Bispecific antibody-NK cell conjugate combining CD16A and Trop2 and its uses

Abstract

The present application provides a bispecific antibody-NK cell conjugate that binds to the CD16A molecule and Trop2, a bispecific antibody that binds to the CD16A molecule and Trop2, a pharmaceutical composition comprising the bispecific antibody-NK cell conjugate or the bispecific antibody, and their uses.

Description

Field of the Invention

[0001] The present invention generally relates to the fields of genetic engineering and biopharmaceuticals; specifically, the present application relates to bispecific antibody-natural killer cell (NK cell) conjugates that bind to the CD16A molecule and Trop2, bispecific antibodies that bind to the CD16A molecule and Trop2, pharmaceutical compositions comprising the bispecific antibody-NK cell conjugates or the bispecific antibodies, and their uses. Background of the Invention

[0002] CD16A, also known as FcγRIIIA, is a receptor that plays an important role in the immune system and is present on natural killer cells (NK cells). This receptor has a transmembrane domain and a cytoplasmic tail and is associated with proteins carrying immunoreceptor tyrosine-based activation motifs (ITAMs). It is mainly expressed on NK cells, macrophages, monocytes, and certain subsets of T cells. One of the main functions of CD16A is to mediate antibody-dependent cell cytotoxicity (ADCC) through low-affinity interactions with the Fc region of human immunoglobulin G (IgG). In this process, CD16A can recognize and bind to the Fc region of IgG that binds to tumor cell-associated antigens, thereby triggering the activation of NK cells or other immune cells expressing CD16A, resulting in the killing of tumor cells.

[0003] The Trop2 target, also known as human trophoblast cell surface antigen 2, is a cell surface glycoprotein encoded by the Tacstd2 gene in the chromosomal region 1p32 and belongs to the GA733 protein family. The high expression of Trop2 in a variety of human epithelial cancers and its close association with malignant behaviors such as tumor growth, invasion, and metastasis. First, Trop2 is overexpressed in a variety of solid tumors, including but not limited to breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer, etc. There have been reports on targeted therapies against Trop2 in the hope of achieving control of a variety of malignant tumors. Second, Trop2 plays a key role in tumor growth. Studies have found that the overexpression of Trop2 is closely related to malignant characteristics such as the proliferation, invasion, and metastasis of tumor cells. Trop2 can promote the proliferation of tumor cells by upregulating the expression of the proliferation marker Ki-67, and promote tumor invasion and metastasis by enriching RACK1 on the cell membrane to reduce the binding of fibronectin to integrin β-1.

[0004] Antibody therapy and cell therapy have become hotspots in the current field of cancer treatment, and related explorations and research are urgently needed in this field. Summary of the Invention

[0005] In a first aspect, the present application provides a bispecific antibody-natural killer cell (NK cell) conjugate, wherein

[0006] The bispecific antibody comprises a first antigen-binding region that binds to human trophoblast cell surface antigen 2 (Trop2) and a second antigen-binding region that binds to the CD16A molecule;

[0007] The bispecific antibody is conjugated to the NK cell by antigen-antibody binding of its second antigen-binding region to the CD16A molecule on the NK cell.

[0008] In some embodiments of the first aspect, the NK cells are obtained from in vitro culture and expansion of NK cells derived from peripheral blood mononuclear cells (PBMC);

[0009] The NK cells are obtained from in vitro culture and expansion of NK cells derived from umbilical cord blood;

[0010] The NK cells are obtained from in vitro culture and expansion of an NK cell line; or

[0011] The NK cells are obtained from in vitro induction, culture, and expansion of induced pluripotent stem cells (iPSC) or mesenchymal stem cells.

[0012] In a second aspect, the present application provides a bispecific antibody that comprises a first antigen-binding region that binds to human trophoblast cell surface antigen 2 (Trop2) and a second antigen-binding region that binds to the CD16A molecule.

[0013] In some embodiments of the first or second aspect, the first antigen-binding region comprises:

[0014] HCDR1 as shown in SEQ ID NO:1,

[0015] HCDR2 as shown in SEQ ID NO:2,

[0016] HCDR3 as shown in SEQ ID NO:3,

[0017] LCDR1 as shown in SEQ ID NO:4,

[0018] LCDR2 as shown in SEQ ID NO:5, and

[0019] LCDR3 as shown in SEQ ID NO:6; and / or

[0020] The second antigen-binding region comprises:

[0021] HCDR1 as shown in SEQ ID NO:7,

[0022] HCDR2 as shown in SEQ ID NO:8,

[0023] The HCDR3 as shown in SEQ ID NO:9,

[0024] the LCDR1 as shown in SEQ ID NO:10,

[0025] the LCDR2 as shown in SEQ ID NO:11, and

[0026] the LCDR3 as shown in SEQ ID NO:12;

[0027] wherein, the amino acid sequence of the HCDR is defined according to Kabat.

[0028] In some embodiments of the first or second aspect, the first antigen-binding region comprises a heavy chain variable region and a light chain variable region, and the amino acid sequence of the heavy chain variable region of the first antigen-binding region is as shown in SEQ ID NO:13 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region of the first antigen-binding region is as shown in SEQ ID NO:14 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in SEQ ID NO:14.

[0029] In some embodiments of the first or second aspect, the second antigen-binding region comprises a heavy chain variable region and a light chain variable region, and the amino acid sequence of the heavy chain variable region of the second antigen-binding region is as shown in SEQ ID NO:15 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region of the second antigen-binding region is as shown in SEQ ID NO:16 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in SEQ ID NO:16.

[0030] In some embodiments of the first or second aspect, the first antigen-binding region is in the form of a single-chain antibody (scFv); and / or the second antigen-binding region is in the form of an scFv.

[0031] In some embodiments of the first or second aspect, the first antigen-binding region is linked to the second antigen-binding region by a peptide linker.

[0032] In some embodiments of the first or second aspect, the first antigen-binding region comprises a heavy-chain variable region and a light-chain variable region, and the heavy-chain variable region is linked to the light-chain variable region by a peptide linker.

[0033] In some embodiments of the first or second aspect, the second antigen-binding region comprises a heavy-chain variable region and a light-chain variable region, and the heavy-chain variable region is linked to the light-chain variable region by a peptide linker.

[0034] In some embodiments of the first or second aspect, the bispecific antibody comprises a first antigen-binding region having the amino acid sequence shown in SEQ ID NO: 17 and a second antigen-binding region having the amino acid sequence shown in SEQ ID NO: 18.

[0035] In a third aspect, the present application provides a pharmaceutical composition comprising the bispecific antibody-NK cell conjugate of the first aspect or the bispecific antibody of the second aspect, and a pharmaceutically acceptable carrier;

[0036] Optionally, the pharmaceutical composition is provided in the form of a sterile fresh preparation or a sterile frozen preparation.

[0037] In a fourth aspect, the present application provides the use of the bispecific antibody-NK cell conjugate of the first aspect, the bispecific antibody of the second aspect, or the pharmaceutical composition of the third aspect in the preparation of a drug for preventing or treating tumors in an individual. Brief Description of the Drawings

[0038] Figure 1 Shows the purity results after NK cell expansion, where LYM is the abbreviation for lymphocytes.

[0039] Figure 2 Shows the conjugation efficiency of the bispecific antibody with NK cells; the conjugation efficiency of the isotype control antibody with NK cells, and the right figure represents the conjugation efficiency of the bispecific antibody against CD16A and Trop2 with NK cells.

[0040] Figure 3 Shows the in vitro killing results of the bispecific antibody-NK cell conjugate against MCF7 cells.

[0041] Figure 4Shows the results of the ADCC effect mediated by bispecific antibodies.

[0042] Figure 5 Shows the results of the anti-tumor efficacy experiment in mice with bispecific antibody-NK cell conjugates.

[0043] Figure 6 Shows the clinical treatment effect of bispecific antibody-NK cell conjugates.

[0044] Sequence description

[0045] SEQ ID NO: 1-6 show the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the first antigen-binding region that binds to human trophoblast cell surface antigen 2 (Trop2), respectively.

[0046] SEQ ID NO: 7-12 show the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the second antigen-binding region that binds to the CD16A molecule, respectively.

[0047] SEQ ID NO: 13-14 show the amino acid sequences of the heavy chain variable region and the light chain variable region of the first antigen-binding region that binds to Trop2, respectively.

