Biacore 8k instrument, and the affinity constant KD was obtained based on the binding and dissociation rates. The affinity data are shown in Table 8.

[0191] Table 8: SPR analysis of the affinity of candidate antibodies to human CD3δε protein dimer

[0192]

[0193] II. Identification of antibody binding activity to the N-terminal peptide epitope of CD3ε

[0194] Human CD3δε protein dimer and CD3ε-N27-BSA were coated in 96-well ELISA plates at concentrations of 0.2μg / mL and 0.5μg / mL, respectively, 50μL / well, and incubated at 4°C overnight. The next day, after washing with PBST 4 times, 150μL of 5% skim milk powder blocking solution was added and incubated at room temperature for half an hour; after washing with PBST 4 times, antibodies diluted with 3% skim milk powder were added and incubated at room temperature for half an hour; after washing with PBST 4 times, 50μL of Anti-Human IgG HRP (1:10000 dilution) was added and incubated at room temperature for half an hour; after washing with PBST 4 times, 50μL of TMB color development solution was added, and color was developed at room temperature in the dark for 5 minutes. 50μL of ELISA stop solution was added to each well to terminate the reaction, and the absorbance value was measured at a wavelength of 450nm using an ELISA reader. The results are as follows Figure 1 and Figure 2 shown.

[0195] The results show that the CD3-specific binding antibody provided in the present application can bind to the CD3δε protein dimer but not to the N-terminal polypeptide epitope of CD3ε, while the reference antibody ERY974 can bind to both of them significantly, indicating that the candidate antibody has a different binding epitope from the reference antibody, that is, it binds to a position on the CD3δε protein other than the N-terminal polypeptide.

[0196] III. T cell activation activity assay of M132-7E1 antibody and M132-Hu285 antibody

[0197] The ovarian cancer COV434 cell line was purchased from Shanghai Fuheng Biotechnology. The tool cell COV434-AMHR2 overexpressing AMHR2 antigen was constructed for subsequent T cell activation and anti-tumor activity experiments; the liver cancer cell HepG2 cells (derived from CCTCC) endogenously expressing GPC3 were used for subsequent T cell activation and anti-tumor activity experiments.

[0198] The Luciferase reporter gene assay was used to analyze the activation activity of candidate antibodies on the Jurkat-NFAT-Luc Effector Cells cell signaling pathway in the presence of target cells expressing AMHR2 or GPC3 antigens. Specific operation: HepG2 cells (derived from CCTCC) in the logarithmic growth phase or COV434-AMHR2 cells overexpressing AMHR2 were digested with cell digestion solution, centrifuged and resuspended with cell culture medium and the density was adjusted to 1×10 5 Cells were added to a white 96-well flat-bottom plate at 100 µL / well and incubated at 37°C and 5% CO2 for 18 h. The next day, the supernatant was removed and assay buffer was added to dilute the cells to a density of 3 × 10 6 Jurkat-NFAT-Luc Effector Cells with a concentration of 50 μL / well were added; the antibody (53.33 nM, 6-fold gradient dilution, 7 concentration points) was added, 50 μL / well; the cells were incubated at 37°C and 5% CO2 for 6 hours. The luciferase colorimetric solution (Biyuntian, RG052M) was taken out and thawed 20 to 30 minutes in advance and equilibrated to room temperature; the 96-well plate was taken out and equilibrated at room temperature for 5 to 10 minutes, and the colorimetric solution was added, 50 μL / well; the plate was incubated at room temperature in the dark for 10 minutes; the relative luminescence intensity value was read on a multifunctional microplate reader. The results are as follows: Figure 3 and Figure 4 The statistical results are shown in Tables 9 and 10.

[0199] Table 9

[0200]

[0201] Table 10

[0202]

[0203] The results showed that when CD3 antibody was combined with anti-AMHR2 or anti-GPC3 antibody to form a bispecific antibody, it had significant T cell activation activity in the presence of tumor cells (ovarian cancer or liver cancer cell lines).

[0204] IV. Determination of antitumor activity of M132-7E1 antibody and M132-Hu285 antibody

[0205] PBMC cells were used as effector cells. Antibodies lysed target cells by inducing T-cell-mediated cell killing (TDCC), and the TDCC effect of candidate antibodies was reflected by detecting the release of cellular lactate dehydrogenase (LDH). Specific operation: PBMC cells were resuscitated (purchased from Miaoshun Biotechnology); the next day, Huh-7 cells (Procell, CL-0120) or COV434-AMHR2 cells grown to the logarithmic growth phase were adjusted to a cell density of 4×10 in 1640 medium (Gibco, 11835030). 5 / mL, add to 96-well U-bottom plate, 50µL / well; add cell density of 2×10 6 PBMC cells / mL, 100µL / well; add antibody (100nM, 10-fold gradient dilution, 6 concentration points), 50µL / well; incubate at 37℃, 5% CO2 for 24 hours. According to the method of LDH detection kit (Promega, G1780), 50µL / well of the supernatant of the test well was aspirated and tested for OD490. The results are as follows Figure 5 and Figure 6 The statistical results are shown in Table 11 and Table 12.

[0206] Table 11

[0207]

[0208] Table 12

[0209]

[0210] The results show that the M132-7E1 antibody and the M132-Hu285 antibody of the present application have high killing activity against tumor target cells after forming bispecific antibodies with different anti-tumor target antibodies.

