Anti-AMHR2*CD3 bispecific antibody as well as preparation method and application thereof

Abstract

The invention relates to an anti-AMHR2 * CD3 bispecific antibody and preparation and application thereof in the field of biological medicine. In the bispecific antibody, a heavy chain variable region of a CD3 binding structural domain has HCDR1 to HCDR3 as shown in SEQ ID NO.7 to 9 or SEQ ID NO.7, 24 and 9, and a light chain variable region of the CD3 binding structural domain has LCDR1 to LCDR3 as shown in SEQ ID NO.10 to 12; a heavy chain variable region of the AMHR2 binding structural domain has HCDR1 to HCDR3 as shown in sequences SEQ ID NO.13 to 15, and a light chain variable region of the AMHR2 binding structural domain has LCDR1 to LCDR3 as shown in sequences SEQ ID NO.16 to 18. The invention provides a novel anti-AMHR2 * CD3 bispecific antibody, and a novel solution is provided for detection and treatment of AMHR2 positive tumors and cancers.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to an anti-AMHR2xCD3 bispecific antibody and its preparation and use. BACKGROUND

[0002] Anti-Mullerian hormone receptor 2 (AMHR2) belongs to the transforming growth factor-β (TGF-β) superfamily members, and its natural ligand is anti-Mullerian hormone (AMH / MIS). After AMH binds to AMHR2, it can form a complex with type I receptors (such as ALK2 / ACVR1), thereby activating the downstream SMAD signaling pathway (such as SMAD1 / 5 / 8), and regulating the proliferation, differentiation and apoptosis of cells through this pathway.

[0003] Tumor studies have shown that AMHR2 is highly expressed in a variety of cancers, including ovarian cancer, endometrial cancer, cervical cancer, breast cancer, and prostate cancer, etc.; while its expression in normal tissues is strictly limited. This significant difference in expression between tumor tissues and normal tissues makes AMHR2 a very potential specific target in the field of cancer treatment, and targeting AMHR2 can achieve precise killing of AMHR2-positive tumor cells, providing a new direction for the treatment of AMHR2-positive tumors and cancers.

[0004] At present, Murlentamab (GM102) is the first AMHR2-targeting antibody drug to enter clinical research, and its main mechanism of action is to activate antibody-dependent NK cell-mediated cytotoxicity (ADCC) and antibody-dependent macrophage-mediated cell killing (ADCP), thereby specifically killing tumor cells expressing AMHR2. In the first I / II phase clinical study of GM102, a total of 68 patients with advanced or recurrent AMHR2-positive ovarian cancer, granulosa cell tumor (GCT), cervical cancer and endometrial cancer were included. The results of the study showed that: when treated with a single drug, only 1 case of GCT patient achieved partial remission; when combined with chemotherapy drugs, 44% (4 / 9 cases) of patients responded to treatment. In terms of safety, no serious adverse events related to Murlentamab were observed. However, there are currently very limited effective AMHR2 antibody drugs, and therefore there is an urgent need to develop new AMHR2-targeting drugs to improve the killing activity of AMHR2-positive tumor cells and provide new strategies for more efficient treatment of AMHR2-positive cancers.

[0005] CD3, as the core component of T cell receptor (TCR) complex, is composed of CD3 epsilon, gamma, delta and zeta chains, and undertakes the TCR signal transduction function. Among them, CD3 epsilon is highly expressed on the surface of T cells and participates in the early activation signal, becoming a common target of bispecific antibodies. CD3 plays a key role in the activation process of T cells and is an important molecular basis for the realization of T cell function.

[0006] AMHR2 x CD3 bispecific antibodies can simultaneously bind AMHR2 tumor antigens and CD3, and recruit T cells to the periphery of tumor cells. After binding to CD3, it can simulate the signal transmission process of TCR-MHC-antigen peptide complex, activate the cytotoxic function of T cells (such as releasing perforin, granzyme, etc.), thereby efficiently exerting anti-tumor effect. Based on this, the development of anti-AMHR2 x CD3 bispecific antibody drugs opens up a new way for the treatment of AMHR2 positive tumors, which has important significance for the treatment of AMHR2 positive tumors. The T cell-mediated cytotoxicity relied on by bispecific antibody drugs is different from the ADCC and ADCP mechanisms of existing drugs, and can be used as a completely new strategy for treating AMHR2 positive solid tumors, enriching cancer treatment methods, especially for patients who are resistant to traditional treatment methods. It is expected to overcome the short board of the limited efficacy of existing AMHR2 single-target antibody drugs. The existing drug Murlentamab has a low response rate for single drug treatment, while the bispecific antibody drug can precisely guide T cells to AMHR2 positive tumor cells by simultaneously binding AMHR2 and CD3, and with the help of the strong killing ability of T cells, it is expected to greatly improve the killing activity of tumor cells and improve the treatment response rate, bringing new hope to patients who do not respond well to existing treatment methods. SUMMARY

[0007] Based on this, one or more embodiments of the present application provide an anti-AMHR2 x CD3 bispecific antibody and its preparation and use. Including the following technical solutions:

[0008] One or more embodiments of the present application provide an anti-AMHR2 x CD3 bispecific antibody, which satisfies the following conditions:

[0009] The heavy chain variable region of the CD3 binding domain has HCDR1 to HCDR3 with the sequence as shown in SEQ ID NO. 7 to 9 or HCDR1 to HCDR3 as shown in SEQ ID NO. 7, 24 and 9, and the light chain variable region has LCDR1 to LCDR3 with the sequence as shown in SEQ ID NO. 10 to 12; and

[0010] The heavy chain variable region of the AMHR2 binding domain has HCDR1 to HCDR3 with sequences as shown in SEQ ID NO. 13 to 15, and the light chain variable region has LCDR1 to LCDR3 with sequences as shown in SEQ ID NO. 16 to 18.

[0011] In some embodiments of the present application, the bispecific antibody has a heavy chain variable region with sequence as shown in SEQ ID NO. 5 or 21 and a light chain variable region with sequence as shown in SEQ ID NO. 6 or 22.

[0012] In some embodiments of the present application, the bispecific antibody has a heavy chain variable region with sequence as shown in SEQ ID NO. 19 and a light chain variable region with sequence as shown in SEQ ID NO. 20.

[0013] In some embodiments of the present application, the bispecific antibody is a 2+1 valency bispecific antibody.