[0048] SEQ ID NO: 15-16 show the amino acid sequences of the heavy chain variable region and the light chain variable region of the second antigen-binding region that binds to the CD16A molecule, respectively.

[0049] SEQ ID NO: 17-18 show the amino acid sequences of the first antigen-binding region that binds to Trop2 and the second antigen-binding region that binds to the CD16A molecule, respectively.

[0050] SEQ ID NO: 19 shows the amino acid sequence of peptide linker 2.

[0051] SEQ ID NO: 20 shows the amino acid sequence of peptide linker 1.

[0052] SEQ ID NO: 21 shows the amino acid sequence of peptide linker 3.

[0053] SEQ ID NO: 22 shows the amino acid sequence of the histidine purification tag H6.

[0054] SEQ ID NO: 23 shows the amino acid sequence of the bispecific antibody against Trop2 and the CD16A molecule.

[0055] SEQ ID NO:24 shows the amino acid sequence of a bispecific antibody against Trop2 and CD16A molecules containing the histidine purification tag H6. DETAILED DESCRIPTION OF THE INVENTION

[0056] Unless otherwise indicated, the practice of this application employs conventional techniques of molecular biology, microbiology, cell biology, biochemistry, and immunology.

[0057] Unless otherwise indicated, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0058] DEFINITIONS

[0059] As used herein, the term "individual" or "subject" refers to a mammal, such as a human, but may also be other animals, such as wild animals, domestic animals, or laboratory animals (such as orangutans, monkeys, rats, mice, rabbits, guinea pigs, marmots, ground squirrels, etc.).

[0060] As used herein, the term "antigen" is a predetermined target to which an antibody can selectively bind. Examples of antigens include, but are not limited to, polypeptides, sugars, nucleic acids, lipids, haptens, or other naturally occurring or synthetic compounds.

[0061] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, etc. As used herein, "antibody" includes not only intact (i.e., full-length) antibodies, but also their binding fragments (e.g., Fab, Fab’, F(ab’)2, Fv), variants thereof, fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, bispecific antibodies, linear antibodies, single-chain antibodies, single-domain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configurations of immunoglobulin molecules containing the antigen recognition site with the desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0062] Typically, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain contains a variable heavy region (VH) and first, second, and third constant regions (CH1, CH2, and CH3). Each light chain contains a variable light region (VL) and a constant region (CL). The full-length antibody can be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), but the antibody does not need to belong to any specific class. Immunoglobulins can be designated into different classes based on the amino acid sequence of the constant region of the heavy chain. Typically, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further differentiated into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The constant regions of the heavy chains corresponding to different immunoglobulin classes are designated as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.

[0063] As used herein, the term "bispecific antibody" refers to an antibody that has the ability to bind two epitopes simultaneously. The two epitopes can be on different antigens or on the same antigen. Bispecific antibodies can have various structural configurations. For example, a bispecific antibody can be obtained by linking two scFv fragments through a linker.

[0064] The antigen-binding region can comprise a variable heavy region (VH), a variable light region (VL), or both. Each of VH and VL typically contains three complementarity-determining regions CDR1, CDR2, and CDR3.

[0065] It is well known to those skilled in the art that the complementarity-determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of the antibody. There are two common ways to define the CDR sequences of VH or VL, namely the Kabat definition and the Chothia definition. (See, for example, Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991)

[15] ; A1-Lazikani et al., J. Mol. Biol. 273:927-948 (1997)

[16] ; and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989))

[17] For the variable region sequences of a given antibody, the CDR region sequences in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition. In the embodiments of the present application, the Kabat definition is used to define the CDR sequences.

[0066] For the variable region sequences of a given antibody, the CDR region sequences in the variable region sequences can be analyzed in various ways. For example, the online software Abysis can be used for determination (http: / / www.abysis.org / ).

[0067] For a general antibody, examples of the antigen-binding region include but are not limited to: (1) Fab fragment, which can be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) F(ab’)2 fragment, which can be a divalent fragment having two Fab’ fragments, and the two Fab’ fragments are connected by a disulfide bridge in the hinge region (i.e., a dimer of Fab’); (3) Fv fragment having the VL and VH domains of a single arm of the antibody; (4) single-chain Fv (scFv), which can be a single polypeptide chain composed of a VH domain and a VL domain via a peptide linker; (5) (scFv)2, which can contain two VH domains and two VL domains connected by a peptide linker, and the two VL domains are combined with the two VH domains via disulfide bridges; and (6) single-domain antibody form.

[0068] In the construction of bispecific antibodies, the "antigen-binding region" includes but is not limited to the single-chain antibody (scFv) form, Fab fragment form, and / or single-domain antibody form.

[0069] As used herein, the term "single-chain antibody (scFv, single chain fragment variable)" refers to an antibody in a single-chain structure generally constructed by genetic engineering techniques, which contains a polypeptide chain of a heavy-chain variable region (VH) and a light-chain variable region (VL). A flexible linker is usually designed between the heavy-chain variable region and the light-chain variable region so that the heavy-chain variable region and the light-chain variable region can fold into a correct conformation capable of binding an antigen.

[0070] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigen epitope.

[0071] As used herein, the term "pharmaceutical composition" refers to a combination of at least one drug and optionally a pharmaceutically acceptable carrier or excipient that are combined to achieve a particular purpose. In certain embodiments, the pharmaceutical composition includes combinations that are separated in time and / or space, provided that they can act together to achieve the purposes of the present application. For example, the components contained in the pharmaceutical composition (such as the antibody-cell conjugate according to the present application) can be administered to an individual as a whole or separately. When the components contained in the pharmaceutical composition are administered to an individual separately, the components can be administered to the individual simultaneously or sequentially. The pharmaceutical composition according to the present application can include conventional components of cell culture, particularly NK cell culture, to maintain the activity of NK cells in the conjugate. The pharmaceutically acceptable carrier can also include water, buffered aqueous solutions, isotonic saline solutions such as PBS (phosphate buffered saline), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol, or polyalkylene glycols such as polypropylene glycol, triglycerides, etc. The pharmaceutical composition or pharmaceutical preparation according to the present application can be administered by any suitable route, such as intravenous administration, intradermal, subcutaneous, intramuscular injection, etc. The composition according to the present application can contain wetting agents, emulsifying agents, or buffering substances as additives.

[0072] As used herein, the term "therapeutically effective amount" or "effective amount" refers to a dose sufficient to demonstrate its beneficial effect on the individual to whom it is administered. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the condition being treated. The prescription of treatment (such as the determination of dosage, etc.) is ultimately the responsibility of the general practitioner and other physicians and depends on their decision-making, usually considering the disease being treated, the individual condition of the patient, the site of delivery, the method of administration, and other factors known to the physician.

[0073] As used herein, the term "tumor" refers to a neoplasm or solid lesion formed by abnormal cell growth. Tumors can be benign, pre-malignant, or malignant.

[0074] As used herein, the term "malignant tumor" refers to or describes a physiological condition in a mammal that is typically characterized by unregulated cell growth. Exemplary malignant tumors include: cancer, solid tumors, melanoma, sarcoma, hematologic malignancies, germ cell tumors, and embryonal tumors. More specific examples of malignant tumors include: lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, or squamous cell lung cancer), breast cancer, pancreatic cancer, gastric cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial or uterine cancer, kidney cancer, bladder cancer, skin cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urothelial cancer, rectal cancer, salivary gland cancer, squamous cell carcinoma (e.g., squamous epithelial cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, B-cell lymphoma, brain cancer, head and neck cancer, renal cell cancer, esophageal cancer, Hodgkin lymphoma, neuroblastoma, multiple myeloma, and associated metastases.

