Bispecific antibody-NK cell conjugates binding CD16A and CD70 and uses thereof

Abstract

The present application provides a bispecific antibody-NK cell conjugate that binds to CD16A molecules and CD70 molecules, a bispecific antibody that binds to CD16A molecules and CD70 molecules, 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 biomedicine; specifically, the present application relates to a bispecific antibody-natural killer cell (NK cell) conjugate that binds to CD16A molecules and CD70 molecules, a bispecific antibody that binds to CD16A molecules and CD70 molecules, a pharmaceutical composition comprising the bispecific antibody-NK cell conjugate or the bispecific antibody, 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 an immunoreceptor tyrosine-based activation motif (ITAM)-carrying protein. It is mainly expressed on NK cells, macrophages, monocytes, and certain T cell subsets. One of the main functions of CD16A is to mediate antibody-dependent cellular cytotoxicity (ADCC) through low-affinity interaction with the Fc segment of human immunoglobulin G (IgG). In this process, CD16A can recognize and bind to the Fc segment of IgG bound to tumor cell-associated antigens, thereby triggering the activation of NK cells or other immune cells expressing CD16A, leading to the killing of tumor cells.

[0003] The CD70 molecule is a type II transmembrane protein belonging to the tumor necrosis factor receptor (TNFR) superfamily, also known as TNFRSF7. Under normal physiological conditions, CD70 is primarily expressed on activated T cells, B cells, and mature dendritic cells. It interacts with its receptor CD27, regulating the activation, proliferation, and differentiation of T and B cells, thus playing a crucial role in the immune response.

[0004] However, under pathological conditions, such as tumor development, the expression pattern of CD70 changes. It is highly expressed in a variety of tumor cells, including solid tumors such as lung cancer and renal cell carcinoma, as well as certain hematological malignancies. In renal clear cell carcinoma, the expression rate of CD70 is as high as over 90%, making it a specific marker for this type of cancer. The high expression of CD70 on the surface of tumor cells may be related to their use of this molecule to control tumor-infiltrating lymphocytes expressing CD27, thereby achieving immune evasion. This mechanism suggests that CD70 may play a key role in the occurrence and progression of tumors.

[0005] In terms of antibody drugs, for example, Cusatuzumab (ARGX-110) is a monoclonal antibody targeting CD70 that has demonstrated certain efficacy in clinical trials. It can bind to CD70 and produce multiple anti-cancer effects, including blocking CD70-CD27 signal transduction, inhibiting tumor cell proliferation, and killing CD70-positive cancer cells through complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC).

[0006] In terms of ADC drugs, SGN-75 is a humanized CD70 antibody-drug conjugate that has shown an inhibitory effect on renal cancer cell proliferation in vitro and prolonged mouse survival in animal models. However, the drug's efficacy and safety in clinical practice require further verification.

[0007] Antibody therapy and cell therapy have become hot topics in the field of cancer treatment, and related exploration and research are also urgently needed in this field. SUMMARY OF THE INVENTION

[0008] In a first aspect, the present application provides a bispecific antibody-natural killer cell (NK cell) conjugate, wherein

[0009] The bispecific antibody comprises a first antigen-binding region that binds to a CD70 molecule and a second antigen-binding region that binds to a CD16A molecule;

[0010] The bispecific antibody is coupled to the NK cell via antigen-antibody binding between its second antigen-binding region and the CD16A molecule on the NK cell.

[0011] In some embodiments of the first aspect, the NK cells are obtained from in vitro culture and expansion of peripheral blood mononuclear cell (PBMC)-derived NK cells;

[0012] The NK cells are obtained from the in vitro culture and expansion of NK cells derived from umbilical cord blood;

[0013] The NK cells are obtained from the in vitro culture and expansion of NK cell lines; or

[0014] The NK cells are obtained from in vitro induction, culture and expansion of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (ESCs).

[0015] In a second aspect, the present application provides a bispecific antibody comprising a first antigen-binding region that binds to a CD70 molecule and a second antigen-binding region that binds to a CD16A molecule.

[0016] In some embodiments of the first or second aspect, the first antigen binding region comprises:

[0017] HCDR1 as shown in SEQ ID NO: 1,

[0018] HCDR2 as shown in SEQ ID NO: 2,

[0019] HCDR3 as shown in SEQ ID NO: 3,

[0020] LCDR1 as shown in SEQ ID NO: 4,

[0021] LCDR2 as shown in SEQ ID NO: 5, and

[0022] LCDR3 as shown in SEQ ID NO: 6; and / or

[0023] The second antigen binding region comprises:

[0024] HCDR1 as shown in SEQ ID NO: 7,

[0025] HCDR2 as shown in SEQ ID NO: 8,

[0026] HCDR3 as shown in SEQ ID NO:9,

[0027] LCDR1 as shown in SEQ ID NO: 10,

[0028] LCDR2 as shown in SEQ ID NO: 11, and

[0029] LCDR3 as shown in SEQ ID NO: 12;

[0030] The amino acid sequence of HCDR is defined according to Kabat.

