Anti-CD30 and CD16a bispecific antibody and application thereof

Abstract

The invention discloses a bispecific antibody which comprises a first protein functional region targeting CD30 and a second protein functional region targeting CD16a, the first protein functional region comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3. The invention also discloses nucleic acid for coding the antibody, a recombinant expression vector, a transformant, a preparation method of the antibody, an antibody drug conjugate, a pharmaceutical composition or a kit containing the antibody drug conjugate, and applications of the nucleic acid, the recombinant expression vector and the transformant in preparation of drugs for preventing and / or treating tumors. The bispecific antibody provided by the invention can effectively activate the NK cells and can effectively target human tumor cell related antigen CD30 protein, mediate the killing activity of the NK cells to target cells, and induce and further enhance the tumor inhibition effect of the NK cells to tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of antibody drugs, specifically relating to a bispecific antibody against CD30 and CD16a and its applications. Background Technology

[0002] CD30 is a member of the tumor necrosis factor receptor superfamily (TNFRSF), specifically TNFRSF8. CD30 is a type I transmembrane protein, consisting of an intracellular domain (188 amino acids), a transmembrane domain, and a cysteine-rich extracellular domain (362 amino acids). CD30 is mainly expressed on the surface of activated T cells, B cells, and NK cells. Its expression level is low under physiological conditions, but high in disease states, such as various lymphomas including anaplastic large cell lymphoma (ALCL) and chronic Hodgkin's lymphoma (CHL), where nearly 100% of tumor cells express CD30. Furthermore, it is expressed to varying degrees in other lymphoma subtypes, including diffuse large B-cell lymphoma (DLBCL), primary mediastinal (thymic) large B-cell lymphoma (PMBL), mycosis fungoides (MF), lymphomatoid papulosis (LyP), and various other types of peripheral T-cell lymphoma (PTCL). In light of this, an increasing number of studies are utilizing it as a therapeutic target for lymphoma. Although the CD30-targeting ADC drug, injectable brentuximab vedotin (Adcetris), has been approved for the treatment of CD30-positive primary cutaneous anaplastic large cell lymphoma or mycosis fungoides that have received prior systemic therapy, the efficacy of anti-CD30 antibodies alone in practice is not significant.

[0003] Natural killer (NK) cells are an important component of the body's innate immunity, serving as the first line of defense against pathogens and malignant tumors, and playing a crucial role in anti-tumor activity. NK cell surface receptors fall into two main categories: KIRs (Killer Cell Inhibitory Receptors) and NCRs (Natural Cytotoxic Receptors), whose activation is regulated by the coordination and balance between activating and inhibitory receptors. NK cells primarily achieve their killing function through the following mechanisms: 1) Secreting killing mediators: cytotoxic lysing particles such as perforin, granzymes, and granzymes induce apoptosis in target cells. 2) Mediating antibody-dependent cytotoxicity (ADCC): NK cells express FcγR receptors, which mediate the killing of target cells by binding to the Fc fragment of IgG antibodies already bound to the surface of virus-infected cells and tumor cells. This is one of the main mechanisms of action in antibody-based anti-tumor therapy and infection control. 3) Secreting cytokines, such as lymphokines, which interact with other immune cells to form an immune response. 4) Killing effect through expression of membrane TNF family molecules: NK cells can induce apoptosis of target cells by binding to target cell membrane ligands through membrane TNF family molecules (FASL, TRAIL, mTNF, etc.).

[0004] Human IgG Fc receptors are classified into three main classes: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγRII comprises three subclasses: IIa, IIb, and IIc, while FcγRIII comprises two subclasses: IIIa and IIIb. The receptor most relevant to activating innate immune cells with cytotoxic functions (such as NK cells) is IIIa (CD16a). CD16a is expressed on the surface of NK cells, monocytes, and macrophages, and also in small amounts on the surface of dendritic cells (DCs). It is an activating receptor and can mediate strong NK cell activation.

[0005] NK cell-based adoptive immunotherapy for tumor treatment has achieved some success in clinical practice, but some problems remain, such as insufficient specificity of NK cells in recognizing tumor cells or inadequate anti-tumor effects. Currently, strategies to improve the specificity of NK cell recognition and anti-tumor effects mainly include chimeric antigen receptor (CAR)-NK cells, T cell receptor (TCR)-NK cells, and bispecific or multispecific NK cell engager (NKCE) therapies.

[0006] NK cell connectors are engineered antibodies that use a monoclonal antibody structure as a backbone and target two or more tumor-associated antigens (TAAs) or effector cell receptors. They can connect NK cells and tumor cells, effectively activating NK cells and enhancing their cytotoxicity and cytokine production while targeting tumor cells, thus endowing NK cells with the ability to treat different types of tumors.

[0007] Current preclinical studies and clinical trials indicate that NKCEs are a promising next-generation tumor immunotherapy approach. However, the structure of NKCEs requires further research and improvement. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention discloses a bispecific antibody against CD30 and CD16a and its applications.

[0009] Specifically, a first aspect of the present invention provides a bispecific antibody comprising a first protein functional region targeting CD30 and a second protein functional region targeting CD16a; wherein:

[0010] The first protein functional region comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO:1, HCDR2 with an amino acid sequence as shown in SEQ ID NO:2, and HCDR3 with an amino acid sequence as shown in SEQ ID NO:3. The light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO:7, LCDR2 with an amino acid sequence as shown in SEQ ID NO:8, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:9.

[0011] Alternatively, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:4, HCDR2 as shown in SEQ ID NO:5, and HCDR3 as shown in SEQ ID NO:6; the light chain variable region comprises LCDR1 as shown in SEQ ID NO:10, LCDR2 as shown in SEQ ID NO:11, and LCDR3 as shown in SEQ ID NO:12.

[0012] In some specific implementations, the first protein functional region is selected from the following:

[0013] (1) The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:43, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:44;

[0014] Alternatively, the heavy chain variable region may contain an amino acid sequence as shown in SEQ ID NO:47, and the light chain variable region may contain an amino acid sequence as shown in SEQ ID NO:48;

[0015] Alternatively, the heavy chain variable region may contain an amino acid sequence as shown in SEQ ID NO:51, and the light chain variable region may contain an amino acid sequence as shown in SEQ ID NO:52;

[0016] (2) The heavy chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:43; the light chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:44;

[0017] Alternatively, the heavy chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:47; the light chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:48.