[0014] In some embodiments of the present application, the bispecific antibody has a Fab / Fab-scFv Fc structure.

[0015] In some embodiments of the present application, one antigen binding arm of the bispecific antibody binds to AMHR2, and the other antigen binding arm binds to AMHR2 and CD3.

[0016] In some embodiments of the present application, the bispecific antibody has a heavy chain with sequence as shown in SEQ ID NO. 2 and a light chain with sequence as shown in SEQ ID NO. 4.

[0017] In some embodiments of the present application, the bispecific antibody has a heavy chain with sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 23, and a light chain with sequence as shown in SEQ ID NO. 4.

[0018] One or more embodiments of the present application provide a nucleic acid molecule encoding the specific antibody.

[0019] One or more embodiments of the present application provide a vector comprising the nucleic acid molecule.

[0020] One or more embodiments of the present application provide a cell expressing the bispecific antibody, or comprising the nucleic acid molecule or the vector.

[0021] One or more embodiments of the present application provide a method of constructing the cell, comprising the step of introducing the nucleic acid molecule or the vector into a host cell.

[0022] One or more embodiments of the present application provide a method for preparing the bispecific antibody, the method comprising the step of culturing the cell.

[0023] One or more embodiments of the present application provide use of the bispecific antibody, the nucleic acid molecule, the vector or the cell in the preparation of a medicament or a detection product.

[0024] One or more embodiments of the present application provide a medicament comprising the bispecific antibody.

[0025] One or more embodiments of the present application provide a detection product comprising the bispecific antibody.

[0026] One or more embodiments of the present application provide a method for detecting AMHR2 or / and CD3 in a sample to be tested, the method determining the presence of AMHR2 or / and CD3 in the sample to be tested by an immunological binding reaction using the bispecific antibody as a detection antibody.

[0027] One or more embodiments of the present application provide a method for treating AMHR2-positive tumors, the method comprising administering to a subject a therapeutically effective amount of the bispecific antibody or the medicament.

[0028] The details of one or more embodiments of the present application are set forth in the accompanying description, which makes apparent to those skilled in the art other features, objectives and advantages of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the accompanying drawings needed to be used in the description of the embodiments will be briefly introduced. Obviously, the accompanying drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0030] Figure 1 Figure 1 is a structural schematic diagram of the hCR04-BA7 bispecific antibody.

[0031] Figure 2 Figure 2 is the detection result of the binding activity of the hCR04-BA7 bispecific antibody to human AMHR2 overexpressing cell lines.

[0032] Figure 3 Figure 3 is the detection result of the binding activity of the hCR04-BA7 bispecific antibody to human T cells.

[0033] Figure 4The results show the antitumor activity assay of the hCR04-BA7 bispecific antibody.

[0034] Figure 5 The results are from the hCR04-BA7 bispecific antibody cytokine release assay.

[0035] Figure 6 The results are from the assay of target cell cytokine release of the hCR04-BA7 bispecific antibody.

[0036] Figure 7 This is a statistical graph showing the tumor volume and body weight of CD34+HSC immune reconstituted mice in the control and treatment groups.

[0037] Figure 8 This is a statistical graph showing the tumor volume and body weight of mice in the control and treatment groups of PBMC immune reconstitution mice. Detailed Implementation

[0038] The present application will be further described in detail below with reference to the accompanying drawings, 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.

[0039] 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. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0040] the term

[0041] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0042] 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 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 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").

[0043] 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.

[0044] 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.

[0045] 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.

[0046] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0047] 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.

[0048] 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.

[0049] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0050] 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.

[0051] 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.

[0052] 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℃.

[0053] 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.

[0054] One or more embodiments of this application provide an anti-AMHR2×CD3 bispecific antibody, wherein the bispecific antibody satisfies the following conditions:

[0055] The heavy chain variable region of the CD3 binding domain has sequences HCDR1 to HCDR3 as shown in SEQ ID NO. 7 to 9 or HCDR1 to HCDR3 as shown in SEQ ID NO. 7, 24 and 9, and the light chain variable region has sequences LCDR1 to LCDR3 as shown in SEQ ID NO. 10 to 12; and

[0056] The heavy chain variable region of the AMHR2 binding domain has HCDR1 to HCDR3 as shown in SEQ ID NO. 13 to 15, and the light chain variable region has LCDR1 to LCDR3 as shown in SEQ ID NO. 16 to 18.

[0057] The CD3-binding domain and AMHR2-binding domain of this application may have the aforementioned CDRs or derivative fragments having the aforementioned CDRs. The derivative fragments are formed by replacing amino acids at no more than six sites relative to their corresponding CDRs (“conservative modification” or “conservative substitution”), retaining the biological activity consistent with their corresponding complementarity-determining regions. For example, the derivative fragments may replace one amino acid with another, or one amino acid with multiple (e.g., two) amino acids, at sites 1, 2, 3, 4, 5, or 6 of their corresponding complementarity-determining regions.

[0058] In the CDRs provided in this application, the derived fragments (conserved variants) refer to polypeptides formed by replacing one, two, or three amino acids with amino acids of similar or related properties compared to the amino acid sequence of the antibody in this application. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0059] Table A

[0060] Original residue Representative substitution Preferred substitution Ala (A) Val; Leu; lie Val Arg (R) Lys; Gin; Asn Lys Asn (N) Gin; His; Lys; Arg Gin Asp (D) Glu Glu Cys (C) Ser Ser Gin (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro; Ala Ala His (H) Asn; Gin; Lys; Arg Arg lie (I) Leu; Val; Met; Ala; Phe Leu Leu (L) lie; Val; Met; Ala; Phe lie Lys (K) Arg; Gin; Asn Arg Met (M) Leu; Phe; lie Leu Phe (F) Leu; Val; lie; Ala; Tyr Leu Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) lie; Leu; Met; Phe; Ala Leu

[0061] "Conservative modification" or "conservative substitution" 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), allowing for frequent alterations without changing the protein's biological activity. Those skilled in the art will recognize that, in general, the substitution of a single amino acid in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987), Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)). Furthermore, substitutions of structurally or functionally similar amino acids are unlikely to disrupt biological activity.

[0062] The "antibody" mentioned in this application refers to immunoglobulins. A complete antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain of immunoglobulins differ, thus their antigenicity also differs. Based on this, immunoglobulins can be divided into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, based on differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in its heavy chain, it can be further divided into different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. The light chains are classified as κ chains or λ chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ chain or a λ chain.