[0075] As used herein, the term "solid tumor" refers to a palpable mass that can be detected by clinical examinations such as radiography, CT scan, B-ultrasound, or palpation. Clinically diagnosed solid tumors are divided into two types: malignant and benign. Malignant solid tumors include: lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, or lung squamous cell carcinoma), breast cancer, pancreatic cancer, gastric cancer, prostate cancer, ovarian cancer, cervical cancer, endometrial or uterine cancer, kidney cancer, bladder cancer, skin cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, salivary gland cancer, squamous cell carcinoma (e.g., squamous epithelial cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, B-cell lymphoma, brain cancer, head and neck cancer, esophageal cancer (e.g., esophageal squamous cell carcinoma), urothelial cancer, oral cancer (e.g., oral squamous cell carcinoma), sarcoma (e.g., synovial sarcoma), childhood Hodgkin lymphoma: lymphocyte-predominant type, nodular sclerosis type, mixed cell type, lymphocyte depletion type; childhood non-Hodgkin lymphoma: prelymphoblastic lymphoma, small non-cleaved cell lymphoma (Burkitt / non-Burkitt lymphoma), diffuse large B-cell lymphoma, anaplastic large cell lymphoma, etc.; childhood kidney tumors: Wilms tumor, renal clear cell carcinoma, renal rhabdoid tumor, renal clear cell sarcoma, primitive neuroectodermal tumor of the kidney, etc.; childhood neuroblastoma: neuroblastoma, ganglioneuroblastoma, ganglioneuroma; childhood extragonadal germ cell tumors: mature teratoma, immature teratoma, endodermal sinus tumor (yolk sac tumor), seminoma, dysgerminoma, choriocarcinoma, embryonal carcinoma, etc.; osteosarcoma and chondrosarcoma; childhood rhabdomyosarcoma: embryonal type, alveolar type, pleomorphic type, etc.; soft tissue sarcoma: fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, angiosarcoma, lymphangiosarcoma, malignant schwannoma, alveolar soft part sarcoma, epithelioid sarcoma, clear cell sarcoma, malignant melanoma, synovial sarcoma, desmoplastic small round cell tumor, etc.; Ewing family sarcoma: Ewing sarcoma, primitive neuroectodermal tumor; childhood liver tumors: hepatoblastoma (embryonal type, fetal type, undifferentiated type), hepatocellular carcinoma; retinoblastoma; other tumors: posterior fossa medulloblastoma, nasopharyngeal carcinoma, papillary thyroid carcinoma, thymoma, lung blastoma, pancreaticoblastoma, insulinoma, ileocecal carcinoid, mesothelioma, etc. Benign solid tumors include: lymphangioma, hemangioma, thyroglossal duct cyst, etc.

[0076] The killing activity of NK cells mainly occurs through:

[0077] 1) Direct lysis of target cells: NK cells release cytotoxic granules such as perforin and granzyme through exocytosis, activating the caspase pathway to induce necrosis or apoptosis of target cells;

[0078] 2) Secreting cytokines: Cytokine-mediated killing. NK cells can synthesize and secrete various cytokines, such as IFN-γ, TNF-α, IL-1, IL-5, IL-8, IL-10, and G-CSF, etc., to induce apoptosis of target cells;

[0079] 3) Inducing apoptosis: Activated NK cells express Fas (CD95) ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) molecules, inducing apoptosis of CD95 + target cells and TRAIL receptor-positive target cells through a cascade reaction of endogenous enzymes;

[0080] 4) ADCC: Antibody-dependent cell-mediated cytotoxicity;

[0081] 5) Immune checkpoint pathway: Expressing programmed death receptor 1 (PD-1), cytotoxic T lymphocyte-associated protein 4 (CTLA4), etc., and playing a role by inhibiting immune checkpoints.

[0082] The above multiple mechanisms of action, as well as the potential for application as a universal product and reliable safety, make NK cell therapy an attractive immunotherapy.

[0083] Activation of the CD16A molecule can also promote the maturation, activation, and cytokine production of NK cells, and these cytokines play key roles in immune responses and inflammatory processes. Therefore, activating the function of NK cells by targeting the CD16A molecule may be an effective immunotherapy strategy, especially in combating cancer and viral infections. Generally speaking, the CD16A molecule, as a key activating receptor on NK cells, plays a central role in mediating ADCC and enhancing NK cell function. Activating the CD16A molecule with specific antibodies can significantly enhance the killing ability of NK cells against tumor cells and virus-infected cells, providing new possibilities and research directions for immunotherapy.

[0084] Trop2, fully known as Trophoblast Cell-Surface Antigen 2, is overexpressed in a variety of solid tumors and plays a key role in tumor growth.

[0085] The inventors of the present application have conducted in-depth research in the field of cellular immunotherapy. In particular, for tumor treatment, a bispecific antibody-NK cell conjugate has been developed, which comprises a first antigen-binding region that binds to Trop2 and a second antigen-binding region that binds to the CD16A molecule. The second antigen-binding region that binds to the CD16A molecule binds to the CD16A molecule expressed on the NK cell to activate the NK cell, and the first antigen-binding region that binds to Trop2 binds to target cells expressing Trop2 (such as Trop2-positive tumor cells), thereby establishing an interaction between the target cell and the NK cell. The target cell is killed through antibody-dependent cell cytotoxicity (ADCC) to achieve the purpose of treating diseases (such as tumors). In many aspects of the present application, novel bispecific antibody-NK cell conjugates comprising a first antigen-binding region that binds to Trop2 and a second antigen-binding region that binds to the CD16A molecule, bispecific antibodies comprising a first antigen-binding region that binds to Trop2 and a second antigen-binding region that binds to the CD16A molecule, pharmaceutical compositions comprising the bispecific antibody-NK cell conjugate or the bispecific antibody, nucleic acid molecules encoding the bispecific antibody, vectors comprising the nucleic acid molecules, host cells comprising the nucleic acid molecules or vectors, methods for preparing and purifying the bispecific antibody, and medical and biological applications of the bispecific antibody-NK cell conjugate and the bispecific antibody are provided.

[0086] In a first aspect, the present application provides a bispecific antibody-NK cell conjugate, wherein

[0087] the bispecific antibody comprises a first antigen-binding region that binds to human trophoblast cell surface antigen 2 (Trop2) and a second antigen-binding region that binds to the CD16A molecule;

[0088] the bispecific antibody is conjugated to the NK cell through antigen-antibody binding of its second antigen-binding region to the CD16A molecule on the NK cell.

[0089] In some embodiments of the first aspect, the NK cells are obtained from in vitro culture and expansion of NK cells derived from peripheral blood mononuclear cells (PBMC), which is also an exemplary method in the examples of this application. PBMC is one of the main sources of NK cells, with the advantages of relatively easy collection, easy in vitro expansion, and no toxic side effects. However, the proportion of NK cells in PBMC is only 10%-15%. Methods for expanding PBMC-derived NK cells include using a combination of cytokines, feeder layer cells, or membrane particles to stimulate the in vitro expansion of NK cells, and these different expansion systems show different levels of NK cell expansion efficiency. In some embodiments, one or several cytokines are used to maintain or activate the activity of natural killer cells during culture. In some embodiments, one or several immunoglobulins or fusion proteins are used to inhibit the proliferation of B cells, macrophages, and other immune cells.

[0090] In some embodiments of the first aspect, the PBMC can be obtained from apheresis peripheral blood lymphocytes of allogeneic healthy donors.

[0091] In some embodiments of the first aspect, the NK cells are obtained from in vitro culture and expansion of NK cells derived from umbilical cord blood. Generally, there are two different methods to obtain a large number of NK cells from umbilical cord blood. One method is to expand NK cells in umbilical cord blood, and the other method is to induce umbilical cord blood CD34 + hematopoietic stem / progenitor cells to differentiate into NK cells and then expand them.

[0092] In some embodiments of the first aspect, the NK cells are obtained from in vitro culture and expansion of NK cell lines. As an example, NK-92 is the first NK cell-based immunotherapy approved by the FDA for clinical trials and is a homogeneous immortalized NK lymphoma cell line.

[0093] In some embodiments of the first aspect, the NK cells are obtained from in vitro induction, culture, and expansion of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells.

[0094] In some embodiments of the first aspect, the phenotype of the NK cells is CD3 - / CD16 + CD56 + .

[0095] In a second aspect, the present application provides a bispecific antibody comprising a first antigen-binding region that binds to Trop2 and a second antigen-binding region that binds to the CD16A molecule.

[0096] In some embodiments of the second aspect, the bispecific antibody is capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC).

[0097] In some embodiments of the first or second aspect, the first antigen-binding region comprises:

[0098] HCDR1 as shown in SEQ ID NO:1,

[0099] HCDR2 as shown in SEQ ID NO:2,

[0100] HCDR3 as shown in SEQ ID NO:3,

[0101] LCDR1 as shown in SEQ ID NO:4,

[0102] LCDR2 as shown in SEQ ID NO:5, and

[0103] LCDR3 as shown in SEQ ID NO:6;

[0104] wherein, the amino acid sequence of the HCDR is defined according to Kabat.