[0018] Alternatively, the heavy chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:51; the light chain variable region comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:52.

[0019] In some implementations, the first protein functional region is an IgG structure.

[0020] In some specific implementations, the constant region of the first protein functional region is selected from at least one of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA, IgM, IgD and IgE constant regions and their related variants.

[0021] In some specific implementations, the constant region of the first protein functional region is human IgG1.

[0022] In some embodiments, the second protein functional region comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 with the amino acid sequence shown in SEQ ID NO:13, HCDR2 with the amino acid sequence shown in SEQ ID NO:14, and HCDR3 with the amino acid sequence shown in SEQ ID NO:15; and the light chain variable region comprises LCDR1 with the amino acid sequence shown in SEQ ID NO:16, LCDR2 with the amino acid sequence shown in SEQ ID NO:17, and LCDR3 with the amino acid sequence shown in SEQ ID NO:18.

[0023] In some implementations, the second protein functional region is an scFv structure.

[0024] In some specific embodiments, the heavy chain variable region of the scFv contains an amino acid sequence as shown in SEQ ID NO:55.

[0025] In some specific embodiments, the light chain variable region of the scFv contains an amino acid sequence as shown in SEQ ID NO:56.

[0026] In some specific implementations, the heavy chain variable region and the light chain variable region of the scFv are connected by a Linker, which is (G4S). n Where n = 1-4, for example n = 3.

[0027] In some specific implementations, the scFv contains an amino acid sequence as shown in SEQ ID NO:61.

[0028] In some specific implementations, the light chains of the second protein functional region and the first protein functional region are connected, preferably with the N-terminus of the light chain of the second protein functional region connected to the C-terminus of the light chain of the first protein functional region.

[0029] In some specific implementations, the first and second protein functional regions are connected by a linker, wherein the linker is (G4S). n n = 1-4, for example n = 3.

[0030] In some specific implementations, the bispecific antibody comprises four polypeptide chains; wherein,

[0031] One of the polypeptide chains contains an amino acid sequence as shown in SEQ ID NO:64, and the other polypeptide chain contains an amino acid sequence as shown in SEQ ID NO:65;

[0032] Alternatively, one of the polypeptide chains contains an amino acid sequence as shown in SEQ ID NO:68, and the other polypeptide chain contains an amino acid sequence as shown in SEQ ID NO:69.

[0033] In some of the above implementation schemes, the amino acid sequences of the listed CDRs are determined according to the Kabat definition rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)), Kabat et al. (1987) based on antibody sequence variability, AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Those skilled in the art will understand that, unless otherwise specified, the terms “CDR” and “complementary determination region” for a given antibody or its region (e.g., variable region) should be understood to encompass the complementary determination region defined by any of the above-mentioned known schemes, or the complementary determination region defined by any other definition rule based on the antibody of the present invention.

[0034] A second aspect of the present invention provides a nucleic acid molecule that encodes the bispecific antibody as described in any of the first aspects of the present invention.

[0035] In some specific embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding HCDR1-3 as shown in SEQ ID NO:19-21 and a nucleotide sequence encoding LCDR1-3 as shown in SEQ ID NO:25-27;

[0036] Alternatively, nucleotide sequences encoding HCDR1-3 as shown in SEQ ID NO:22-24 and nucleotide sequences encoding LCDR1-3 as shown in SEQ ID NO:28-30.

[0037] In some specific embodiments, the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO:45-46;

[0038] Alternatively, the nucleic acid molecule may contain a nucleotide sequence as shown in SEQ ID NO:49-50;

[0039] Alternatively, the nucleic acid molecule may contain a nucleotide sequence as shown in SEQ ID NO:43-54.

[0040] In some specific embodiments, the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO:66-67;

[0041] Alternatively, the nucleic acid molecule may contain a nucleotide sequence as shown in SEQ ID NO:70-71.

[0042] A third aspect of the present invention provides a recombinant expression vector comprising the nucleic acid molecule described in any of the second aspects of the present invention.

[0043] In some specific implementations, the recombinant expression vector is any one or a combination of at least two of the following: viral vector, plasmid, DNA fragment, RNA, etc.

[0044] In some specific implementations, the recombinant expression vector is a plasmid.

[0045] A fourth aspect of the present invention provides a transformant characterized in that it contains a nucleic acid molecule as described in any one of the second aspects of the present invention or a recombinant expression vector as described in the third aspect of the present invention in a host cell.

[0046] In some specific implementations, the host cell is a mammalian cell.

[0047] A fifth aspect of the present invention provides a method for preparing a bispecific antibody as described in any one of the first aspects of the present invention, characterized in that it comprises culturing a transformant as described in the fourth aspect of the present invention and obtaining the bispecific antibody from the culture.

[0048] A sixth aspect of the present invention provides a genetically modified cell, characterized in that it comprises a bispecific antibody as described in any one of the first aspects of the present invention.

[0049] A seventh aspect of the present invention provides an antibody-drug conjugate, characterized in that the antibody-drug conjugate comprises a cytotoxic agent or tag, and a bispecific antibody as described in any of the first aspects of the present invention.

[0050] The eighth aspect of the present invention provides a pharmaceutical composition or a kit containing the same, characterized in that the pharmaceutical composition or the kit containing the same comprises at least one of the bispecific antibody of any one of the first aspects of the present invention, the genetically modified cell of the sixth aspect of the present invention, or the antibody-drug conjugate of the seventh aspect of the present invention, and a pharmaceutically acceptable carrier, excipient, or diluent.

[0051] In some specific embodiments, the pharmaceutical composition or the kit containing it further comprises one or more of the group consisting of hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

[0052] The ninth aspect of the present invention provides the use of a bispecific antibody as described in any of the first aspects of the present invention, a nucleic acid molecule as described in any of the second aspects of the present invention, a recombinant expression vector as described in the third aspect of the present invention, a transformant as described in the fourth aspect of the present invention, a gene-modified cell as described in the sixth aspect of the present invention, an antibody-drug conjugate as described in the seventh aspect of the present invention, or a pharmaceutical composition or a cassette containing the present invention as described in the eighth aspect of the present invention in the preparation of a medicament for the prevention and / or treatment of tumors.

[0053] In some specific implementations, the tumor is a CD30-positive tumor.

[0054] In some specific implementations, the tumor is selected from the following: human degenerative large cell lymphoma, mycosis fungoides, T-cell lymphoma, and Hodgkin lymphoma.