[0063] The sequence of approximately 110 amino acids near the N-terminus of both the antibody heavy and light chains varies considerably and is known as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are called the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved backbone regions (FR). The three hypervariable regions determine the antibody's specificity and are also called complementarity-determining regions (CDR). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0064] A biantibody is an antibody fragment in which scFv or Fab is dimerized, and it is an antibody fragment with bivalent antigen-binding activity. In bivalent antigen-binding activity, the two antigens can be the same or different.

[0065] Bispecific antibodies and multispecific antibodies are antibodies that can bind to two or more antigens or antigenic determinants.

[0066] dsFv is obtained by linking polypeptides in which one amino acid residue in each VH and VL is replaced by a cysteine ​​residue via disulfide bonds between cysteine ​​residues. The amino acid residues to be replaced by cysteine ​​residues can be selected based on the prediction of the antibody's three-dimensional structure using known methods (e.g., Protein Engineering, 7, 697 (1994)).

[0067] The term "amino acid difference" or "amino acid mutation" refers to an alteration or mutation of amino acids in a variant protein or polypeptide compared to the original protein or polypeptide. This includes the insertion, deletion, or substitution of one, two, three, or more amino acids in the original protein or polypeptide.

[0068] The term "antibody framework" or "FR region" refers to a portion of the variable domain VL or VH that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain. Essentially, it is a variable domain without a CDR.

[0069] The terms "complementarity-determining region," "CDR," or "hypervariant region" refer to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitates antigen binding. Typically, three CDRs (HCDR1, HCDR2, HCDR3) exist in each heavy chain variable region, and three CDRs (LCDR1, LCDR2, LCDR3) exist in each light chain variable region. The amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the "Kabat" numbering rule (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering rule (see Al-Lazikani et al., (1997) JMB 27 3: 927-948), and the Immunogenetics (IMGT) numbering rule (Leefranc). MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003), etc. For example, for the classical format, following Kabat rules, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Following Chothia rules, the CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acids in VL are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acids in VL are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the CDR amino acids in VL are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3). The amino acid residues are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). Combining the CDR definitions from Kabat and Chothia, the CDR consists of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) from human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) from human VL. Following the IMGT rules, the CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), while those in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3).Following the IMGT rules, the CDR region of an antibody can be determined using the IMGT / DomainGap Align procedure.

[0070] The term "epitope" or "antigenic determinant" refers to a site on an antigen that is bound to an immunoglobulin or antibody. Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a distinctive spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996).

[0071] Amino acid sequence “identity” refers to the percentage of amino acid residues in a first sequence that are identical to those in a second sequence when aligning amino acid sequences (introducing gaps where necessary) to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity. To determine the percentage of amino acid sequence identity, alignment can be performed in a variety of ways within the scope of the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0072] In some embodiments of this application, the bispecific antibody has a heavy chain variable region with a sequence as shown in SEQ ID NO. 5 (or a sequence having at least 80% identity with SEQ ID NO. 5, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and a light chain variable region with a sequence as shown in SEQ ID NO. 6 (or a sequence having at least 80% identity with SEQ ID NO. 6, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%).

[0073] In some embodiments of this application, the bispecific antibody has a heavy chain variable region with a sequence as shown in SEQ ID NO. 21 (or a sequence having at least 80% identity with SEQ ID NO. 21, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and a light chain variable region with a sequence as shown in SEQ ID NO. 22 (or a sequence having at least 80% identity with SEQ ID NO. 22, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%).

[0074] In some embodiments of this application, the bispecific antibody has a heavy chain variable region as shown in SEQ ID NO. 19 (or a sequence having at least 80% identity with SEQ ID NO. 19, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and a light chain variable region as shown in SEQ ID NO. 20 (or a sequence having at least 80% identity with SEQ ID NO. 20, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%).

[0075] In some embodiments of this application, the bispecific antibody is a 2+1 valence bispecific antibody.

[0076] In some embodiments of this application, the bispecific antibody has a Fab / Fab-scFv Fc structure.

[0077] In some embodiments of this application, one antigen-binding arm of the bispecific antibody binds AMHR2, and the other antigen-binding arm binds both AMHR2 and CD3.

[0078] Optionally, the bispecific antibody has a heavy chain with a sequence as shown in SEQ ID NO.2 (or a sequence having at least 80% identity with SEQ ID NO.2, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and a light chain with a sequence as shown in SEQ ID NO.4 (or a sequence having at least 80% identity with SEQ ID NO.4, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%).

[0079] Optionally, the bispecific antibody has a heavy chain with a sequence as shown in SEQ ID NO. 3 (or a sequence having at least 80% identity with SEQ ID NO. 3, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) or SEQ ID NO. 23 (or a sequence having at least 80% identity with SEQ ID NO. 23, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), or a heavy chain with a sequence as shown in SEQ ID NO. 4 (or a sequence having at least 80% identity with SEQ ID NO. 23, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%), or a heavy chain with a sequence as shown in SEQ ID NO. 4 (or a sequence having at least 80% identity with SEQ ID NO. 23, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 99%), or a heavy chain with a sequence as shown in SEQ ID NO. 4 (or a heavy chain with at least 80% identity with SEQ ID NO. 23, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 98%, 99%), or a heavy chain with a sequence as shown in SEQ ID NO. 4 (or a heavy chain with NO.4 contains sequences with at least 80% identity, such as the light chains shown in the examples 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%).

[0080] The bispecific antibody hCR04-BA7 in the embodiments of this application has the sequence shown in SEQ ID NO.2, SEQ ID NO.4, and SEQ ID NO.3, and its structure... Figure 1 As shown.

[0081] One or more embodiments of this application provide a nucleic acid molecule that encodes the specific antibody.

[0082] As used herein, the term "nucleic acid molecule" refers to DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0083] The nucleic acid molecules in this application primarily refer to isolated nucleic acid molecules. "Isolated" means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. Generally, the term "isolated" is not intended to mean the complete absence of these materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds described herein.

[0084] One or more embodiments of this application provide a vector comprising the nucleic acid molecule described above.