[0105] In some embodiments of the first or second aspect, the second antigen-binding region comprises:

[0106] HCDR1 as shown in SEQ ID NO:7,

[0107] HCDR2 as shown in SEQ ID NO:8,

[0108] HCDR3 as shown in SEQ ID NO:9,

[0109] LCDR1 as shown in SEQ ID NO:10,

[0110] LCDR2 as shown in SEQ ID NO:11, and

[0111] LCDR3 as shown in SEQ ID NO:12;

[0112] wherein, the amino acid sequence of the HCDR is defined according to Kabat.

[0113] In some embodiments of the first or second aspect, the amino acid sequence of the heavy chain variable region of the first antigen-binding region is as shown in SEQ ID NO:13 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in SEQ ID NO:13.

[0114] In some embodiments described in the first or second aspect, the amino acid sequence of the heavy chain variable region of the first antigen-binding region differs from the amino acid sequence shown in SEQ ID NO: 13 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions.

[0115] In some embodiments described in the first or second aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 13 can be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids while still maintaining a similar function of the heavy chain variable region of the first antigen-binding region.

[0116] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids are added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 13, and the resulting amino acid sequence still maintains a similar function of the heavy chain variable region of the first antigen-binding region.

[0117] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids can be added or deleted from a region other than the C-terminal or N-terminal of the amino acid sequence shown in SEQ ID NO: 13, provided that the altered amino acid sequence substantially maintains a similar function of the heavy chain variable region of the first antigen-binding region.

[0118] In some embodiments described in the first or second aspect, the amino acid sequence of the light chain variable region of the first antigen-binding region is as shown in SEQ ID NO: 14 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO: 14.

[0119] In some embodiments described in the first or second aspect, the amino acid sequence of the light chain variable region of the first antigen-binding region differs from the amino acid sequence shown in SEQ ID NO: 14 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions.

[0120] In some embodiments described in the first or second aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 14 may also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining the function similar to that of the light chain variable region of the first antigen-binding region.

[0121] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 14, and the resulting amino acid sequence still maintains the function similar to that of the light chain variable region of the first antigen-binding region.

[0122] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may be added or deleted from a region other than the C-terminal or N-terminal of the amino acid sequence shown in SEQ ID NO: 14, as long as the altered amino acid sequence substantially maintains the function similar to that of the light chain variable region of the first antigen-binding region.

[0123] In some embodiments described in the first or second aspect, the amino acid sequence of the heavy chain variable region of the first antigen-binding region is as shown in SEQ ID NO: 13 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO: 13, and the amino acid sequence of the light chain variable region of the first antigen-binding region is as shown in SEQ ID NO: 14 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO: 14.

[0124] In some embodiments described in the first or second aspect, the amino acid sequence of the heavy chain variable region of the first antigen-binding region is as shown in SEQ ID NO: 13 and the amino acid sequence of the light chain variable region of the first antigen-binding region is as shown in SEQ ID NO: 14.

[0125] In some embodiments described in the first or second aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding region is as shown in SEQ ID NO:15 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:15.

[0126] In some embodiments described in the first or second aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding region differs from the amino acid sequence shown in SEQ ID NO:15 by about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions and / or additions.

[0127] In some embodiments described in the first or second aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:15 can be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still maintaining a similar function of the heavy chain variable region of the second antigen-binding region.

[0128] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids are added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:15, and the resulting amino acid sequence still maintains a similar function of the heavy chain variable region of the second antigen-binding region.

[0129] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can be added or deleted from a region other than the C-terminal or N-terminal of the amino acid sequence shown in SEQ ID NO:15, as long as the modified amino acid sequence substantially maintains a similar function of the heavy chain variable region of the second antigen-binding region.

[0130] In some embodiments described in the first or second aspect, the amino acid sequence of the light chain variable region of the second antigen-binding region is as shown in SEQ ID NO:16 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence shown in SEQ ID NO:16.

[0131] In some embodiments described in the first or second aspect, the amino acid sequence of the light chain variable region of the second antigen-binding region differs from the amino acid sequence shown in SEQ ID NO: 16 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions.

[0132] In some embodiments described in the first or second aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 16 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids while still maintaining a similar function of the light chain variable region of the second antigen-binding region.

[0133] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids can also be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 16, and the resulting amino acid sequence still maintains a similar function of the light chain variable region of the second antigen-binding region.

[0134] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, or more amino acids can also be added or deleted from a region other than the C-terminal or N-terminal of the amino acid sequence shown in SEQ ID NO: 16, as long as the altered amino acid sequence substantially maintains a similar function of the light chain variable region of the second antigen-binding region.

[0135] In some embodiments described in the first or second aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding region is as shown in SEQ ID NO: 15 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO: 15, and the amino acid sequence of the light chain variable region of the second antigen-binding region is as shown in SEQ ID NO: 16 or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO: 16.

[0136] In some embodiments described in the first or second aspect, the amino acid sequence of the heavy chain variable region of the second antigen-binding region is as shown in SEQ ID NO: 15 and the amino acid sequence of the light chain variable region of the second antigen-binding region is as shown in SEQ ID NO: 16.

[0137] In some embodiments described in the first or second aspect, the first antigen-binding region is in the form of a single-chain antibody (scFv).

[0138] In some embodiments described in the first or second aspect, the second antigen-binding region is in the form of a single-chain antibody (scFv).

[0139] In some embodiments described in the first or second aspect, the first antigen-binding region and the second antigen-binding region are linked by a peptide linker. In some embodiments, the peptide linker is a GS-type flexible peptide linker. In some embodiments, the peptide linker is (G4S)n or (G2S)n, where n is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or an integer or integer range between any two of the above values. In some embodiments, the peptide linker is (G4S)n or (G2S)n, where n is an integer from 1 to 5, such as 1, 2, 3, 4, or 5. In some specific embodiments, the peptide linker is GGGGS (SEQ ID NO: 19).

[0140] In some embodiments described in the first or second aspect, the first antigen-binding region comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region are linked by a peptide linker. In some embodiments, the peptide linker is a GS-type flexible peptide linker. In some embodiments, the peptide linker is (G4S)n or (G2S)n, where n is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or an integer or integer range between any two of the above values. In some embodiments, the peptide linker is (G4S)n or (G2S)n, where n is an integer from 1 to 5, such as 1, 2, 3, 4, or 5. In some specific embodiments, the peptide linker is GGGGSGGGGSGGGGS (SEQ ID NO: 20).

[0141] In some embodiments described in the first or second aspect, the second antigen-binding region comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region are linked by a peptide linker. In some embodiments, the peptide linker is a GS-type flexible peptide linker. In some embodiments, the peptide linker is (G4S)n or (G2S)n, where n is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or an integer or integer range between any two of the above values. In some embodiments, the peptide linker is (G4S)n or (G2S)n, where n is an integer from 1 to 5, such as 1, 2, 3, 4, or 5. In some specific embodiments, the peptide linker is GGSGGSGGSGGSGGS (SEQ ID NO:21).

[0142] In some embodiments described in the first or second aspect, the bispecific antibody has the following linking pattern: CD16A VL1-linker1-CD16A VH1-linker2-Trop2 VL2-linker3-Trop2 VH2. In some embodiments, the peptide linker 1, the peptide linker 2, or the peptide linker 3 are the same. In some embodiments, the peptide linker 1, the peptide linker 2, or the peptide linker 3 are different. In some embodiments, the peptide linker 1, the peptide linker 2, or the peptide linker 3 is a GS-type flexible peptide linker. In some embodiments, the peptide linker 1, the peptide linker 2, or the peptide linker 3 is (G4S)n or (G2S)n, where n is an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or an integer or integer range between any two of the above values. In some embodiments, the peptide linker 1, the peptide linker 2, or the peptide linker 3 is (G4S)n or (G2S)n, where n is an integer from 1 to 5, such as 1, 2, 3, 4, or 5. In some specific embodiments, the peptide linker 1 is GGGGSGGGGSGGGGS (SEQ ID NO:20). In some specific embodiments, the peptide linker 2 is GGGGS (SEQ ID NO:19). In some specific embodiments, the peptide linker 3 is GGSGGSGGSGGSGGS (SEQID NO:21).