[0055] The tenth aspect of the present invention provides the use of a bispecific antibody as described in any of the first aspects of the present invention, a nucleic acid molecule as described in any of the second aspects of the present invention, a recombinant expression vector as described in the third aspect of the present invention, a transformant as described in the fourth aspect of the present invention, a gene-modified cell as described in the sixth aspect of the present invention, an antibody-drug conjugate as described in the seventh aspect of the present invention, or a pharmaceutical composition or a cassette containing the thereof as described in the eighth aspect of the present invention in the treatment of tumors.

[0056] In some specific implementations, the tumor is a CD30-positive tumor.

[0057] In some specific implementations, the tumor is selected from the following: human degenerative large cell lymphoma, mycosis fungoides, T-cell lymphoma, and Hodgkin lymphoma.

[0058] The eleventh aspect of the present invention provides a method for treating tumors using a bispecific antibody as described in any of the first aspects of the present invention, a nucleic acid molecule as described in any of the second aspects of the present invention, a recombinant expression vector as described in the third aspect of the present invention, a transformant as described in the fourth aspect of the present invention, a gene-modified cell as described in the sixth aspect of the present invention, an antibody-drug conjugate as described in the seventh aspect of the present invention, or a pharmaceutical composition or a kit containing the thereof as described in the eighth aspect of the present invention.

[0059] In some specific implementations, the tumor is a CD30-positive tumor.

[0060] In some specific implementations, the tumor is selected from the following: human degenerative large cell lymphoma, mycosis fungoides, T-cell lymphoma, and Hodgkin lymphoma.

[0061] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0062] The reagents and raw materials used in this invention are all commercially available.

[0063] To better understand this invention, the following are definitions of some commonly used terms. Definitions of other terms are listed throughout the detailed embodiments section.

[0064] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 10% less than the specified numeric value and an upper limit of 10% greater than the specified numeric value.

[0065] When the term “and / or” is used to connect two or more options, it should be understood to mean any one of the options or any two or more of the options.

[0066] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations consisting of the mentioned elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0067] In this invention, the terms "antibody," "full-length antibody," "complete antibody," and "conventional antibody" are used interchangeably to refer to glycoproteins comprising at least two heavy chains (HC) and two light chains (LC) linked together by disulfide bonds. Each heavy chain of a full-length antibody consists of a heavy chain variable region (abbreviated as VH in this invention) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL in this invention) and a light chain constant region (abbreviated as CL in this invention). The light chain constant region consists of one domain: CL. Mammalian heavy chains are classified into α, δ, ε, γ, and μ heavy chains. Mammalian light chains are classified into λ or κ light chains. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are immunoglobulins (Ig) A, IgD, IgE, IgG, and IgM. Complete antibodies form a "Y" shape. The stem of Y consists of the second and third constant regions (and, for IgE and IgM, a fourth constant region) of two heavy chains joined together, with disulfide bonds (interchain) forming in the hinge. Heavy chains γ, α, and δ have constant regions consisting of three tandem (in a row) Ig domains, and hinge regions for increased flexibility; heavy chains μ and ε have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the "CH2 domain" and the "CH3 domain," respectively. Each arm of Y includes a variable region of a single heavy chain that binds to a single light chain and a first constant region (CH1). The "Fc" region consists of two heavy chain segments containing the CH2 and CH3 domains of the antibody, held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain.

[0068] The term "variable region" or "variable domain" refers to the domain in the antibody heavy or light chain involved in antibody binding to the antigen. VH and VL each contain four conserved frame regions (FRs) and three complementarity-determining regions (CDRs). The term "complementarity-determining region" or "CDR" refers to the region within the variable domain that primarily facilitates antigen binding; "frame" or "FR" refers to the variable domain residues other than the CDR residues. VH contains three CDR regions: HCDR1, HCDR2, and HCDR3; VL contains three CDR regions: LCDR1, LCDR2, and LCDR3. Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0069] In this invention, the scFv (single chain antibody fragment) VH and VL domains are linked by a linker (also called a linker) to form a polypeptide chain. The VL and VH domains pair to form a monovalent molecule by enabling them to generate linkers that act as single polypeptide chains [see, for example, Bird et al., Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988)]. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating G4S amino acid sequence or a variant thereof. For example, linkers with the amino acid sequence (G4S)4 or (G4S)3 can be used, but variants thereof may also be used.

[0070] The terms "bispecific antibody" and "multispecific antibody," used in their broadest sense, encompass antibodies that have specificity for two or more epitopes. These multispecific antibodies include, but are not limited to: antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH-VL unit has specificity for two or more epitopes; antibodies having two or more VL and VH regions, each VH-VL unit binding to a different target or a different epitope of the same target; and antibodies having two or more single variable regions, each single variable region binding to a different target or a different epitope of the same target.

[0071] The term "epitope" refers to a region on an antigen that can specifically bind to an antibody. Epitopes can be formed from a continuous string of amino acids (linear epitopes) or contain discontinuous amino acids (conformal epitopes), for example, due to the folding of the antigen (i.e., the tertiary folding of an antigen as a protein). The difference between conformational and linear epitopes is that antibody binding to a conformational epitope is lost in the presence of a denaturing solvent. An epitope contains at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Screening for antibodies that bind to a specific epitope (i.e., those that bind to the same epitope) can be performed using methods routine in the art, such as, but not limited to, alanine scanning, peptide blotting (see Meth. Mol. Biol. 248 (2004) 443-463).

[0072] The term "specific binding" refers to an antibody binding to an antigen or an epitope within that antigen with a higher affinity than it binds to other antigens or epitopes. Typically, antibodies bind with an affinity of approximately 1 × 10⁻⁶. -7 M or smaller (e.g., about 1×10⁻⁶) -8 M or smaller, approximately 1×10 -9 M or smaller, approximately 1×10 -10 M or smaller, approximately 1×10 -11 M or smaller, or about 1×10 -12 The equilibrium dissociation constant (KD) of an antibody (M or less) binds to an antigen or an epitope within the antigen. In some embodiments, the KD of antibody binding to an antigen is 10% or 1% of the KD of antibody binding to a nonspecific antigen (e.g., BSA, casein). KD can be measured using standard procedures, such as by... Surface plasmon resonance assays are used to measure this. However, antibodies that specifically bind to antigens or epitopes within antigens may exhibit cross-reactivity with other related antigens, for example, cross-reactivity with the same antigens from other species (homologous) (such as humans or monkeys, such as cynomolgus (Cyno) and chimpanzee (Chimp)) or common marmoset (Callithrix jacchus) (Marmoset).