[0085] The term "vector," also known as "expression vector," refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop to which an additional DNA segment can be linked. In another embodiment, the vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. The vectors disclosed herein are capable of autonomous replication in host cells that have been introduced into them (e.g., bacterial vectors with bacterial origins of replication and augmented mammalian vectors) or can be integrated into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome (e.g., non-augmented mammalian vectors). In general, vectors may be selected from, but are not limited to, mammalian cell viruses, bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, or combinations thereof.

[0086] One or more embodiments of this application provide a cell that expresses the bispecific antibody, or the cell includes the nucleic acid molecule or the vector.

[0087] The term "cell," also known as "host cell," refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant, or animal cells. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of *Escherichia coli* or *Salmonella*; members of the Bacillaceae family, such as *Bacillus subtilis*; *Pneumococcus*; *Streptococcus*; and *Haemophilus influenzae*. Suitable microorganisms include *Saccharomyces cerevisiae* and *Pichia pastoris*. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.

[0088] The terms “cell,” “cell line,” and “cell culture” used herein are used interchangeably, and all such names include progeny. Therefore, “transformant” and “transformed cell” include primary test cells and cultures derived from them, regardless of passage number. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells. Where different names are intended, the context will be clear.

[0089] One or more embodiments of this application provide a method for constructing the cell described above, the method comprising the step of introducing the nucleic acid molecule or the vector into a host cell.

[0090] One or more embodiments of this application provide a method for preparing the bispecific antibody, the method comprising the step of culturing the cells.

[0091] The antibody or antigen-binding fragments of this application can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect host cells. As a more preferred prior art, mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site in the Fc region. Stable clones are obtained by expressing the target antibody. Positive clones are scaled up in a bioreactor to produce antibodies. The culture medium secreting the antibody can be purified using conventional techniques, such as using an A or G Sepharose FF column with adjusted buffer. Non-specifically bound components are washed away. The bound antibody is then eluted using a pH gradient, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0092] Methods for producing and purifying antibodies and antigen-binding fragments are well-known in the prior art, such as those described in Cold Spring Harbor's Guide to Antibody Laboratory Techniques, Chapters 5-8 and 15. For example, the resulting antibodies can be renatured, purified, and sequenced using conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibodies or antigen-binding fragments described in this application are genetically engineered to add one or more human FR regions to a non-human CDR region. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website (http: / / imgt.cines.fr) by comparing against the IMGT Human Antibody Variable Region Germplasm Database and MOE software, or from the journal *Immunoglobulins*, 2001 ISBN012441351.

[0093] One or more embodiments of this application provide the use of the described bispecific antibody, the described nucleic acid molecule, the described vector, or the described cell in the preparation of drugs or detection products.

[0094] One or more embodiments of this application provide a drug comprising the bispecific antibody described above.

[0095] It is understood that the medicines in this application include pharmaceutically acceptable carriers.

[0096] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in a formulation for delivering antibody or antigen-binding fragments. Carriers can be anti-adhesion agents, adhesives, coatings, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption delay agents, wetting agents, emulsifiers, buffers, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffer solutions, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0097] This application includes a pharmaceutical agent for treating diseases associated with positive cells of target antigens (e.g., CD3 and AMHR2), said pharmaceutical agent comprising the CD3 and AMHR2 antibodies of this application or antigen-binding fragments thereof as active ingredients.

[0098] One or more embodiments of this application provide a detection product, the detection product comprising the aforementioned bispecific antibody.

[0099] This application involves a bispecific antibody in the test product competitively binding to CD3 and AMHR2 in the test sample.

[0100] When the term "competition" is used in the context of competing antigen-binding proteins for the same epitope, it refers to competition between antigen-binding proteins, which is determined by an assay in which the antigen-binding protein to be detected (e.g., an antibody or a functional fragment thereof) prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., an antigen or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another. These assays include: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assays (see, for example, Stahli et al., 1983, Methods in Enzymology 9: 242-253); solid-phase direct biotin-avidin EIA (see, for example, Kirkland et al., 1986, J. Immunol. 137: 3614-3619), solid-phase direct labeling assays, and solid-phase direct labeling sandwich assays (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); and assays using I-1 2. 5. Solid-phase direct labeling of RIAs (see, for example, Morel et al., 1988, Molec. Immunol. 25: 7-15); solid-phase direct biotin-avidin EIAs (see, for example, Cheung et al., 1990, Virology 176: 546-552); and directly labeled RIAs (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). Typically, the assay involves using a solid surface or cell to bind purified antigen loaded with either an unlabeled detection antigen-binding protein or a labeled reference antigen-binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cell in the presence of the detection antigen-binding protein. Typically, the detection antigen-binding protein is present in excess. Antigen-binding proteins identified by competitive assays (competitive antigen-binding proteins) include: antigen-binding proteins that bind to the same epitope as a reference antigen-binding protein; and antigen-binding proteins that bind to a neighboring epitope sufficiently close to the binding epitope of the reference antigen-binding protein, wherein the two epitopes spatially prevent each other from binding. Further details regarding methods for determining competitive binding are provided in the embodiments herein. Typically, when an excess of a competing antigen-binding protein is present, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of the specific binding of the reference antigen-binding protein to the common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0101] One or more embodiments of this application provide a method for detecting AMHR2 and / or CD3 in a sample to be tested. The detection method uses the bispecific antibody as the detection antibody to determine whether the sample to be tested contains AMHR2 and / or CD3 through an immune binding reaction.

[0102] The detection method described in this application can be a detection method for diagnostic purposes or a detection method for non-diagnostic purposes.

[0103] This application relates to methods for immunodetection or determination of target antigens (e.g., AMHR2, CD3), reagents for immunodetection or determination of target antigens (e.g., AMHR2, CD3), methods for immunodetection or determination of cells expressing target antigens (e.g., AMHR2, CD3), and diagnostic agents for diagnosing diseases associated with target antigen (e.g., AMHR2, CD3) positive cells, comprising, as active ingredients, antibodies or antibody fragments that specifically recognize the target antigen (e.g., AMHR2, CD3) and bind to the amino acid sequence or its three-dimensional structure in the extracellular region.

[0104] In this application, the method for detecting or measuring the amount of a target antigen (e.g., AMHR2, CD3) can be any known method. For example, it includes immunoassay or assay methods. Immunoassay or assay methods are methods that use labeled antigens or antibodies to detect or measure the amount of antibody or antigen. Examples of immunoassay or assay methods include radiolabeled antibody methods (RIA), enzyme immunoassays (EIA or ELISA), fluorescence immunoassays (FIA), luminescent immunoassays, Western blotting, physicochemical methods, etc. The aforementioned diseases associated with, for example, AMHR2, CD3-positive cells can be diagnosed by detecting or measuring cells expressing AMHR2, CD3 using the antibodies or antibody fragments of this application.