[0031] 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 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical 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 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: The sequence shown in NO:14 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.

[0032] 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 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical 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 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence shown in SEQ ID NO: The sequence shown in NO:16 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.

[0033] 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 a scFv.

[0034] In some embodiments of the first or second aspect, the first antigen binding region and the second antigen binding region are connected by a peptide linker.

[0035] 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 and the light chain variable region are connected by a peptide linker.

[0036] 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 and the light chain variable region are connected by a peptide linker.

[0037] In some embodiments of the first or second aspect, the bispecific antibody comprises a first antigen-binding region with an amino acid sequence as shown in SEQ ID NO: 17 and a second antigen-binding region with an amino acid sequence as shown in SEQ ID NO: 18.

[0038] 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;

[0039] Optionally, the pharmaceutical composition is provided in the form of a sterile fresh preparation or a sterile frozen preparation.

[0040] In a fourth aspect, the present application provides 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 medicament for preventing or treating tumors in an individual. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The purity results after NK cell expansion are shown, where LYM is the abbreviation for lymphocyte.

[0042] Figure 2 The diagram shows the coupling efficiency of bispecific antibodies to NK cells; the left graph represents the coupling efficiency of isotype control antibodies to NK cells, and the right graph represents the coupling efficiency of anti-CD16A and CD70 bispecific antibodies to NK cells.

[0043] Figure 3 The results show the in vitro killing of 786-O cells by the bispecific antibody-NK cell conjugate.

[0044] Figure 4 The results of the ADCC effect mediated by the bispecific antibody are shown.

[0045] Figure 5 The results of the mouse tumor-bearing efficacy experiment of the bispecific antibody-NK cell conjugate are shown.

[0046] Sequence Description

[0047] SEQ ID NOs: 1-6 respectively show the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 of the first antigen-binding region of the CD70 molecule.

[0048] SEQ ID NOs: 7-12 respectively 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.

[0049] SEQ ID NOs: 13-14 respectively 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 the CD70 molecule.

[0050] SEQ ID NOs: 15-16 respectively 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.

[0051] SEQ ID NOs: 17-18 respectively show the amino acid sequences of the first antigen-binding region that binds to the CD70 molecule and the second antigen-binding region that binds to the CD16A molecule.

[0052] SEQ ID NO: 19 shows the amino acid sequence of peptide linker 2.

[0053] SEQ ID NO: 20 shows the amino acid sequence of peptide linker 1.

[0054] SEQ ID NO: 21 shows the amino acid sequence of peptide linker 3.

[0055] SEQ ID NO: 22 shows the amino acid sequence of the histidine purification tag H6.

[0056] SEQ ID NO: 23 shows the amino acid sequence of a bispecific antibody against CD70 and CD16A molecules.

[0057] SEQ ID NO: 24 shows the amino acid sequence of a bispecific antibody against CD70 and CD16A molecules comprising a histidine purification tag H6. Detailed Description of the Invention

[0058] The practice of the present application employs, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, and immunology within the skill of the art.

[0059] Unless otherwise specified, the terms used in this application have the meanings commonly understood by those skilled in the art.

[0060] definition

[0061] 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, livestock, or experimental animals (e.g., gorillas, monkeys, rats, mice, rabbits, guinea pigs, woodchucks, ground squirrels, etc.).

[0062] 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.

[0063] The term "antibody" as used herein refers to an immunoglobulin molecule that can specifically bind 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, and the like. "Antibodies" as used herein include not only complete (i.e., full-length) antibodies, but also binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), variants thereof, fusion proteins comprising 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 comprising antigen recognition sites of desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0064] Typically, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and the first, second, and third constant regions (CH1, CH2, and CH3). Each light chain contains a light chain variable region (VL) and a constant region (CL). A full-length antibody can be of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), but antibodies need not belong to any particular class. Immunoglobulins can be assigned to different classes based on the antibody amino acid sequence of the constant region of the heavy chain. Generally, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of the different classes of immunoglobulins are well known.

[0065] As used herein, the term "bispecific antibody" refers to an antibody that has the ability to bind to two antigenic epitopes simultaneously. The two antigenic epitopes can be on different antigens or on the same antigen. Bispecific antibodies can have a variety of structural configurations. For example, a bispecific antibody can be obtained by connecting two scFv fragments via a linker.