[0073] The term "affinity" refers to the overall strength of the non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its bound ligand (e.g., an antigen). Unless otherwise specified, as used herein, "affinity" refers to internal binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its ligand Y can generally be expressed by the dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein. The term "kassoc" or "ka" refers to the association rate of a particular antibody-antigen interaction, while the term "kdis" or "kd" as used herein refers to the dissociation rate of a particular antibody-antigen interaction. As used herein, the term "KD" refers to the dissociation constant, which is derived from the ratio of kd to ka (i.e., kd / ka) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods well established in the art. Methods for determining antibody KD include using biosensing systems, such as systems measuring surface plasmon resonance, or by bio-layer interferometry (BLI).

[0074] The term "EC50," also known as the half-maximal effect concentration, refers to the antibody concentration that produces a 50% maximum effect.

[0075] The term "IC50," also known as half-inhibitory concentration, refers to the concentration of a drug or inhibitor required to inhibit a specified biological process or a component of that process (such as an enzyme, receptor, or cell) by half. In competitive ELISA, it is an important indicator characterizing competitive inhibitory ability.

[0076] The term "vector" refers to a device that enables a target gene to reach its destination, including viral and non-viral vectors. Viral vectors are virus-based gene vectors, primarily including lentivirus (LV) vectors, adenovirus (AdV) vectors, and adeno-associated virus (AAV) vectors. Viral vectors can carry foreign genes and package them into viral particles, thereby mediating the transfer and expression of foreign genes. Non-viral vectors include lipid nanoparticles (LNPs), inorganic nanoparticles, polymer nanoparticles, and peptide vectors. LNPs mainly consist of four components: ionizable cationic phospholipids, neutral accessory phospholipids, cholesterol, and PEGylated lipids, which can effectively encapsulate various nucleic acid molecules. One type of non-viral vector is a "plasmid," which is a circular double-stranded DNA loop that can link to additional DNA segments. Some vectors can replicate autonomously in the host cells they are introduced into (e.g., bacterial vectors with bacterial origins of replication and free mammalian vectors). Other vectors (such as non-attachment mammalian vectors) can integrate into the host cell's genome after introduction into the host cell and thereby replicate along with the host genome. Furthermore, some vectors can direct the expression of genes to which they are efficiently linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Typically, expression vectors useful in recombinant DNA technology exist in the form of plasmids. However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), also serve equivalent functions.

[0077] The term "nucleic acid molecule" is intended to include both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded and can be cDNA. RNA molecules include messenger RNA (mRNA), a class of single-stranded ribonucleic acid molecules transcribed from one strand of DNA as a template, carrying genetic information and directing protein synthesis. mRNA can contain one or more base modifications, such as pseudouridine (ψ), 5-methylcytidine (m5C), N6-methyladenosine (m6A), 5-methyluridine (m5U), or 2-thiouridine (s2U).

[0078] The term "polypeptide" refers to a chain containing at least two consecutively linked amino acid residues, with no upper limit on chain length. One or more amino acid residues in a protein may contain modifications, such as, but not limited to, glycosylation, phosphorylation, or disulfide bonds. A "protein" may include one or more polypeptides.

[0079] The term "host cell" refers to a cell in which the vector can proliferate and whose DNA can be expressed; said cell can be a prokaryotic or eukaryotic cell. The term also includes any progeny of the tested host cell. It should be understood that not all progeny are identical to the parent cell, as mutations can occur during replication; such progeny are included.

[0080] The term "purification" and its grammatical variations are used to indicate the complete or partial removal of at least one impurity from a mixture containing proteins and one or more impurities, reducing the content of impurities in the composition, thereby increasing the purification level of the protein in the composition.

[0081] The terms "targeting CD30" and "targeting CD16a" refer to antibodies (or protein functional regions) that can bind to human CD30 or human CD16a with sufficient affinity, making them usable as diagnostic and / or therapeutic agents targeting human CD30 or CD16a. In some embodiments, antibodies that bind to human CD30 or human CD16a may have the following dissociation constants (KD): < about 1 μM, < about 100 nM, < about 10 nM, < about 1 nM, < about 0.1 nM, < about 0.01 nM, or < about 0.001 nM (e.g., 10). -8 M or smaller, such as 10 -8 M to 10 -12 M, for example, 10 -9 M to 10 -10 M). In some embodiments, anti-CD30 or CD16a antibodies bind to conserved antigenic epitopes in CD30 or CD16a from different species. The positive and progressive effects of this invention are: the bispecific antibody provided by this invention can effectively activate NK cells and mediate the killing activity of NK cells against target cells by effectively targeting the human tumor cell-associated antigen CD30 protein, thereby inducing and further enhancing the tumor-suppressing effect of NK cells on tumor cells. Attached Figure Description

[0082] Figure 1 The image shows the structure of the NK cell adaptor antibody of the CD30xCD16a bispecific antigen-binding molecule of this application.

[0083] Figure 2This application demonstrates the effect of the NK cell connector antibody molecules in inducing NK cell activation. The ability of SEQN10-2 and SEQN10-4 antibodies to mediate NK cell activation was evaluated by measuring the expression of the NK cell surface activation marker CD107a, using human NK cells as effector cells, Karpas 299 (…). Figure 2 A) and TALL-104 ( Figure 2 B) were the target cells, with an effector-to-target ratio of 2:1. After co-incubating the effector-to-target cells for 4 hours, the expression of CD107a was detected by flow cytometry to assess the ability of SEQN10-2 and SEQN10-4 antibodies to mediate NK cell activation.

[0084] Figure 3 This application demonstrates the NK cell connector antibody molecule-mediated NK cell killing activity against target cells. Human NK cells were used as effector cells, and TALL-104 cells were used as target cells, with an effector-to-target ratio of 2:1. Figure 3 A) and 10:1 ( Figure 3 B) After co-incubating the target cells for 6 hours, the killing activity of NK cells mediated by SEQN10-2 and SEQN10-4 antibodies against the target cells was evaluated.