[0105] To detect cells expressing peptides, known immunoassay methods can be used, with immunoprecipitation, fluorescent cell staining, and immunohistochemical staining being preferred. Alternatively, fluorescent antibody staining using the FMAT8100HTS (Applied Biosystem) can be employed.

[0106] The application does not impose any particular restrictions on the test sample used to detect or measure the target antigen (e.g., AMHR2, CD3), as long as it has the potential to contain cells expressing the target antigen (e.g., AMHR2, CD3), such as tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium.

[0107] Depending on the required diagnostic method, diagnostic reagents containing the monoclonal antibody or antibody fragment thereof of this application may also contain reagents for performing antigen-antibody reactions or for detecting reactions. Reagents for performing antigen-antibody reactions include buffers, salts, etc. Reagents for detection include those commonly used in immunoassay or assay methods, such as labeled second antibodies that recognize the monoclonal antibody, its antibody fragments, or conjugates, and substrates corresponding to the labeled antibodies.

[0108] One or more embodiments of this application provide a treatment method for AMHR2-positive tumors, the treatment method comprising administering a subject a therapeutically effective amount of the bispecific antibody or the drug.

[0109] "Administration," "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. "Administration," "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. "Administration," "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.

[0110] "Treatment" means administering an oral or topical therapeutic agent, such as a composition containing any of the antibodies or antigen-binding fragments of this application, 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 symptoms of one or more diseases to induce the regression of such symptoms or inhibit 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 application (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.

[0111] "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 present 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 in a patient.

[0112] AMHR2-positive tumors include gynecological tumors such as ovarian cancer, endometrial cancer, and uterine sarcoma, as well as other solid tumors such as prostate cancer and breast cancer, and rare tumors such as tumors originating from the Miller duct.

[0113] 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.

[0114] 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.

[0115] Example 1

[0116] 1. Animal immunization

[0117] CD3δε protein dimers were purchased from Peptasys (catalog number: CDD-H52W1). A peptide fragment containing the N-terminal 27 amino acid sequence of the human CD3ε protein was synthesized: QDGNEEMGGITQTPYKVSISGTTVILT (SEQ ID NO.1). Its C-terminus was conjugated to the widely used carrier proteins keyforaminifera hemocyanin (KLH) or bovine serum albumin (BSA) by adding a cysteine ​​residue, yielding CD3ε-N27-KLH or CD3ε-N27-BSA. The conjugation of KLH was intended to elicit a significant immune response.

[0118] Select 4-6 week old Balb / c (albino lab rat) or SJL mice. Administer a mixture of antigen (CD3δε protein dimer, CD3ε-N27-KLH, or CD3ε-N27-BSA) and adjuvant (TiterMax or Sigma adjuvant system) via subcutaneous injection at multiple sites, once every 2-3 weeks, for a total of 3-4 immunizations. Based on serum titer, select high-titer animals 3 days before spleen collection for a pulse immunization via intraperitoneal injection of the antigen solution. Collected mouse spleen cells were processed using a single B-cell selection platform to obtain the antibody light chain and heavy chain variable region sequences. The expression vector constructed using PCR was co-transfected into 293T cells, and the cell culture supernatant was collected.

[0119] 2. ELISA and FCM screening

[0120] Human CD3δε protein dimer, CD3ε-N27-BSA, or cynomolgus CD3δε protein dimer (purchased from Pypsis, catalog number: CDD-C52W4) were coated into 96-well ELISA plates at 100 μL / well and incubated overnight at 4°C. The next day, after washing four times with PBST, 150 μL of 5% skim milk blocking buffer was added, and the plates were incubated at 37°C for 1 hour; after washing four times with PBST, 100 μL of transfection supernatant was added, and the plates were incubated at 37°C for 1 hour; after washing four times with PBST, 100 μL of Anti-Human IgG HRP (1:10000 dilution) was added, and the plates were incubated at 37°C for 0.5 hours; after washing four times with PBST, 50 μL of TMB chromogenic buffer was added, and the plates were incubated at 37°C for 8 minutes. The reaction was terminated by adding 50 μL of ELISA stop solution to each well, and the absorbance was measured at 450 nm using a microplate reader.

[0121] Take Jurkat-NFAT-Luc Effector Cells and use 2×10 6 Cells were resuspended at 1 / mL in FACS buffer (PBS containing 4% FBS). 50 μL of the resuspended solution was added to each well of a 96-well U-plate, centrifuged at 300g for 5 minutes, the supernatant was discarded, and 100 μL of transfection supernatant was added. The cells were incubated at 4°C for 1 hour. Cells were washed three times with FACS buffer, and after centrifugation, 100 μL of APC-labeled anti-human IgG antibody was added to each well. Cells were incubated at 4°C for 30 minutes, washed three times, the supernatant was discarded, and the cells were resuspended in 150 μL of FACS buffer for flow cytometry analysis.

[0122] Based on the ELISA and flow cytometry binding activity of the transfection supernatant (containing the corresponding antibody protein), this application screened out clone M137 that binds to human CD3δε protein dimer or CD3ε-N27-BSA and has Jurkat cell binding activity while also satisfying the requirement of cross-binding activity with cynomolgus CD3δε protein dimer. Its ELISA and FCM results are shown in Table 1.

[0123] Table 1

[0124]

[0125] 3. Construction and preparation of bispecific antibodies targeting AMHR2 and CD3

[0126] The CD3 antibody-positive clone (M137 clone) obtained from the above screening was combined with the humanized anti-Mullerian Hormone Receptor Type 2 (AMHR2) antibody 7E1 to construct a bispecific antibody with a "2+1" configuration (denoted as CR04-BA7). The structure of the bispecific antibody is shown below. Figure 1The sequences are shown in Table 2. In the bispecific antibody, the sequence of the AMHR2-terminal heavy chain is shown in SEQ ID NO.2, the sequence of the AMHR2-terminal heavy chain + CD3-terminal ScFv is shown in SEQ ID NO.3, and the sequence of the AMHR2-terminal light chain is shown in SEQ ID NO.4.