[0143] In some embodiments described in the first or second aspect, the bispecific antibody comprises a first antigen-binding region that binds to Trop2 and a second antigen-binding region that binds to the CD16A molecule, wherein the first antigen-binding region comprises the amino acid sequence shown in SEQ ID NO:17.

[0144] In some embodiments described in the first or second aspect, the amino acid sequence of the first antigen-binding region differs from the amino acid sequence shown in SEQ ID NO: 17 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions.

[0145] In some embodiments described in the first or second aspect, the amino acid sequence of the first antigen-binding region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology with the amino acid sequence shown in SEQ ID NO: 17.

[0146] In some embodiments described in the first or second aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 17 can also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids while still maintaining a similar function of the first antigen-binding region.

[0147] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can also be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO: 17, and the resulting amino acid sequence still maintains a similar function of the first antigen-binding region.

[0148] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids can also be added or deleted from a region other than the C-terminal or N-terminal of the amino acid sequence shown in SEQ ID NO: 17, as long as the altered amino acid sequence substantially maintains a similar function of the first antigen-binding region.

[0149] In some embodiments described in the first or second aspect, the bispecific antibody comprises a first antigen-binding region that binds to Trop2 and a second antigen-binding region that binds to the CD16A molecule, wherein the second antigen-binding region comprises the amino acid sequence shown in SEQ ID NO: 18.

[0150] In some embodiments described in the first or second aspect, the amino acid sequence of the second antigen-binding region differs from the amino acid sequence shown in SEQ ID NO: 18 by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions.

[0151] In some embodiments described in the first or second aspect, the amino acid sequence of the second antigen-binding region has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homology with the amino acid sequence shown in SEQ ID NO:18.

[0152] In some embodiments described in the first or second aspect, the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:18 may also be truncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids, while still maintaining the function of the second antigen-binding region similar thereto.

[0153] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added to the C-terminal or N-terminal region of the amino acid sequence shown in SEQ ID NO:18, and the resulting amino acid sequence still maintains the function of the second antigen-binding region similar thereto.

[0154] In some embodiments described in the first or second aspect, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or more amino acids may also be added or deleted in a region other than the C-terminal or N-terminal of the amino acid sequence shown in SEQ ID NO:18, as long as the altered amino acid sequence substantially maintains the function of the second antigen-binding region similar thereto.

[0155] In some embodiments described in the first or second aspect, the bispecific antibody comprises a first antigen-binding region having an amino acid sequence as shown in SEQ ID NO:17 and a second antigen-binding region having an amino acid sequence as shown in SEQ ID NO:18.

[0156] In some embodiments described in the first or second aspect, the amino acid sequence of the bispecific antibody is as shown in SEQ ID NO:23.

[0157] In some embodiments described in the first or second aspect, a purification tag, such as a histidine tag HHHHHH (SEQ ID NO:22), is linked to the N-terminus of the bispecific antibody. In some embodiments, the amino acid sequence of the bispecific antibody is as shown in SEQ ID NO:24.

[0158] In a third aspect, the present application provides a pharmaceutical composition comprising the bispecific antibody-NK cell conjugate described in the first aspect or the bispecific antibody described in the second aspect, and a pharmaceutically acceptable carrier.

[0159] When conditions permit, the pharmaceutical composition can be directly provided to the patient in the form of a sterile fresh preparation. If long-term storage (e.g., for multiple administrations) or transportation is required, the pharmaceutical composition can be provided in the form of a sterile frozen preparation and thawed and resuscitated before use.

[0160] In some embodiments of the third aspect, the pharmaceutical composition comprises sodium chloride and / or human serum albumin.

[0161] In some embodiments of the third aspect, the pharmaceutical composition comprises trehalose, sucrose, dextran, DMSO, or any combination thereof.

[0162] In some embodiments of the third aspect, the pharmaceutical composition is used for preventing or treating tumors in an individual.

[0163] In some embodiments of the third aspect, the tumor is a tumor with high Trop2 expression (Trop2 + ). In some embodiments, a tumor with high Trop2 expression (Trop2 + ) means that at least 60% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor with high Trop2 expression (Trop2 + ) means that at least 70% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor with high Trop2 expression (Trop2 + ) means that at least 80% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor with high Trop2 expression (Trop2 + ) means that at least 90% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor with high Trop2 expression (Trop2 + ) means that at least 95% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor with high Trop2 expression (Trop2 + ) means that at least 98% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor with high Trop2 expression (Trop2 + ) means that at least 99% of the tumor cells in the tumor cell population express Trop2.

[0164] In some embodiments of the third aspect, the tumor is a solid tumor.

[0165] In some embodiments of the third aspect, the tumor is a hematological tumor.

[0166] In some embodiments of the third aspect, the tumor is a malignant tumor.

[0167] In some embodiments of the third aspect, the tumor is cancer.

[0168] In some embodiments of the third aspect, the tumor is selected from: lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma or squamous cell carcinoma of the lung), breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, cervical cancer, endometrial or uterine cancer, kidney cancer, bladder cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, salivary gland cancer, squamous cell carcinoma (e.g., squamous epithelial cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, B-cell lymphoma, brain cancer and head and neck cancer, and metastases of the above cancers.

[0169] In a fourth aspect, the present application provides the use of the bispecific antibody-NK cell conjugate described in the first aspect, the bispecific antibody described in the second aspect, or the pharmaceutical composition described in the third aspect in the preparation of a drug for preventing or treating a tumor in an individual.

[0170] In some embodiments of the fourth aspect, the tumor is a tumor in which tumor cells highly express Trop2 (Trop2 + ). In some embodiments, a tumor in which tumor cells highly express Trop2 (Trop2 + ) means that at least 60% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor in which tumor cells highly express Trop2 (Trop2 + ) means that at least 70% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor in which tumor cells highly express Trop2 (Trop2 + ) means that at least 80% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor in which tumor cells highly express Trop2 (Trop2 + ) means that at least 90% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor in which tumor cells highly express Trop2 (Trop2 + ) means that at least 95% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor in which tumor cells highly express Trop2 (Trop2 + ) means that at least 98% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor in which tumor cells highly express Trop2 (Trop2 + ) means that at least 99% of the tumor cells in the tumor cell population express Trop2.

[0171] In some embodiments of the fourth aspect, the tumor is a solid tumor.

[0172] In some embodiments of the fourth aspect, the tumor is a hematological tumor.

[0173] In some embodiments of the fourth aspect, the tumor is a malignant tumor.

[0174] In some embodiments of the fourth aspect, the tumor is cancer.

[0175] In some embodiments of the fourth aspect, the tumor is selected from: lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma or squamous cell carcinoma of the lung), breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, cervical cancer, endometrial or uterine cancer, kidney cancer, bladder cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, salivary gland cancer, squamous cell carcinoma (e.g., squamous epithelial cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, B-cell lymphoma, brain cancer, and head and neck cancer, and metastases of the above cancers.

[0176] In a fifth aspect, the present application provides a method for preventing or treating a tumor in an individual, which comprises administering to an individual in need an effective amount of the bispecific antibody-NK cell conjugate described in the first aspect, the bispecific antibody described in the second aspect, or the pharmaceutical composition described in the third aspect.

[0177] In some embodiments of the fifth aspect, the tumor is a tumor in which tumor cells highly express Trop2 (Trop2 + ). In some embodiments, a tumor in which tumor cells highly express Trop2 (Trop2 + ) means that at least 60% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor in which tumor cells highly express Trop2 (Trop2 + ) means that at least 70% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor in which tumor cells highly express Trop2 (Trop2 + ) means that at least 80% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor in which tumor cells highly express Trop2 (Trop2 + ) means that at least 90% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor in which tumor cells highly express Trop2 (Trop2 + ) means that at least 95% of the tumor cells in the tumor cell population express Trop2. In some embodiments, a tumor in which tumor cells highly express Trop2 (Trop2 +The tumor of ) refers to a tumor in which at least 98% of the tumor cells in the tumor cell population express Trop2. In some embodiments, the tumor cells highly express Trop2 (Trop2 + ) The tumor of ) refers to a tumor in which at least 99% of the tumor cells in the tumor cell population express Trop2.

[0178] In some embodiments of the fifth aspect, the tumor is a solid tumor.

[0179] In some embodiments of the fifth aspect, the tumor is a hematological tumor.