[0085] Figure 4 This demonstrates the tumor-suppressing effect of the NK cell adaptor antibody molecule of this application in the NVG-hIL15 mouse Hut-78 subcutaneous tumor-bearing model. Figure 4 A shows the curve of tumor volume change over time after drug administration (mean ± SEM, mm). 3 ), Figure 4 B represents the tumor weight (mean ± SEM, g) of mice in each group at the experimental endpoint. Detailed Implementation

[0086] To provide a more comprehensive understanding and application of the present invention, it will be described in detail below with reference to embodiments and accompanying drawings. These embodiments are merely illustrative and not intended to limit the scope of the invention. The scope of the invention is specifically defined by the appended claims. Experimental methods not specifically described in the following embodiments are performed according to conventional methods and conditions, or as selected according to the trade description.

[0087] Example 1. Preparation and screening of recombinant rabbit monoclonal antibodies targeting human CD30

[0088] In this embodiment, New Zealand white rabbits were immunized with human CD30 antigen to isolate antigen-specific B lymphocytes. Positive B lymphocytes were screened using ELISA and FACS methods, and the rabbit monoclonal antibody sequence was obtained by gene cloning.

[0089] For the first immunization, an equal volume of complete adjuvant and antigen was emulsified, with an immunization dose of 300 μg / rabbit. Subsequent immunizations used an equal volume of incomplete adjuvant and antigen emulsified, with an immunization dose of 150 μg / rabbit, administered subcutaneously in the back. A total of four immunizations were performed. Serum titers were detected using an indirect enzyme-linked immunosorbent assay (ELISA). Peripheral blood was aseptically collected from rabbits, and peripheral blood mononuclear cells (PBMCs) were separated using Ficoll density gradient centrifugation. B lymphocytes were isolated using human CD30 immunomagnetic beads. The isolated B lymphocytes were diluted and cultured in 96-well cell culture plates for 6 days. The culture supernatant was collected for antibody screening using ELISA. After transient transfection of expi293 cells with the human CD30 antigen vector, the supernatant of ELISA-positive B lymphocytes was screened by flow cytometry (FACS) to obtain 1E9 positive clones. RNA corresponding to the positive antibody was extracted from the B lymphocytes and reverse transcribed into cDNA. Primers were designed to amplify the variable region genes of the antibody heavy or light chain. The amplified antibody heavy and light chain variable region genes were constructed into expression vectors and transiently transfected into expi293 cells for expression. The obtained antibodies were subjected to FACS binding function detection. The variable region gene sequence of the rabbit monoclonal antibody 1E9 was obtained by gene sequencing, as shown in Table 10 (SEQ ID NO: 45–SEQ ID NO: 46), and its corresponding amino acid sequence is shown in Table 10 (SEQ ID NO: 43–SEQ ID NO: 44). The amino acid sequence and DNA sequence of the CDR region defined according to the Kabat principle are shown in Tables 6 and 7.

[0090] Example 2. Humanization of rabbit antibody targeting human CD30

[0091] This embodiment uses the CDR transplantation method to humanize rabbit-derived hu1E9 antibody. The V region sequence of the maternal antibody was compared with the database to determine the human germline with the highest similarity. After defining the maternal antibody CDR and framework regions, reversion mutations were designed based on the differential sites in the framework region. After gene synthesis, plasmid construction, protein expression, purification, and identification, the humanized hu1E9 antibody protein was obtained. The antigen-binding activity of the humanized antibody was detected by flow cytometry. The real-time interaction between the humanized antibody and human CD30 was detected using the surface plasmon resonance (SPR) principle, and its affinity kinetic constant was calculated. The detection results are shown in Table 1. D The value is 3.09E-10(M). The amino acid sequences of the heavy and light chains of the humanized antibody are detailed in Table 10, as shown in SEQ ID NO: 51–SEQ ID NO: 52, and the DNA sequences are shown in SEQ ID NO: 53–SEQ ID NO: 54.

[0092] Table 1. Affinity kinetics results of humanized 1E9 antibody (hu1E9)

[0093]

[0094] Example 3. Design of NK cell adaptor antibody (NKCE) targeting CD30

[0095] This embodiment provides an NK cell adaptor antibody that binds to a bispecific antigen-binding molecule that binds to both NK cell surface receptors (such as CD16a) and tumor-associated antigens (TAAs). The TAA-targeting portion of the bispecific antigen-binding molecule targets tumor cells expressing the tumor-associated antigen CD30, while the NK cell surface receptor CD16a-targeting portion activates NK cells. By simultaneously binding to CD30 on tumor cells and the NK cell surface receptor CD16a, the NK cell adaptor antibody directs NK cell activity towards tumor cells, promoting the targeted killing (cell lysis) ability of activated NK cells against tumor cells.

[0096] The CD30xCD16a bispecific antibody provided in this embodiment contains four polypeptide chains (I, II, III, IV), as follows: Figure 1 As shown in the figure. Polypeptide chains I and II are the heavy chains of the anti-CD30 monoclonal antibody, while polypeptide chains III and IV are the fusion of the CD30-targeting antibody light chain with the single-chain variable region scFv of the CD16a-targeting antibody. That is, the C-terminus of the CD30-targeting light chain is fused to the N-terminus of the CD16a-targeting scFv structure via a linker peptide.

[0097] Two different anti-CD30 antibody sequences were selected. One was derived from the humanized anti-CD30 antibody hu1E9 described in Examples 1 and 2, and the other was derived from the anti-CD30 antibody huHRS-3 disclosed in the published literature "Functional humanization of an anti-CD30 Fab fragment for the immunotherapy of Hodgkin's lymphoma using an in vitro evolution approach," whose heavy and light chain variable region amino acid sequences are shown in SEQ ID NO:47 and 48. Following the above-described linkage method, the C-terminus of the light chain targeting CD30 was fused to the N-terminus of the scFv structure targeting CD16a via a linker peptide. Two CD30xCD16a bispecific antibodies, SEQN10-2 and SEQN10-4, were constructed.

[0098] The sequence information of the CD30xCD16a bispecific antibody provided in this embodiment is shown in Table 12.

[0099] Example 4. Expression, purification, and affinity detection of NK cell adaptor antibodies.