[0127] Table 2. AMHR2×CD3 Bispecific Antibody

[0128] Antibody name AMHR2 end heavy chain AMHR2 end heavy chain + CD3 end ScFv AMHR2 end light chain CR04-BA7 SEQ ID NO. 2 SEQ ID NO. 23 SEQ ID NO. 4

[0129] After confirming the bispecific antibody sequences, large-scale culture was performed. Plasmid extraction was then carried out using an endotoxin-free large-scale extraction kit (ZYMORESEARCH, D4201-A), following the manufacturer's instructions. Each antibody heavy chain expression plasmid and light chain expression plasmid were co-transfected into CHO-S cells. After culturing 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%.

[0130] 4. Affinity determination of CR04-BA7 bispecific antibody against human CD3δε protein and AMHR2

[0131] Surface plasmon resonance (SPR) technology was used. Approximately 20-30 RU of amino-coupled antigen hCD3DE was conjugated to a CM5 chip, binding different concentrations of antibody: 50, 25, 12.5, 6.25, 3.125, 1.56, and 0 nM, with a repeat concentration of 3.125 nM. Alternatively, approximately 20 RU of amino-coupled antigen hAMHR2 was conjugated to a CM5 chip, binding different concentrations of antibody: 200, 100, 50, 25, 12.5, 6.25, 3.125, and 0 nM for 100 s. Dissociation was performed for 180 s, followed by regeneration with Glycine 1.5 for 30 s. The affinity constant KD was obtained based on the binding and dissociation rates. Affinity data are shown in Table 3.

[0132] Table 3. Affinity results of SPR analysis between candidate antibodies and human CD3δε protein dimer and AMHR2 protein.

[0133]

[0134] 5. Humanization of candidate antibodies

[0135] Antibody humanization: The light chain and heavy chain variable regions of M137 were homology-aligned with human IgG germline sequences. IGHV1-69*01 / IGHJ4*01 was selected as the heavy chain CDR transplantation template, and the CDR regions (HCDR1, HCDR2, and HCDR3) of the M137 heavy chain were transplanted into the backbone region of IGHV1-69*01 / IGHJ4*01. IGKV4-1*01 / IGKJ4*01 was selected as the light chain CDR transplantation template, and the CDR regions (LCDR1, LCDR2, and LCDR3) of the M137 light chain were transplanted into the backbone region of IGKV4-1*01 / IGKJ4*01. Reversion mutations were performed at specific sites in the backbone region, and high-risk amino acid NG combinations in HCDR2 were mutated to remove them, resulting in the hM137 humanized antibody variable region of the heavy chain. The sequence of the humanized heavy chain variable region is shown in SEQ ID. As shown in NO.5, the sequence of the light chain variable region is shown in SEQ ID NO.6, as shown in Table 4. The heavy and light chain genes of the humanized hM137 antibody were synthesized and cloned into the expression vector pCDNA3.4. The antibody hCR04-BA7 was prepared according to the method described in section 3, and its sequence is shown in Table 5.

[0136] Table 4

[0137]

[0138] Table 5

[0139] Antibody name AMHR2 end heavy chain AMHR2 end heavy chain + CD3 end ScFv AMHR2 end light chain hCR04-BA7 SEQ ID NO. 2 SEQ ID NO. 3 SEQ ID NO. 4

[0140] 6. hCR04-BA7 Bispecific Antibody Affinity Assay

[0141] 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, 100, 50, 25, 12.5, 6.25, 3.125, 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.

[0142] 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, 25, 12.5, 6.25, 3.125, 1.56, 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.

[0143] Table 6

[0144]

[0145] Table 7

[0146]

[0147] 7. Binding activity of hCR04-BA7 bispecific antibody to human AMHR2 overexpressing cell lines

[0148] This embodiment primarily uses flow cytometry to detect the binding activity of bispecific antibodies to human AMHR2 overexpressing cell lines. Specific method description: Logarithmically growing COV434-hAMHR2 cells (COV434 cells were purchased from Fuheng Biotechnology, CAT#FH0724; unless otherwise specified, COV434 refers to this cell line) were harvested and centrifuged at 300×g for 5 minutes at room temperature. The supernatant was discarded, and the cells were washed once with analytical buffer (PBS + 2% FBS) and the cell density was adjusted to 1E6 cells / mL. 100 μL of the prepared cell suspension was added to each well of a 96-well U-plate, followed by 100 μL of diluted antibody solution (initial concentration 320 nM, 5-fold serial dilution, 10 concentration points). After mixing, the plates were incubated at 4°C for 1 hour. Centrifuge at 300×g for 5 minutes 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) per well, and incubate at 4°C for 0.5 hours. Centrifuge at 300×g for 5 minutes at 4°C, discard the supernatant, wash cells twice with analysis buffer, and finally resuspend cells in analysis buffer. Measure the mean fluorescence signal (MFI) on a BD flow cytometer.

[0149] See results Figure 2 The results showed that the hCR04-BA7 bispecific antibody had significant binding activity to hAMHR2-positive tumor cells.

[0150] 8. Binding activity of hCR04-BA7 bispecific antibody to human T cells

[0151] This embodiment primarily uses flow cytometry to detect the binding activity of bispecific antibodies to human T cells. Specific method description: One day prior to the test, thaw frozen PBMC cells (Miaoshun Biotechnology: catalog number Y1701), adjust the density to 2E6 cells / mL with culture medium (1640 + 10% FBS + 1% PS), and culture overnight. The next day, collect the cells, resuspend them in analytical buffer (DPBS containing 2% FBS solution), count them, and adjust the cell density to 2E6 cells / mL for later use. Block the prepared cell suspension with a human FC block. Add 50 μL of the blocked cell suspension to each well of a 96-well U-plate, then add 50 μL of diluted antibody solution (initial concentration 320 nM, 3-fold serial dilution, 11 concentration points) to each well. Mix well and incubate at 4°C for 1 hour. Centrifuge at 300×g for 5 minutes at 4°C, discard the supernatant, wash cells twice with analysis buffer, add 100 μL / well of a mixed antibody solution of PE anti-human CD4 (Biolegend, catalog number: 344606), APC anti-human CD8 (Biolegend, catalog number: 344722), and goat anti-human IgG Fc specific-FITC (Sigma, catalog number: F9512-1ML), and incubate at 4°C for 0.5 hours. Centrifuge at 300×g for 5 minutes at 4°C, discard the supernatant, wash cells twice with analysis buffer, and finally resuspend cells in analysis buffer. Measure the mean fluorescence signal (MFI) on a BD flow cytometer.