[0180] In some embodiments of the fifth aspect, the tumor is a malignant tumor.

[0181] In some embodiments of the fifth aspect, the tumor is cancer.

[0182] In some embodiments of the fifth aspect, the tumor is selected from: lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma or squamous cell carcinoma of the lung), breast cancer, pancreatic cancer, gastric cancer, ovarian cancer, cervical cancer, endometrial or uterine cancer, kidney cancer, bladder cancer, colon cancer, liver cancer, prostate cancer, peritoneal cancer, hepatocellular carcinoma, glioblastoma, urinary tract cancer, rectal cancer, salivary gland cancer, squamous cell carcinoma (e.g., squamous epithelial cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, B-cell lymphoma, brain cancer and head and neck cancer, and metastases of the above cancers.

[0183] The present application also provides a nucleic acid molecule encoding the bispecific antibody described in the second aspect, a vector comprising the nucleic acid molecule, and a host cell comprising the nucleic acid molecule or the vector. In other aspects, the present application also provides a method for producing the bispecific antibody described in the second aspect. In some embodiments, the method for producing the bispecific antibody described in the second aspect includes culturing a host cell to express the nucleic acid molecule. In some embodiments, the method for producing the bispecific antibody described in the second aspect further includes recovering the bispecific antibody from the host cell culture medium.

[0184] It should be understood that the above detailed description is only to make those skilled in the art more clearly understand the content of the present application, and is not intended to limit in any way. Those skilled in the art can make various changes and modifications to the embodiments.

[0185] The following examples are for illustrative purposes only and are not intended to limit the scope of the present application. Examples

[0186] Example 1: Preparation of Bispecific Antibody

[0187] 1.1 Gene Design and Cloning

[0188] Gene synthesis: According to the determined antibody fragment sequence, the corresponding gene was synthesized. The amino acid sequence of the bispecific antibody against Trop2 and CD16A (CD16A VL1 - linker 1 - CD16A VH1 - linker 2 - Trop2 VL2 - linker 3 - Trop2 VH2 - His) is as follows: SYVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGAIEPMYGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSSGGGGSMKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVPTFGGGTKLEIKGGSGGSGGSGGSGGSMGGWSWIFLFLLSETAGVLSEVHLQQSGPELVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGYIYPNNGGTGYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAREGLWDYFGGSFDYWGQGTTLTVSSHHHHHH (SEQ ID NO:24).

[0189] Constructing the expression vector: The synthesized gene was inserted into the pET22b plasmid.

[0190] 1.2 Amplification and culture of the bacterial strain

[0191] (1) Take 100 μL of the bacterial strain BL21(DE3) / pET22b - Trop2 - CD16 and add it to 10 mL of LB medium (containing kanamycin). Culture at 37 °C and 150 rpm for 6 - 7 h.

[0192] (2) Add the bacterial liquid obtained in step (1) to 100 mL of LB medium (containing kanamycin). Culture overnight at 37 °C and 150 rpm.

[0193] (3) Add the bacterial solution obtained in step (2) to 2 L of LB medium (containing kanamycin), culture at 37 °C and 150 rpm for 6 - 7 h, then cool down to 32 °C, and at the same time add IPTG to a final concentration of 0.2 mmol / L, continue to culture for 15 h, and centrifuge to collect the supernatant.

[0194] 1.3 The collected supernatant after centrifugation was ultrafiltered and concentrated to 250 mL using a membrane package with a molecular weight cut-off of 10 kDa.

[0195] 1.4 Purification

[0196] 1.4.1 Purification by Ni column

[0197] Column volume: 10 mL

[0198] Flow rate: 3 mL / min

[0199] Equilibration buffer: 20 mM PB, pH 7.0

[0200] Elution buffer: 20 mM PB, 0.5 M NaCl, 0.5 M imidazole, pH 7.0

[0201] Purification method: After loading the sample, rinse with the equilibration buffer until UV280 reaches the baseline, and elute in one step with the elution buffer. Adjust the eluate to pH 5.0.

[0202] 1.4.2 Purification by SPFF

[0203] Column volume: 15 mL

[0204] Flow rate: 3 mL / min

[0205] Equilibration buffer: 50 mM PB, pH 5.0

[0206] Elution buffer: 50 mM PB, 1 M NaCl, pH 5.0

[0207] Purification method: Dilute the eluate from Ni column purification 10-fold with the equilibration buffer and load the sample. Rinse with the equilibration buffer until UV280 reaches the baseline, and elute in one step with the elution buffer;

[0208] Exchange the buffer and concentrate to 1 / 10 of the initial volume using an ultrafiltration tube with a molecular weight cut-off of 10 kDa.

[0209] Example 2: Preparation of bispecific antibody-NK cell conjugate

[0210] 2.1 NK cell culture

[0211] 2.1.1 Isolation of human peripheral blood mononuclear cells (PBMC)

[0212] Collect blood (50 mL each) from healthy volunteers. All volunteers have signed the informed consent form. The inclusion criteria for volunteers are as follows:

[0213] I. Basic criteria:

[0214] 1. Aged 18 - 45 years old, gender is not restricted.

[0215] 2. Without severe chronic diseases or active infections.

[0216] 3. Without a history of malignant tumors.

[0217] 4. Without a history of long - term or recent use of immunosuppressants.

[0218] 5. Without a history of drug, alcohol, or other substance abuse.

[0219] 6. Not pregnant or lactating women.

[0220] II. Hematological criteria:

[0221] 1. Hemoglobin, white blood cell count, and platelet count are within the normal range.

[0222] 2. Liver function and renal function tests are normal.

[0223] III. Immunological criteria:

[0224] 1. NK cell activity is normal.

[0225] 2. Without known immunodeficiency or autoimmune diseases.

[0226] IV. Infectious disease screening:

[0227] 1. Viral screening for HIV, HBV, HCV, HTLV - 1 / 2, CMV, etc. is negative.

[0228] 2. Screening for infectious diseases such as syphilis and tuberculosis is negative.

[0229] V. Family history / genetic history:

[0230] 1. Without familial hereditary diseases with aggregation, such as hemophilia, cystic fibrosis, etc.

[0231] 2. Without a history of immediate family members (parents, siblings) having malignant tumors at an early age.

[0232] 3. Without clear hereditary diseases or gene mutations related to the research.

[0233] (1) Transfer peripheral blood: Use a pipette to transfer the donor's peripheral blood into a 50 - mL centrifuge tube.

[0234] (2) Centrifugation: Balance the centrifuge tubes, centrifuge at 800 g, with an acceleration rate of 6 and a deceleration rate of 6 for 15 min.

[0235] (3) Transfer of plasma: Transfer the upper-layer plasma obtained by centrifugation into a 50 mL centrifuge tube and store it at 4°C.

[0236] (4) Dilution: Dilute the peripheral blood with sodium chloride injection at a volume ratio of 2:1 and mix well.

[0237] (5) Layer addition and sample loading: Add 15 mL of separation liquid to each 50 mL centrifuge tube. The volume ratio of the diluted blood to the separation liquid is 2:1. Centrifuge at 800 g, with an acceleration rate of 6 and a deceleration rate of 3 for 20 min.

[0238] (6) Extraction of mononuclear cells: After centrifugation, there is an obvious layering in the centrifuge tube. Aspirate the cell suspension in the mononuclear cell layer and transfer it into a 50 mL centrifuge tube.

[0239] (7) Washing: Resuspend with sodium chloride injection in a 50 mL centrifuge tube, centrifuge at 300 g, with an acceleration rate of 9 and a deceleration rate of 7 for 10 min, and discard the supernatant after centrifugation.

[0240] (8) Re-washing: Resuspend with sodium chloride injection in a 50 mL centrifuge tube, mix well and count (reserve 1 mL for counting before culture), centrifuge at 400 g, with an acceleration rate of 6 and a deceleration rate of 6 for 10 min. Discard the supernatant after centrifugation. The precipitate is the mononuclear cells, which are collected for standby.

[0241] 2.1.2 Magnetic bead purification of NK cells in PBMC:

[0242] (1) Resuspend the mononuclear cell precipitate obtained in Section 2.1.1 with separation liquid to 1×10 8 cells / mL.

[0243] (2) Add 100 μL of CD56 + sorting magnetic beads to each milliliter of the resuspended liquid and incubate in a horizontal shaker at 2 - 8°C for 15 min.