[0066] The antigen binding region may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of VH and VL typically contains three complementarity determining regions: CDR1, CDR2, and CDR3.

[0067] 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 an antibody. There are two common definitions of VH or VL CDR sequences: 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 sequence 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 CDR sequences are defined using Kabat.

[0068] For a given antibody variable region sequence, the CDR region sequence in the variable region sequence can be analyzed in a variety of ways, for example, it can be determined using the online software Abysis (http: / / www.abysis.org / ).

[0069] For general antibodies, examples of antigen-binding regions include, but are not limited to: (1) a Fab fragment, which can be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) a F(ab')2 fragment, which can be a bivalent fragment having two Fab' fragments connected by a disulfide bridge in the hinge region (i.e., a dimer of Fab'); (3) an Fv fragment having the VL and VH domains of a single arm of an antibody; (4) a single-chain Fv (scFv), which can be a single polypeptide chain consisting of a VH domain and a VL domain via a peptide connector; (5) (scFv)2, which can comprise two VH domains connected by a peptide connector and two VL domains, the two VL domains being combined with the two VH domains via a disulfide bridge; and (6) single-domain antibody formats.

[0070] In bispecific antibody construction, the "antigen binding region" includes, but is not limited to, single chain antibody (scFv) format, Fab fragment format and / or single domain antibody format.

[0071] As used herein, the term "single-chain antibody (scFv)" refers to an antibody with a single-chain structure, typically constructed using genetic engineering techniques, comprising a single polypeptide chain consisting of a heavy chain variable region (VH) and a light chain variable region (VL). A flexible linker is typically designed between the heavy and light chain variable regions to facilitate folding of the heavy and light chain variable regions into the correct conformation for antigen binding.

[0072] 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.

[0073] 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 is combined together to achieve a specific purpose. In certain embodiments, the pharmaceutical composition includes a combination separated in time and / or space, as long as it can work together to achieve the purpose of this application. For example, the ingredients contained in the pharmaceutical composition (such as the antibody-cell conjugate according to the present application) can be applied to an individual as a whole, or separately. When the ingredients contained in the pharmaceutical composition are applied separately to an individual, the ingredients can be applied to the individual simultaneously or sequentially. The pharmaceutical composition according to the present application can include conventional components for cell culture, especially NK cell culture, to maintain the activity of NK cells in the conjugate. Pharmaceutically acceptable carriers 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 appropriate route, such as intravenous administration, intradermal, subcutaneous, intramuscular injection, etc. The composition according to the present application may contain a wetting agent, an emulsifier or a buffer substance as an additive.

[0074] As used herein, the term "therapeutically effective amount" or "effective amount" refers to a dose sufficient to show benefit to 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 individual condition and severity of the condition being treated. The prescription of treatment (e.g., determination of dosage, etc.) is ultimately the responsibility of and is relied upon by general practitioners and other physicians, generally taking into account the disease being treated, the individual patient's condition, the site of delivery, the method of administration, and other factors known to the physician.

[0075] 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.

[0076] As used herein, the term "malignancy" refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Exemplary malignancies include carcinomas, solid tumors, melanomas, sarcomas, hematological tumors, germ cell tumors, and blastomas. More specific examples of malignant tumors include renal cell carcinoma (e.g., clear cell renal cell carcinoma), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, or lung squamous cell carcinoma), breast cancer, pancreatic cancer, stomach 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 cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, brain cancer, head and neck cancer, esophageal cancer, nasopharyngeal cancer, thymoma, glioblastoma, lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, or peripheral T-cell lymphoma), leukemia (e.g., acute myeloid leukemia), neuroblastoma, multiple myeloma, and related metastases.