[0152] See results Figure 3 The results showed that the hCR04-BA7 bispecific antibody had binding activity against human primary T cells.

[0153] 9. Assay of antitumor activity of hCR04-BA7 antibody

[0154] PBMCs were used as effector cells. Antibodies induced T-cell-mediated cellular cytotoxicity (TDCC), causing target cell lysis. The TDCC effect of candidate antibodies was reflected by detecting the release of cellular lactate dehydrogenase (LDH). Specific procedures included: resuscitating PBMCs; the following day, adjusting the cell density of COV434-hAMHR2 cells grown to 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. 6PBMC cells (cells / mL), 100 μL / well; antibody (50 nM, 10-fold serial dilution, 6 concentration points), 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.

[0155] The results are as follows Figure 4 As shown in Table 8, the statistical results are as follows.

[0156] Table 8

[0157] EC50 PBMC donor 1 (Miaoyun Biological: Cat. No. P123120918C) 1.047 pM PBMC donor 2 (Miaoyun Biological: Cat. No. Y1701) 0.373 pM

[0158] The results showed that the hCR04-BA7 antibody of this application has significant killing activity against tumor target cells.

[0159] 10. hCR04-BA7 Bispecific Antibody Cytokine Release Assay

[0160] Cytokine levels were detected using CBA. Specific procedures: PBMC cells were resuscitated; the following day, COV434-hAMHR2 cells grown to the logarithmic growth phase were adjusted to a cell density of 4 × 10⁻⁶ cells using 1640 medium. 5 Add 50 μL / well to a 96-well U-bottom plate; the cell density is 2 × 10⁶ cells / mL. 6 PBMC cells were collected at 100 μL / well; antibody (50 nM, 10-fold serial dilution, 6 concentration points) was added at 50 μL / well; and incubated at 37°C and 5% CO2 for 24 hours. Cytokine levels were measured according to the Human Th1 / Th2 Cytokine kit II (manufacturer: BD, catalog number: 551809) manufacturer's instructions.

[0161] The results are as follows Figure 5 As shown in the figure. PBMC donor 1, purchased from Miaoshun Biotechnology, catalog number P123120918C. Results showed that the hCR04-BA7 bispecific antibody, in the presence of both tumor target cells and PBMCs, triggered the release of cytokines such as IL2, IL6, IL10, TNFα, and IFNγ.

[0162] 11. hCR04-BA7 Bispecific Antibody Target-Free Cell Cytokine Release Assay

[0163] Cytokine levels were detected using CBA. Specific procedures: PBMC cells (derived from Miaoshun Biotechnology) were resuscitated; the following day, cells were added at a density of 2×10⁶. 6PBMC cells (number cells / mL), 100 μL / well; antibody (500 nM, 10-fold serial dilution, 3 concentration points), 100 μL / well; incubate at 37°C and 5% CO2 for 24 hours. Cytokine levels were measured according to the Human Th1 / Th2 Cytokine kit II (manufacturer: BD, catalog number: 551809) manufacturer's instructions.

[0164] The results are as follows Figure 6 As shown in the figure. PBMC donor 1, purchased from Miaoshun Biotechnology, catalog number P123120918C. Results showed that the hCR04-BA7 bispecific antibody produced almost no release of IL2, IL6, and IL10 cytokines in the absence of tumor target cells, and the release of TNFα, IFNγ, and other cytokines was significantly lower than when tumor target cells were present.

[0165] 12. CD34+HSC immune reconstitution mouse subcutaneous COV434-AMHR2 tumor model

[0166] Female HSC-NOG-EXL mice (Vitaliva) 12-13 weeks after immune reconstitution were selected and subcutaneously inoculated with COV434-AMHR2 human ovarian cancer cells (2.5 × 10⁻⁶). 6 / each). When the average tumor volume reaches 150mm. 3 Mice were administered the drug via tail vein injection at a dose of 3 mg / kg, given once weekly (QW), for a total of two administrations. Tumor volume and body weight were measured during the administration. In contrast to the antibody-treated group, control mice were subcutaneously injected with PBS (phosphate-balanced saline).

[0167] See results Figure 7 The hCR04-BA7 molecule effectively inhibited tumor growth, with a tumor inhibition rate of 81.8% at the study endpoint. As shown in Table 9, 6 hours after administration, one mouse in each of the control group and the hCR04-BA7 treatment group showed elevated IL-6 levels (C), and no mouse deaths occurred in the study.

[0168] Table 9

[0169]

[0170] 13. PBMC-reconstituted mouse subcutaneous COV434 tumor model

[0171] Select 6-9 week old NOG mice (Guangdong Vital River Laboratory Animal Technology Co., Ltd.), and inject each mouse via tail vein with 5×10 6 One PBMC cell (Miaoshun Biotechnology: catalog number P123120918C) was subcutaneously inoculated with COV434-AMHR2 human ovarian cancer cells (2.5×10⁻⁶ cells) 7 days later. 6 / each), until the average tumor volume reaches 150-160mm. 3 When the hCD45+% (hCD45+ / (hCD45+mCD45+)) in the peripheral blood of mice was ≥0.3%, the drug was administered via tail vein injection. The control ADCC-enhanced monoclonal antibody 3C23K was administered at a dose of 5 mg / kg. The experimental groups BA7 (low, medium, and high doses) were administered 0.04 mg / kg, 0.2 mg / kg, and 1 mg / kg, respectively, at a dosing frequency of QW, for a total of two administrations. Tumor volume and body weight were measured in mice. Compared to the antibody-treated group, the model group mice were subcutaneously injected with PBS (phosphate-balanced saline) and denoted as "G1 Vehicle".

[0172] The results are as follows Figure 8 hCR04-BA7 molecules effectively inhibited tumor growth. The tumor inhibition rate of the control drug 3C23K was 33%, while the tumor inhibition rates of the low, medium and high dose groups of BA7 were 43%, 67% and 76%, respectively.

[0173] >SEQ ID NO.1: QDGNEEMGGITQTPYKVSISGTTVILT.