[0244] (3) Resuspend with sodium chloride injection in a 50 mL centrifuge tube, centrifuge at 300 g, with an acceleration rate of 9 and a deceleration rate of 7 for 10 min, and discard the supernatant after centrifugation.

[0245] (4) Add 500 μL of separation liquid to resuspend the cells for every 1×10 8 cells.

[0246] (5) Add the cell suspension into the separation column placed on the magnetic bracket and discard the liquid flowing through the separation column.

[0247] (6) Wash the separation column 3 times with 30 mL of separation liquid.

[0248] (7) Add 20 mL of eluent to the separation column and elute the purified CD56 + NK cells.

[0249] 2.1.3 Inoculation and culture of NK cells:

[0250] (1) Cell inoculation: Inoculate the purified NK cells into a 75 cm 2 culture flask at an inoculation concentration of approximately 1×10 6 cells / mL. Aspirate and discard the PBS with a pipette, and add 50 mL of complete culture medium (GT551 culture medium containing 500 U / mL of IL-2, 800 U / mL of IL-15, 400 U / mL of IL-21, and 5% autologous plasma), and culture in a 37°C, saturated humidity, 5% CO2 incubator;

[0251] (2) Cell transfer: The cells can be passaged after 3 - 5 days of culture. Observe whether the color of the culture medium turns yellow, and passage when the cell growth state is good under the microscope and the cell concentration ≥ 3×10 6 cells / mL. Transfer the cells, culture medium, and plasma in the original culture flask to a cell culture bag together, and supplement 250 mL of complete culture medium. At the same time, add 50 mL of complete culture medium to the original culture flask and continue to culture, and transfer to the culture bag again the next day. Mark information such as cell coding and culture time on the culture bag, and culture in a 37°C, saturated humidity, 5% CO2 incubator.

[0252] (3) Supplement of culture medium: After cell transfer, observe the color change of the cell suspension. If the cell suspension turns significantly yellow, replenish the liquid with complete culture medium, and the final volume ≤ 1800 mL, and culture in a 37°C, saturated humidity, 5% CO2 incubator.

[0253] (4) Cell counting: Three days before the cells are filled and released, draw 20 mL of culture medium from the cell culture bag with a disposable syringe and send it to the quality control center for blood culture, cell counting, and detection of bacteria, endotoxin, mycoplasma, Gram staining, and cell surface antibodies. After the detection is qualified, the technician notifies the cell release planner to arrange the cell release time.

[0254] 2.2 Conjugation of NK cells with bispecific antibodies:

[0255] (1) Cell collection: Transfer the cell suspension in the culture bag into a 250 mL centrifuge tube, centrifuge at 300 g, acceleration 9, deceleration 7, for 10 min, and discard the supernatant; Collect the cell suspension into a 50 mL centrifuge tube and resuspend it with sodium chloride injection to 45 mL, centrifuge at 300 g, acceleration 9, deceleration 7, for 10 min, and discard the supernatant.

[0256] (2) Bispecific antibody conjugation: Resuspend the injection of sodium chloride in a 50 mL centrifuge tube to 45 mL, add 500 μL of the recombinant anti-Trop2 and CD16A bispecific antibody, and incubate at 4 °C for 30 min in a horizontal shaker; centrifuge at 300 g, acceleration speed 9, deceleration speed 7, for 10 min, and discard the supernatant. Repeat once. Transfer the supernatant to a 50 mL centrifuge tube before encapsulation and store at 4 °C for 48 h for future reference.

[0257] (3) Cell resuspension: Add the injection of sodium chloride to the cells obtained in the previous step, pipette to suspend the cells, and filter through a 100 μm sterile cell filter. According to the cell count before distribution, aliquot into 100 mL transfer bags at 5 × 10 9 cells / 100 mL / bag, and reserve 2 mL for partial detection before aliquoting.

[0258] 2.3 Identification of NK cell phenotype and conjugation efficiency

[0259] Take 4 flow cytometry tubes and number them sequentially as ①, ②, ③, ④. Add 100 - 200 μL of well-mixed cell suspension (cell count, 0.5 - 1 × 10 6 cells / tube), add 10 μL of isotype IgG1-FITC and IgG1-PE antibodies to tube ①, and add 10 μL of CD3-FITC antibody to each of tubes ②, ③, and ④; add 10 μL of CD16-PE antibody to tube ②; add 10 μL of CD56-APC antibody to tube ③; add 10 μL of Trop2-Cy5.5 recombinant protein to tube ④, gently shake to mix, and incubate at room temperature in the dark for 30 min. After incubation, centrifuge at 300 g for 5 min and discard the supernatant. Add 1 mL of PBS buffer to each tube, vortex to mix, centrifuge at 300 g for 5 min, and discard the supernatant. Add 1 mL of PBS buffer to each tube and detect on the machine within 1 hour at 4 °C in the dark.

[0260] Figure 1 The results show that the phenotype of NK cells is CD3 - / CD16 + CD56 + , and the purity of NK cells is 96.59%. Figure 2 The results show that the conjugation efficiency of the isotype control antibody with NK cells is 1.17%, and the conjugation efficiency of the anti-CD16A and Trop2 bispecific antibody with NK cells is 92.36%.

[0261] Example 3: In vitro killing verification of bispecific antibody-NK cell conjugate

[0262] 3.1 Cell seeding

[0263] In a 96-well plate, according to the preset effector-to-target ratio, add the corresponding amounts of bispecific antibody-NK cell conjugates or NK cells and MCF7 cells respectively. Set at least 3 replicates for each effector-to-target ratio to reduce errors. At the same time, set up control wells containing only NK cells, control wells containing only MCF7 cells, and blank medium control wells.

[0264] Gently shake the 96-well plate to evenly distribute the cells.

[0265] 3.2 Co-culture

[0266] Place the 96-well plate in an incubator at 37 °C and 5% CO2, and co-culture NK cells and MCF7 cells for 4 h.

[0267] 3.3 CCK8 assay

[0268] After co-culture, add 10 μL of CCK8 reagent to each well.

[0269] Continue to place the 96-well plate in the incubator for 2 h to allow the CCK8 reagent to fully react with the cells.

[0270] Use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance (OD value) at a wavelength of 450 nm. Record the OD value of each well.

[0271] Figure 3 The results show that the bispecific antibody-NK cell conjugate has stronger cell killing efficiency compared to NK cells.

[0272] Example 4: ADCC effect mediated by bispecific antibody

[0273] 4.1 Cell culture and preparation

[0274] Culture MCF7 target cells to the logarithmic growth phase and inoculate them into a 96-well plate at an inoculation concentration of approximately 1×10 6 cells / mL.

[0275] At the same time, prepare Jurkat effector cells stably expressing the NFAT luciferase reporter gene and FcγRIIIa and culture them to an appropriate density as well.

[0276] 4.2 Preparation of luciferase substrate

[0277] According to the instructions of the luciferase detection kit, prepare the luciferase substrate solution D-luciferin at a concentration of 1 mM.

[0278] 4.3 Binding of bispecific antibody to target cells

[0279] In a 96-well plate containing MCF7 target cells, add the bispecific antibody (final concentration 10 μg / mL), and set up a vehicle control and an isotype IgG antibody control. Each treatment is set up with three replicates to reduce experimental error, and incubate at 37 °C and 5% CO2 for 60 min.

[0280] 4.4 Co-culture of effector cells and target cells

[0281] Mix the target cells conjugated with the bispecific antibody with Jurkat effector cells at a certain ratio. The effector-to-target ratios are 5:1, 1:1, and 1:5, and co-culture at 37 °C and 5% CO2 for 6 h.

[0282] 4.5 Detection of luciferase activity

[0283] After the co-culture is completed, add 100 μL of the luciferase substrate D-luciferin solution.

[0284] Use a multifunctional microplate reader to detect the fluorescence signal and record the relative light units (RLU).

[0285] Figure 4 The results show that the bispecific antibody against CD16A and Trop2 can effectively mediate the ADCC effect.

[0286] Example 5: Tumor inhibition verification of bispecific antibody-NK cell conjugate in an animal model

[0287] 5.1 Experimental preparation:

[0288] Experimental animals: Select NOD / SCID mice. Because of their immunodeficient characteristics, they are suitable for xenograft tumor model experiments.