[0211] 5. Cytokine release assay of M132-7E1 antibody and M132-Hu285 antibody

[0212] CBA was used to detect the cytokine content. Specific operation: PBMC cells (from Miaoshun Biotechnology) were resuscitated; the next day, Huh-7 cells or COV434-AMHR2 cells grown to the logarithmic growth phase were adjusted to a cell density of 4×10 5 / mL, add to 96-well U-bottom plate, 50µL / well; add cell density of 2×10 6PBMC cells / mL, 100µL / well; add antibody (100nM, 10-fold gradient dilution, 6 concentration points), 50µL / well; incubate for 24 hours at 37℃, 5% CO2. According to the instructions of Human Th1 / Th2 Cytokine kit II (manufacturer: BD catalog number: 551809), the cytokine content was detected. The results are as follows Figure 7 and Figure 8 shown.

[0213] The results showed that the cytokine release of M132-Hu285 was significantly lower than that of the reference antibody ERY974, which indicates that the candidate antibody has significantly higher safety than the reference antibody while maintaining its tumor killing activity.

[0214] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A CD3-specific binding protein, characterized in that: The CD3-specific binding protein has a CD3 binding domain; The CD3 binding domain has heavy chain CDR1 to heavy chain CDR3 shown in SEQ ID NO.11 to SEQ ID NO.13, and light chain CDR1 to light chain CDR3 shown in SEQ ID NO.14 to SEQ ID NO.

16.

2. The CD3-specific binding protein according to claim 1, characterized in that: The CD3 binding domain satisfies one or more of the following conditions: (1) the heavy chain variable region is as shown in SEQ ID NO.30; and, (2) The light chain variable region is shown in SEQ ID NO.

29.

3. The CD3-specific binding protein according to claim 1, characterized in that: The CD3 binding domain satisfies one or more of the following conditions: (1) the heavy chain is as shown in SEQ ID NO.3; and (2) The light chain is shown in SEQ ID NO.

4.

4. A bispecific antibody, characterized in that: Include: A first antigen binding domain that specifically binds to CD3 and a second antigen binding domain that specifically binds to AMHR2 or GPC3; The first antigen binding domain that specifically binds to CD3 comprises the CD3-specific binding protein according to any one of claims 1 to 3.

5. The bispecific antibody according to claim 4, characterized in that The second antigen-binding domain has heavy chain CDR1 to heavy chain CDR3 shown in SEQ ID NO.17 to SEQ ID NO.19, and light chain CDR1 to light chain CDR3 shown in SEQ ID NO.20 to SEQ ID NO.

22.

6. The bispecific antibody according to claim 4, characterized in that The second antigen binding domain has a heavy chain variable region as shown in SEQ ID NO.31 and a light chain variable region as shown in SEQ ID NO.

32.

7. The bispecific antibody according to claim 4, characterized in that The second antigen-binding domain has heavy chain CDR1 to heavy chain CDR3 shown in SEQ ID NO.23 to SEQ ID NO.25, and light chain CDR1 to light chain CDR3 shown in SEQ ID NO.26 to SEQ ID NO.

28.

8. The bispecific antibody according to claim 4, characterized in that The second antigen binding domain has a heavy chain variable region shown in SEQ ID NO.33 and a light chain variable region shown in SEQ ID NO.

34.

9. A nucleic acid molecule, characterized in that The method comprises encoding the CD3-specific binding protein according to any one of claims 1 to 3 and the bispecific antibody according to any one of claims 4 to 8.

10. A carrier, characterized in that Comprising the nucleic acid molecule of claim 9.

11. A cell, characterized in that Comprising the nucleic acid molecule of claim 9 or the vector of claim 10.

12. A method for preparing the cell according to claim 11, comprising the step of introducing the nucleic acid molecule or the vector into a target cell.

13. The method for preparing a CD3-specific binding protein according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: Cultivating the cell of claim 11, and isolating the antibody from the resulting culture.

14. The method for preparing a bispecific antibody according to any one of claims 4 to 8, characterized in that: The preparation method comprises the following steps: Cultivating the cell of claim 11, and isolating the antibody from the resulting culture.

15. A CD3 detection product, characterized in that: The detection product comprises the CD3-specific binding protein according to any one of claims 1 to 3 and the bispecific antibody according to any one of claims 4 to 8.

16. A method for detecting CD3 in a sample for non-disease diagnosis purposes, characterized in that: The detection method uses the CD3-specific binding protein of any one of claims 1 to 3 or the bispecific antibody of any one of claims 4 to 8 as a detection antibody, and determines whether the sample to be tested contains CD3 through an immune binding reaction.

17. A drug, characterized in that The drug comprises the CD3-specific binding protein according to any one of claims 1 to 3 or the bispecific antibody according to any one of claims 4 to 8.

18. Use of the CD3-specific binding protein according to any one of claims 1 to 3, the bispecific antibody according to any one of claims 4 to 8, the nucleic acid molecule according to claim 9, the vector according to claim 10 or the cell according to claim 11 in the preparation of a drug for treating tumors; The tumors treated by the drug for treating tumors include one or more of breast cancer, ductal cancer, ovarian cancer, endometrial cancer, pancreatic cancer, thyroid cancer, kidney cancer, bladder cancer, lung cancer and liver cancer.

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