[0100] This embodiment uses a mammalian cell expression system for antibody expression. First, the nucleotide sequences encoding the heavy and light chains of the CD30xCD16a bispecific antibody were codon-optimized. The optimized heavy and light chain nucleotide sequences of the CD30xCD16a bispecific antibody are shown in SEQ ID NO:66 / 67 (SEQN10-2) and SEQ ID NO:70 / 71 (SEQN10-4), respectively. The heavy and light chain nucleotide sequences of the bispecific antibodies SEQN10-2 and SEQN10-4 were synthesized using gene synthesis methods. Then, the synthesized sequences were cloned into the expression vector pCDNA3.1(+) via homologous recombination. The recombinant plasmid containing the nucleotide sequence encoding the specific antibody described in this application was transiently transfected into the expi293F (Gibco) suspension cell line using a PEI (Polysciences) mediated method for transient expression. The cell culture supernatant was harvested by centrifugation.

[0101] The harvested supernatant was used to capture the target protein using an AT Protein A Diamond affinity chromatography column (Borglon). The affinity chromatography purification method was as follows: after capturing the culture supernatant with Protein A affinity packing material, it was washed with phosphate buffer (pH 7.00) containing 0.5 M sodium chloride and 0.1 M glycine solution (pH 5.0), respectively. The target protein was eluted with 0.1 M glycine solution (pH 3.0), and then immediately neutralized to pH 7.0 with 0.3 M disodium hydrogen phosphate and dialyzed into citrate buffer for storage. Antibody purity was analyzed by reducing and non-reducing SDS-PAGE gel electrophoresis and SEC-HPLC; antibody purity was above 85% in all cases.

[0102] The binding affinity of the antibody molecules of this invention to two isotypes of human CD16a 176V (Novoprotein, Cat#: C441) and CD16a 176F (Novoprotein, Cat#: CS11), and human CD30 protein (Novoprotein, Cat#: C643) was detected using the BLI (Bio-Layer Interferometry) method. Specifically, BLI detection was performed using an OctetRED 96 (Pall-ForteBio) instrument. The antigen protein immobilized biosensor was used to perform binding experiments with antibody molecules of different concentrations. The antibody molecules were serially diluted from an initial concentration of 50 nM, for a total of seven dilutions. The immobilized biosensor captured the antibody molecules. 0.02% PBST was used as a negative control. The binding time was 180 s, and the dissociation time was 200 s. After subtracting the negative control from the sample signal, the data was fitted using OctetDataAnalysis HT 12.0 software. K was calculated from the antibody binding and dissociation curves of the concentration gradients. D Values. See Tables 2, 3, and 4 for details. The results show that the CD30xCD16a bispecific antibody constructed in this invention has a high affinity for human CD16a, and the SEQN10-4 bispecific antibody molecule constructed using hu1E9 obtained in Examples 1 and 2 of this invention also has a high binding capacity to the human tumor cell-associated antigen CD30 protein.

[0103] Table 2. Results of binding kinetics between CD30xCD16a bispecific antibody and CD16a(F176)

[0104]

[0105] Table 3. Results of binding kinetics between CD30xCD16a bispecific antibody and CD16a(V176)

[0106]

[0107] Table 4. Results of the binding kinetics assay between CD30xCD16a bispecific antibody and human CD30 protein.

[0108]

[0109] Example 5. In vitro evaluation of the effect of NK cell adaptor antibody on NK cell activation.

[0110] This embodiment uses human NK cells as the evaluation system and evaluates the ability of SEQN10-2 and SEQN10-4 antibodies to mediate NK cell activation by measuring the expression of the NK cell surface activation marker CD107a.

[0111] Specifically, in this embodiment, PBMCs from healthy volunteers were extracted using Ficoll-Paque Plus (GE, Cat#:17-1440-02). First, CD3- cells were collected using CD3-positive magnetic beads (miltenyni, Cat#:130-097-043), and then CD56+ cells were collected using CD56-positive magnetic beads (miltenyni, Cat#:130-097-042). Cells were then processed at a ratio of 1 x 102. 5 Density per cell / well and 5x10 4 NK cells and CD30-positive target cells (human large cell lymphoma cell line Karpas 299 or T-cell lymphoma cell line TALL-104) were added to 96-well U-bottom cell culture plates at cell / well densities. SEQN10-2 and SEQN10-4 were serially diluted with RPMI 1640 complete medium (Gibco, Cat#: C11875500CP): final concentrations were set at 0.001, 0.01, 0.1, 1, and 10 nM. NKCE was added to 96-well U-bottom cell culture plates and incubated with NK cells and target cells at 37°C for 4 h. After washing with STB, the cells were stained with detection antibodies. The following detection antibodies were used:

[0112] BV421 anti-human CD107a antibody: BD Biosciences, Cat#:1349317.

[0113] Then, the proportion and number of target cells in each well of the 96-well plate were detected using a Thermo Fisher Attune NxT flow cytometer: CD107a+ cells among CD3-CD56+ cells.

[0114] Data statistics were performed using GraphPad Prism software. The results are as follows: Figure 2 As shown in A and 2B, compared with the solvent control, after co-incubation of SEQN10-2 and SEQN10-4 with NK cells and target cells for 4 h, the proportion of CD107a-expressing cells in CD3-CD56+ cells was significantly increased, and the increase was dose-dependent with respect to concentration.

[0115] In summary, the antibodies SEQN10-2 and SEQN10-4 provided by this invention, when co-incubated with NK cells, can mediate NK cell activation in a dose-dependent manner.

[0116] Example 6. In vitro evaluation of NK cell adaptor antibody-mediated NK cell killing activity against target cells

[0117] This embodiment evaluates the NK cell adaptor antibodies SEQN10-2 and SEQN10-4-mediated NK cell killing activity against target cells. Human NK cells were used as effector cells, and human acute T-lymphoblastic leukemia cell line TALL-104 was used as target cells. After co-incubating NK cells and target cells with different concentrations of SEQN10-2 and SEQN10-4, the proportion and number of target cells in the system were detected, thereby evaluating the NK cell killing activity mediated by SEQN10-2 and SEQN10-4 against target cells.