[0174] >SEQ ID NO.2 (hCR04-BA7 AMHR2 end heavy chain):

[0175] QVQLVQSGPGLVKPSETLSLTCTVSGDSISSYYWSWIRQPPGKGLEWIGYISYSGSTDYNPSLKSRVTISSVDTSKNQFSLKLNSVTAADTAVYYCARHAY

[0176] REKGQFVPYYFYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVT

[0177] VPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

[0178] NAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0179] >SEQ ID NO.3 (AMHR2 end heavy chain + CD3 end ScFv):

[0180]

[0181] >SEQ ID NO.4 (AMHR2 end light chain):

[0182] DIVMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPNLLIYAASTLQSGVPSRFSGSGSGTEFTLTIISLQPEDFATYYCQQLNSYPLTFGGGT

[0183] KVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0184] >SEQ ID NO.5 (hM137 heavy chain variable region):

[0185]

[0186] >SEQ ID NO.6 (hM137 end light chain variable region):

[0187]

[0188] >SEQ ID NO.7 (CD3 binding domain, HCDR1): DYYMH.

[0189] >SEQ ID NO.8 (CD3 binding domain, HCDR2): WIVPEQGDTRYDPKFQG.

[0190] >SEQ ID NO.9 (CD3 binding domain, HCDR3): DQYGNYFFAH.

[0191] >SEQ ID NO.10 (CD3 binding domain, LCDR1): KSSQSLLNSRTRKNYLA.

[0192] >SEQ ID NO.11 (CD3 binding domain, LCDR2): WASTRES.

[0193] >SEQ ID NO.12 (CD3 binding domain, LCDR3): KQSYTLRT.

[0194] >SEQ ID NO.13 (AMHR2 binding domain, HCDR1): SYYWS.

[0195] >SEQ ID NO.14(AMHR2 binding domain, HCDR2): YISYSGSTDYNPSLKS.

[0196] >SEQ ID NO.15 (AMHR2 binding domain, HCDR3): HAYREKGQFVPYYFYYGMDV.

[0197] >SEQ ID NO.16 (AMHR2 binding domain, LCDR1): RASQGISSYLA.

[0198] >SEQ ID NO.17 (AMHR2 binding domain, LCDR2): AASTLQS.

[0199] >SEQ ID NO.18 (AMHR2 binding domain, LCDR3): QQLNSYPLT.

[0200] >SEQ ID NO.19 (AMHR2 binding domain, heavy chain variable region):

[0201] QVQLVQSGPGLVKPSETLSLTCTVSGDSISSYYWSWIRQPPGKGLEWIGYISYSGSTDYNPSLKSRVTISSVDTSKNQFSLKLNSVTAADTAVYYCARHAY REKGQFVPYYFYYGMDVWGQGTTVTVSS.

[0202] >SEQ ID NO.20 (AMHR2 binding domain, light chain variable region):

[0203] DIVMTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPNLLIYAASTLQSGVPSRFSGSGSGTEFTLTIISLQPEDFATYYCQQLNSYPLTFGGG TKVDIK.

[0204] >SEQ ID NO.21 (CD3 end heavy chain variable region):

[0205]

[0206] >SEQ ID NO.22 (CD3 end light chain variable region):

[0207]

[0208] >SEQ ID NO.23 (AMHR2 end heavy chain + mouse CD3 end ScFv):

[0209] Note: >The N marked in the box in SEQ ID NO.23 is the only site that changed in the six CDR regions before and after humanization of the CD3-terminal antibody 137.

[0210] >SEQ ID NO.24 (Chimeric antibody, CD3 binding domain, HCDR2): WIVPENGDTRYDPKFQG.

[0211] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0212] 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 and drawings can be used to interpret the content of the claims.

Claims

1. An anti-AMHR2×CD3 bispecific antibody, characterized in that, The bispecific antibody meets the following conditions: The heavy chain variable region of the CD3 binding domain has sequences HCDR1 to HCDR3 as shown in SEQ ID NO. 7 to 9 or HCDR1 to HCDR3 as shown in SEQ ID NO. 7, 24 and 9, and the light chain variable region has sequences LCDR1 to LCDR3 as shown in SEQ ID NO. 10 to 12; and The heavy chain variable region of the AMHR2 binding domain has HCDR1 to HCDR3 as shown in SEQ ID NO. 13 to 15, and the light chain variable region has LCDR1 to LCDR3 as shown in SEQ ID NO. 16 to 18. Optionally, the bispecific antibody has a heavy chain variable region as shown in SEQ ID NO. 5 or 21 and a light chain variable region as shown in SEQ ID NO. 6 or 22; Optionally, the bispecific antibody has a heavy chain variable region as shown in SEQ ID NO.19 and a light chain variable region as shown in SEQ ID NO.20; Optionally, the bispecific antibody is a 2+1 valence bispecific antibody; Optionally, the bispecific antibody has a Fab / Fab-scFv Fc structure; Optionally, one antigen-binding arm of the bispecific antibody binds to AMHR2, and the other antigen-binding arm binds to both AMHR2 and CD3; Optionally, the bispecific antibody has a heavy chain with the sequence shown in SEQ ID NO.2 and a light chain with the sequence shown in SEQ ID NO.4; Optionally, the bispecific antibody has a heavy chain with a sequence as shown in SEQ ID NO.3 or SEQ ID NO.23 and a light chain with a sequence as shown in SEQ ID NO.

4.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the specific antibody as described in claim 1.

3. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 2.

4. A cell, characterized in that, The cell expresses the bispecific antibody of claim 1, or the cell comprises the nucleic acid molecule of claim 2 or the vector of claim 3.

5. The method for constructing cells according to claim 4, characterized in that, The construction method includes the step of introducing the nucleic acid molecule of claim 2 or the vector of claim 3 into a host cell.

6. The method for preparing the bispecific antibody according to claim 1, characterized in that, The preparation method includes the step of culturing the cells according to claim 4.

7. The use of the bispecific antibody of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, or the cell of claim 4 in the preparation of drugs or detection products.

8. A drug, characterized in that, The drug includes the bispecific antibody as described in claim 1.

9. The product being tested, characterized in that, The detection product includes the bispecific antibody as described in claim 1.

10. A method for detecting AMHR2 and / or CD3 in a sample to be tested, characterized in that, The detection method uses the bispecific antibody described in claim 1 as the detection antibody to determine whether the sample contains AMHR2 and / or CD3 through an immune binding reaction.

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