[0077] As used herein, the term "solid tumor" refers to a tangible mass that can be palpated by clinical examination such as X-ray film, CT scan, B-ultrasound or palpation. The solid tumors treated clinically are divided into malignant and benign types. Malignant solid tumors include: renal cell carcinoma (for example, renal clear cell carcinoma), lung cancer (for example, 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, 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 (for example, squamous cell carcinoma), vulvar cancer, thyroid cancer, anal cancer, penile cancer, melanoma, lymphoma (for example, Hodgkin lymphoma, non-Hodgkin lymphoma) Childhood Hodgkin lymphoma: lymphocyte-predominant, nodular sclerosis, mixed cell, lymphocyte-depleted; Childhood non-Hodgkin lymphoma: prelymphoblastic lymphoma, small non-cleaved cell lymphoma (Burkitt / non-Burkitt lymphoma), diffuse large B-cell lymphoma, anaplastic large cell lymphoma; Childhood kidney tumor: renal Wilms tumor, renal clear cell carcinoma, renal rhabdoid tumor, renal clear cell sarcoma, renal primitive neuroectodermal tumor, etc.; childhood neuroblastoma: neuroblastoma, ganglioneuroblastoma, ganglioneuroma; childhood extracranial germ cell tumor: 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 neurilemmoma, alveolar soft tissue sarcoma, epithelioid sarcoma, clear cell sarcoma, malignant melanoma, synovial sarcoma, desmoplastic small round cell tumor, etc.; Ewing family sarcomas: Ewing sarcoma, primitive neuroectodermal tumor; childhood liver tumors: hepatoblastoma (embryonic, fetal, undifferentiated), hepatocellular carcinoma; retinoblastoma; other tumors: posterior fossa medulloblastoma, nasopharyngeal carcinoma, papillary thyroid carcinoma, thymoma, pulmonary blastoma, pancreatic blastoma, islet cell tumor, ileocecal carcinoid, mesothelioma, etc. Benign solid tumors include: lymphangioma, hemangioma, thyroglossal duct cyst, etc.

[0078] As used herein, the term "hematologic malignancy" refers to a malignancy that is caused by the uncontrolled growth and proliferation of abnormal cells. In most cases, the origin of these abnormal cells is the bone marrow, which is where blood cells are produced. Exemplary hematologic malignancies include various types of leukemias, multiple myeloma, and malignant lymphomas. More specific examples of hematologic malignancies include acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia, large granular lymphocytic leukemia, juvenile myelomonocytic leukemia, B-cell prolymphocytic leukemia, Burkitt's leukemia, and adult T-cell leukemia, non-Hodgkin's lymphoma, B-cell lymphoma, small lymphocytic lymphoma, lymphoplasmacytic lymphoma, primary macroglobulinemia ( macroglobulinemia), splenic marginal zone lymphoma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, nodal marginal zone B-cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, Burkitt lymphoma, B-cell chronic lymphocytic lymphoma, classical Hodgkin lymphoma, Nodular lymphocyte-predominant Hodgkin lymphoma, adult T-cell lymphoma, extranodal nasal NK / T-cell lymphoma, enteropathy-type T-cell lymphoma, hepatosplenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides, Say-Sher syndrome, primary cutaneous CD30-positive T-cell lymphoproliferative disease, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T-cell lymphoma, unspecified peripheral T-cell lymphoma, and anaplastic large cell lymphoma.

[0079] The killing activity of NK cells is mainly through:

[0080] 1) Direct lysis of target cells: NK cells release cytotoxic granules such as perforin and granzymes through exocytosis, activating the caspase pathway to induce necrosis or apoptosis of target cells;

[0081] 2) Cytokine secretion: Cytokine-mediated killing effect. NK cells can synthesize and secrete a variety of cytokines, such as IFN-γ, TNF-α, IL-1, IL-5, IL-8, IL-10 and G-CSF, to induce target cell apoptosis;

[0082] 3) Induction of apoptosis: Activated NK cells express Fas (CD95) ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) molecules, inducing CD95 +Target cells and TRAIL receptor-positive target cells undergo apoptosis through a cascade of endogenous enzymes;

[0083] 4) ADCC: antibody-dependent cell-mediated cytotoxicity;

[0084] 5) Immune checkpoint pathway: It expresses programmed death receptor 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA4), etc., and exerts its effect by inhibiting immune checkpoints.

[0085] The above multiple mechanisms of action, the potential for application as a universal product, and reliable safety make NK cell therapy an attractive immunotherapy.

[0086] Activation of CD16A molecules can also promote the maturation and activation of NK cells and the production of cytokines, which play a key role in immune responses and inflammatory processes. Therefore, activating the function of NK cells by targeting CD16A molecules may be an effective immunotherapy strategy, especially in the fight against cancer and viral infections. In general, as a key activating receptor on NK cells, CD16A molecules play a core role in mediating ADCC and enhancing NK cell function. Activating CD16A molecules through 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.

[0087] The CD70 molecule is a type II transmembrane protein belonging to the tumor necrosis factor receptor (TNFR) superfamily, also known as TNFRSF7. It is highly expressed in a variety of blood tumors or solid tumors and plays a key role in tumor growth and development.