[0289] Cell preparation: Human breast cancer cell line MCF7, for tumor-bearing; NK cells and bispecific antibody-NK cell conjugates, which are amplified and induced to activate in vitro, for tail vein injection.

[0290] Experimental grouping: The experiment is divided into three groups, with 10 mice in each group, namely the PBS injection group, the NK cell injection group, and the bispecific antibody-NK cell conjugate injection group.

[0291] 5.2 Establishment of tumor-bearing model

[0292] MCF7 cell passage and amplification: Culture and amplify MCF7 cells in vitro to ensure good cell status.

[0293] Cell inoculation: Collect MCF7 cells in the logarithmic growth phase, adjust the cell density, and inoculate subcutaneously into the groin of NOD / SCID mice at 2.5×10 8 cells / kg body weight, and the injection volume is 1 ml.

[0294] Observation of tumor formation: After inoculation, the status of the mice and the growth of the tumors were observed regularly, the changes in tumor volume were recorded, and the major axis length of the tumor was about 1 cm after 7 days.

[0295] 5.3 Administration and evaluation of bispecific antibody-NK cell conjugate

[0296] Intravenous injection via the tail vein: On the 8th day after the mice were inoculated with tumors, PBS, NK cells, and bispecific antibody-NK cell conjugate were respectively administered according to the grouping situation. The dosage of cells administered was 1×10 9 cells / kg body weight.

[0297] Observation and evaluation: The mental state, activity, diet, body weight, tumor size, and survival of the mice were observed regularly.

[0298] Figure 5 The results showed that: compared with NK cells, the bispecific antibody-NK cell conjugate could effectively inhibit the tumor growth in mice and improve the survival rate of mice.

[0299] Example 6: Therapeutic effect of exploratory clinical study of bispecific antibody-NK cell conjugate

[0300] Patient: 54 years old, male

[0301] Admission examination: Chest CT showed: space-occupying in the right lung, bilateral pleural effusion. The patient had a long-term smoking history and could have cough manifestations. Imaging examinations showed that the masses or nodules mostly had lobulation, notch, and spiculation, and often had pleural traction depression manifestations, and could be accompanied by mediastinal and hilar lymphadenectasis. The patient was diagnosed with stage IV non-small cell lung cancer. Through genetic testing, the targetable driver gene mutation was negative. After multiple chemotherapy treatments, drug resistance progressed.

[0302] Figure 6 The results showed that: the therapeutic effect evaluation after 4 cycles of treatment with bispecific antibody-NK cell conjugate was partial remission (PR).

[0303] All publications and patent documents cited in this specification are incorporated herein by reference as if each publication or patent was specifically and individually indicated to be incorporated herein by reference. Without departing from the true spirit and scope of the disclosure of the present application, various changes can be made to the embodiments disclosed in the present application and equivalents can be substituted. Unless otherwise specified in the context, any feature, step, or embodiment of the embodiments of the present disclosure can be combined with any other feature, step, or embodiment.

[0304] Sequence information

[0305] SEQ ID NO:1

[0306] DYYMN

[0307] SEQ ID NO:2

[0308] YIYPNNGGTGYNQKFKG

[0309] SEQ ID NO:3

[0310] EGLWDYFGGSFDY

[0311] SEQ ID NO:4

[0312] RSSQSLVHSNGNTYLH

[0313] SEQ ID NO:5

[0314] KVSNRF

[0315] SEQ ID NO:6

[0316] SQSTHVPT

[0317] SEQ ID NO:7

[0318] SYYMH

[0319] SEQ ID NO:8

[0320] AIEPMYGSTSYAQKFQG

[0321] SEQ ID NO:9

[0322] GSAYYYDFADY

[0323] SEQ ID NO:10

[0324] GGHNIGSKNVH

[0325] SEQ ID NO:11

[0326] QDNKRPS

[0327] SEQ ID NO:12

[0328] QVWDNYSVL

[0329] SEQ ID NO:13

[0330] MGGWSWIFLFLLSETAGVLSEVHLQQSGPELVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGYIYPNNGGTGYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAREGLWDYFGGSFDYWGQGTTLTVSS

[0331] SEQ ID NO:14

[0332] MKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVPTFGGGTKLEIK

[0333] SEQ ID NO:15

[0334] EVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGAIEPMYGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSS

[0335] SEQ ID NO:16

[0336] SYVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVL

[0337] SEQ ID NO:17

[0338] MKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVPTFGGGTKLEIKGGSGGSGGSGGSGGSMGGWSWIFLFLLSETAGVLSEVHLQQSGPELVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGYIYPNNGGTGYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAREGLWDYFGGSFDYWGQGTTLTVSS

[0339] SEQ ID NO:18

[0340] SYVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGAIEPMYGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSS

[0341] SEQ ID NO:19

[0342] GGGGS

[0343] SEQ ID NO:20

[0344] GGGGSGGGGSGGGGS

[0345] SEQ ID NO:21

[0346] GGSGGSGGSGGSGGS

[0347] SEQ ID NO:22

[0348] HHHHHH

[0349] SEQ ID NO:23

[0350] SYVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGAIEPMYGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSSGGGGSMKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVPTFGGGTKLEIKGGSGGSGGSGGSGGSMGGWSWIFLFLLSETAGVLSEVHLQQSGPELVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGYIYPNNGGTGYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAREGLWDYFGGSFDYWGQGTTLTVSS

[0351] SEQ ID NO:24

[0352] SYVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGAIEPMYGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSSGGGGSMKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHVPTFGGGTKLEIKGGSGGSGGSGGSGGSMGGWSWIFLFLLSETAGVLSEVHLQQSGPELVKPGASVKMSCKASGYTFTDYYMNWVKQSHGKSLEWIGYIYPNNGGTGYNQKFKGKATLTVDKSSSTAYMELRSLTSEDSAVYYCAREGLWDYFGGSFDYWGQGTTLTVSSHHHHHH

Claims

1. A bispecific antibody-natural killer cell (NK cell) conjugate, wherein the bispecific antibody comprises a first antigen-binding region that binds to human trophoblast cell surface antigen 2 (Trop2) and a second antigen-binding region that binds to the CD16A molecule; the amino acid sequence of the first antigen-binding region is as shown in SEQ ID NO: 17 and the amino acid sequence of the second antigen-binding region is as shown in SEQ ID NO: 18; the bispecific antibody is conjugated to the NK cell by antigen-antibody binding of its second antigen-binding region to the CD16A molecule on the NK cell.

2. The bispecific antibody-NK cell conjugate according to claim 1, wherein the first antigen-binding region is linked to the second antigen-binding region by a peptide linker.

3. The bispecific antibody-NK cell conjugate according to claim 2, wherein the peptide linker is a GS-type flexible peptide linker.

4. The bispecific antibody-NK cell conjugate according to claim 2, wherein the peptide linker is (G4S)n or (G2S)n, where n is an integer from 1 to 10.

5. The bispecific antibody-NK cell conjugate according to claim 4, wherein n is an integer from 1 to 5.

6. The bispecific antibody-NK cell conjugate according to any one of claims 1-5, wherein the amino acid sequence of the bispecific antibody is as shown in SEQ ID NO: 23 or 24.

7. The bispecific antibody-NK cell conjugate according to any one of claims 1-5, wherein the NK cells are obtained from in vitro culture and expansion of NK cells derived from peripheral blood mononuclear cells (PBMC); the NK cells are obtained from in vitro culture and expansion of NK cells derived from umbilical cord blood; the NK cells are obtained from in vitro culture and expansion of an NK cell line; or the NK cells are obtained from in vitro induction, culture and expansion of induced pluripotent stem cells (iPSC) or mesenchymal stem cells.

8. The bispecific antibody-NK cell conjugate according to any one of claims 1-5, wherein the phenotype of the NK cells is CD3 - / CD16 + CD56 + .

9. A pharmaceutical composition comprising the bispecific antibody-NK cell conjugate according to any one of claims 1-8 and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition is provided in the form of a sterile fresh preparation or a sterile frozen preparation.

11. Use of the bispecific antibody-NK cell conjugate according to any one of claims 1-8 or the pharmaceutical composition according to any one of claims 9-10 in the preparation of a drug for preventing or treating tumors in an individual; wherein the tumors are selected from: lung cancer, breast cancer, and metastases of the above cancers.

12. The use according to claim 11, wherein the lung cancer is non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma or squamous cell carcinoma of the lung.

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