[0118] Specifically, in this embodiment, PBMCs from healthy volunteers were extracted using Ficoll-Paque Plus. First, CD3- cells were collected by sorting with CD3-positive magnetic beads (miltenyni, Cat#:130-097-043), and then CD56+ cells were collected by sorting with CD56-positive magnetic beads (miltenyni, Cat#:130-097-042). Target cells (TALL-104) were resuspended at a density of 1E6 / ml and labeled with violet-cell trace. NK cells and target cells were added to 96-well U-bottom cell culture plates at a ratio of 10:1 or 2:1. SEQN10-2 and SEQN10-4 were serially diluted with RPIM 1640 complete medium and added to the above 96-well U-bottom cell culture plates, with final concentrations set at 0.001, 0.01, 0.1, 1, and 10 nM. After incubation at 37°C for 6 hours, the plates were washed and stained with 7-AAD. The proportion and number of CFSE+ cells in each well were detected using a Thermo Fisher Attune NxT flow cytometer, and the killing efficiency was calculated. The formula for calculating the killing efficiency is as follows:

[0119] Killing efficiency (%) = 100% × [1 - (CFSE + cell count in sample wells) / (CFSE + cell count in solvent control wells)]

[0120] The data was analyzed using GraphPad Prism software. The results are shown below. Figure 3 A and 3B. The results showed that, under both effector-target ratios, SEQN10-2 and SEQN10-4 could mediate the killing of human acute T-lymphoblastic leukemia cell line TALL-104 by NK cells in a dose-dependent manner, and the killing activity was higher than that of anti-CD30 or anti-CD16a monoclonal antibodies at the same molar concentration.

[0121] Example 7. Antitumor effect of NK cell adaptor antibody in NVG-hIL15 mouse Hut-78 subcutaneous tumor-bearing model

[0122] This embodiment evaluates the tumor-suppressing effect of SEQN10-4 in the NVG-hIL15 mouse Hut-78 subcutaneous tumor-bearing model.

[0123] The human immune system was reconstructed in severely immunodeficient mice (NVG-hIL15) by transplanting human NK cells. The human NK cells were derived from PBMCs of healthy volunteers, and CD3+ cells were obtained after sorting with CD3 and CD56 magnetic beads. - CD56 + NK cells. NK cells obtained using this method (PBMC-NK) were directly used for immune reconstitution in NVG-hIL15 mice, or activated and expanded in vitro (huNK) for immune reconstitution in NVG-hIL15 mice. One day prior to inoculation, all animals were irradiated, and 2 × 10⁶ cells were subcutaneously inoculated into the right side of female NVG-hIL15 mice. 6 Hut-78 cells and 1×10 6 NK cells. When the average tumor volume reaches 60 mm. 3 At approximately 10:00 AM, 30 mice were randomly divided into the following 5 groups (6 mice / group) according to the tumor volume: PBS control group (G1); huNK cell group (G2); huNK cell plus SEQN10-4 (5 mg / kg) group (G3); PBMC-NK cell group (G4); and PBMC-NK cell plus SEQN10-4 (5 mg / kg) group (G5).

[0124] Mice in each group were administered the drug intravenously twice a week (on days 1 and 4) for two weeks, for a total of four administrations. Human NK cells were injected via the tail vein one day prior to administration at a dose of 1×10⁻⁶. 6 One cell, injected once.

[0125] Grouping day was designated D0, and the drug administration period was D0-D12. Mouse body weight and tumor volume were measured and recorded two to three times per week. Tumor volume inhibition rate (TGI) was calculated based on the measured tumor volume. TV At the study endpoint, tumors were harvested, weighed, and the tumor weight inhibition rate (TGI) was calculated. TW Drug efficacy is expressed as tumor growth inhibition (TGI), including tumor volume inhibition rate (TGI). TV ) and tumor weight inhibition rate (TGI) TW ).

[0126] The SEQN10-4 antibody was pharmacodynamically evaluated based on clinical observation indicators such as tumor growth inhibition rate, mouse body weight, and 24-hour food / water intake. The animal handling procedures were approved by the International Association for the Management and Use of Laboratory Animals (IACUC) of Shanghai Ruili Biological.

[0127] Tumor volume inhibition rate (TGI) in each group during drug administration TV Changes such as Figure 4As shown in A, at the study endpoint, the TGI of groups G2-G5 was... TV The percentages were 1.38%, 26.5%, 98.0%, and 99.7%, respectively.

[0128] At the study endpoint, tumors were harvested, weighed, and the tumor weight inhibition rate (TGI) was calculated. TW The results are shown in Table 5 and Figure 4 As shown in B.

[0129] Table 5. Endpoint tumor weight and tumor weight inhibition rate (TGI) in each treatment group TW )

[0130]

[0131] Note: Tumor weight is expressed as mean ± SEM; the difference in tumor weight between each treatment group and G1 was statistically analyzed by independent samples t-test (*: P<0.05, ***: P<0.001).

[0132] The results showed that the NK cell adaptor antibody SEQN10-4 could induce and further enhance the tumor-suppressing effect of NK cells on tumor cells.

[0133] Table 6: Amino acid sequence of the heavy and light chain CDR regions of antibodies targeting human CD30

[0134]

[0135]

[0136] Table 7: DNA sequence of the heavy and light chain CDR region of antibody targeting human CD30

[0137]

[0138] Table 8: Amino acid sequence of the heavy and light chain CDR regions of antibodies targeting human CD16a

[0139]

[0140] Table 9: DNA sequence of the heavy and light chain CDR region of antibody targeting human CD16a

[0141]

[0142]

[0143] Table 10: Amino acid sequence and DNA sequence of the variable region of the heavy chain of antibody targeting human CD30

[0144]

[0145]

[0146] Table 11: Amino acid sequence and DNA sequence of the variable region of the heavy light chain of antibody targeting human CD16a

[0147]

[0148]

[0149] Table 12: Full-length amino acid sequence of the light chain and DNA sequence of bispecific antigen-binding molecules

[0150]

[0151]

[0152]

[0153] Table 13: Full-length amino acid sequences of heavy and light chains and DNA sequences of anti-CD30 mAb and anti-CD16a mAb control antibodies

[0154]

[0155]

Claims

1. A bispecific antibody, characterized in that, The bispecific antibody comprises a first protein functional region targeting CD30 and a second protein functional region targeting CD16a; wherein: The first protein functional region comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 with an amino acid sequence as shown in SEQ ID NO:1, HCDR2 with an amino acid sequence as shown in SEQ ID NO:2, and HCDR3 with an amino acid sequence as shown in SEQ ID NO:

3. The light chain variable region comprises LCDR1 with an amino acid sequence as shown in SEQ ID NO:7, LCDR2 with an amino acid sequence as shown in SEQ ID NO:8, and LCDR3 with an amino acid sequence as shown in SEQ ID NO:

9. Alternatively, the heavy chain variable region comprises HCDR1 as shown in SEQ ID NO:4, HCDR2 as shown in SEQ ID NO:5, and HCDR3 as shown in SEQ ID NO:6; the light chain variable region comprises LCDR1 as shown in SEQ ID NO:10, LCDR2 as shown in SEQ ID NO:11, and LCDR3 as shown in SEQ ID NO:

12.

2. The bispecific antibody as described in claim 1, characterized in that, The first protein functional region is selected from the following: (1) The heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO:43, and the light chain variable region contains an amino acid sequence as shown in SEQ ID NO:44; Alternatively, the heavy chain variable region may contain an amino acid sequence as shown in SEQ ID NO:47, and the light chain variable region may contain an amino acid sequence as shown in SEQ ID NO:48; Alternatively, the heavy chain variable region may contain an amino acid sequence as shown in SEQ ID NO:51, and the light chain variable region may contain an amino acid sequence as shown in SEQ ID NO:52; (2) The heavy chain variable region contains an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:43; the light chain variable region contains an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO:44; Alternatively, the heavy chain variable region comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:47; the light chain variable region comprises an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:48; Alternatively, the heavy chain variable region may contain an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:51; and the light chain variable region may contain an amino acid sequence having at least 80% identity with the amino acid sequence shown in SEQ ID NO:

52.

3. The bispecific antibody as described in claim 2, characterized in that, The first protein functional region is an IgG structure; preferably, its constant region is selected from at least one of human IgG1, IgG2, IgG3, IgG4, IgA1, IgA, IgM, IgD and IgE constant regions and their related variants; more preferably, its constant region is human IgG1.

4. The bispecific antibody according to any one of claims 1-3, characterized in that, The second protein functional region comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 with the amino acid sequence shown in SEQ ID NO:13, HCDR2 with the amino acid sequence shown in SEQ ID NO:14, and HCDR3 with the amino acid sequence shown in SEQ ID NO:

15. The light chain variable region comprises LCDR1 with the amino acid sequence shown in SEQ ID NO:16, LCDR2 with the amino acid sequence shown in SEQ ID NO:17, and LCDR3 with the amino acid sequence shown in SEQ ID NO:

18.

5. The bispecific antibody as described in claim 4, characterized in that, The second protein functional region is an scFv structure; Preferably, the heavy chain variable region of the scFv contains an amino acid sequence as shown in SEQ ID NO:55; And / or, the light chain variable region of the scFv contains an amino acid sequence as shown in SEQ ID NO:56; And / or, the heavy chain variable region and the light chain variable region of the scFv are connected by a Linker, the Linker being (G4S). n Where n = 1-4, for example n = 3.

6. The bispecific antibody as described in claim 5, characterized in that, The scFv contains an amino acid sequence as shown in SEQ ID NO:

61.

7. The bispecific antibody according to any one of claims 1-6, characterized in that, The light chains of the second protein functional region and the first protein functional region are connected, preferably with the N-terminus of the light chain of the second protein functional region connected to the C-terminus of the light chain of the first protein functional region; And / or, the first and second protein functional regions are connected by a linker, said linker being (G4S). n n = 1-4, for example n = 3.

8. The bispecific antibody according to any one of claims 1-7, characterized in that, The bispecific antibody comprises four polypeptide chains; among which... One of the polypeptide chains contains an amino acid sequence as shown in SEQ ID NO:64, and the other polypeptide chain contains an amino acid sequence as shown in SEQ ID NO:65; Alternatively, one of the polypeptide chains contains an amino acid sequence as shown in SEQ ID NO:68, and the other polypeptide chain contains an amino acid sequence as shown in SEQ ID NO:

69.

9. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the bispecific antibody as described in any one of claims 1-8.

10. The nucleic acid molecule as described in claim 9, characterized in that, The nucleic acid molecule contains the following nucleotide sequence: Nucleotide sequences encoding HCDR1-3 as shown in SEQ ID NO:19-21 and nucleotide sequences encoding LCDR1-3 as shown in SEQ ID NO:25-27; Alternatively, nucleotide sequences encoding HCDR1-3 as shown in SEQ ID NO:22-24 and nucleotide sequences encoding LCDR1-3 as shown in SEQ ID NO:28-30.

11. The nucleic acid molecule as described in claim 10, characterized in that, The nucleic acid molecule contains a nucleotide sequence as shown in SEQ ID NO:45-46; Alternatively, the nucleic acid molecule may contain a nucleotide sequence as shown in SEQ ID NO:49-50; Alternatively, the nucleic acid molecule may contain a nucleotide sequence as shown in SEQ ID NO:43-54.

12. The nucleic acid molecule as described in claim 11, characterized in that, The nucleic acid molecule contains a nucleotide sequence as shown in SEQ ID NO:66-67; Alternatively, the nucleic acid molecule may contain a nucleotide sequence as shown in SEQ ID NO:70-71.

13. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule according to any one of claims 9-12; Preferably, the recombinant expression vector is any one or a combination of at least two of the following: viral vector, plasmid, DNA fragment, RNA, etc. More preferably, the recombinant expression vector is a plasmid.

14. A transformant, characterized in that, The host cell contains a nucleic acid molecule as described in any one of claims 9-12 or a recombinant expression vector as described in claim 13; Preferably, the host cell is a mammalian cell.

15. A method for preparing a bispecific antibody as described in any one of claims 1-8, characterized in that, It includes culturing the transformant as described in claim 14 and obtaining the bispecific antibody from the culture.

16. A genetically modified cell, characterized in that, It includes the bispecific antibody as described in any one of claims 1-8.

17. An antibody-drug conjugate, characterized in that, The antibody-drug conjugate comprises a cytotoxic agent or tag, and a bispecific antibody as described in any one of claims 1-8.

18. A pharmaceutical composition or a medicine box containing the same, characterized in that, The pharmaceutical composition or the kit containing it comprises at least one of the following: the bispecific antibody of any one of claims 1-8, the genetically modified cell of claim 16, or the antibody-drug conjugate of claim 17; and a pharmaceutically acceptable carrier, excipient, or diluent. Preferably, the pharmaceutical composition or the kit containing it further comprises one or more of the following groups: hormone preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic drugs, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.

19. The use of the bispecific antibody as described in any one of claims 1-8, the nucleic acid molecule as described in any one of claims 9-12, the recombinant expression vector as described in claim 13, the transformant as described in claim 14, the gene-modified cell as described in claim 16, the antibody-drug conjugate as described in claim 17, or the pharmaceutical composition or cassette containing the thereof as described in claim 18, in the preparation of a medicament for the prevention and / or treatment of tumors.

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