[0088] The inventors of this application have conducted in-depth research in the field of cellular immunotherapy, and in particular, have developed a bispecific antibody-NK cell conjugate for tumor treatment, comprising a first antigen binding region that binds to CD70 molecules and a second antigen binding region that binds to CD16A molecules. The conjugate activates NK cells by binding to the second antigen binding region of the CD16A molecule and binding to the CD16A molecules expressed on NK cells, and binds to target cells expressing CD70 molecules (such as CD70-positive tumor cells) by binding to the first antigen binding region of the CD70 molecule, thereby establishing an interaction between the target cells and the NK cells, killing the target cells through antibody-dependent cellular cytotoxicity (ADCC), and achieving the purpose of treating diseases (such as tumors). In various aspects of the present application, provided are novel bispecific antibody-NK cell conjugates comprising a first antigen-binding region that binds to a CD70 molecule and a second antigen-binding region that binds to a CD16A molecule, bispecific antibodies comprising a first antigen-binding region that binds to a CD70 molecule and a second antigen-binding region that binds to a CD16A molecule, pharmaceutical compositions comprising the bispecific antibody-NK cell conjugates or the bispecific antibodies, nucleic acid molecules encoding the bispecific antibodies, vectors comprising the nucleic acid molecules, host cells comprising the nucleic acid molecules or vectors, methods for preparing and purifying the bispecific antibodies, and medical and biological applications of the bispecific antibody-NK cell conjugates and the bispecific antibodies.

[0089] In a first aspect, the present application provides a bispecific antibody-NK cell conjugate, wherein

[0090] The bispecific antibody comprises a first antigen-binding region that binds to a CD70 molecule and a second antigen-binding region that binds to a CD16A molecule;

[0091] The bispecific antibody is coupled to the NK cell via antigen-antibody binding between its second antigen-binding region and the CD16A molecule on the NK cell.

[0092] In some embodiments of the first aspect, the NK cells are obtained from the in vitro culture and expansion of NK cells derived from peripheral blood mononuclear cells (PBMC), which is also an exemplary method in the embodiments of the present application. PBMC is one of the main sources of NK cells, and has the advantages of being relatively easy to collect, easy to expand in vitro, and having no toxic side effects. However, the proportion of NK cells in PBMC is only 10%-15%. The method of expanding NK cells derived from PBMC includes using a combination of cytokines, feeder cells or membrane particles to stimulate the in vitro expansion of NK cells. These different expansion systems show different levels of NK cell expansion efficiency. In some embodiments, one or more cytokines are used to maintain or activate the activity of natural killer cells during culture. In some embodiments, one or more immunoglobulins or fusion proteins are used to inhibit the proliferation of B cells, macrophages and other immune cells.

[0093] In some embodiments of the first aspect, the PBMCs may be derived from apheresis peripheral blood lymphocytes from an allogeneic healthy donor.

[0094] In some embodiments of the first aspect, the NK cells are obtained by in vitro culture and expansion of NK cells derived from umbilical cord blood. There are generally 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 CD34 + Hematopoietic stem / progenitor cells differentiate into NK cells and then expand.

[0095] In some embodiments of the first aspect, the NK cells are obtained from in vitro culture and expansion of an NK cell line. 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.

[0096] 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 (ESCs).

[0097] In some embodiments of the first aspect, the phenotype of the NK cells is CD3- / CD16 + CD56 + .

[0098] In a second aspect, the present application provides a bispecific antibody comprising a first antigen-binding region that binds to a CD70 molecule and a second antigen-binding region that binds to a CD16A molecule.

[0099] In some embodiments of the second aspect, the bispecific antibody is capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC).

[0100] In some embodiments of the first or second aspect, the first antigen binding region comprises:

[0101] HCDR1 as shown in SEQ ID NO: 1,

[0102] HCDR2 as shown in SEQ ID NO: 2,

[0103] HCDR3 as shown in SEQ ID NO: 3,

[0104] LCDR1 as shown in SEQ ID NO: 4,

[0105] LCDR2 as shown in SEQ ID NO: 5, and

[0106] LCDR3 as shown in SEQ ID NO:6;

[0107] The amino acid sequence of HCDR is defined according to Kabat.

[0108] In some embodiments of the first or second aspect, the second antigen binding region comprises:

[0109] HCDR1 as shown in SEQ ID NO: 7,

[0110] HCDR2 as shown in SEQ ID NO: 8,

[0111] HCDR3 as shown in SEQ ID NO:9,

[0112] LCDR1 as shown in SEQ ID NO: 10,

[0113] LCDR2 as shown in SEQ ID NO: 11, and

[0114] LCDR3 as shown in SEQ ID NO: 12;

[0115] The amino acid sequence of HCDR is defined according to Kabat.

[0116] 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.

[0117] 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.

[0118] 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 similar function of the heavy chain variable region of the first antigen binding region.

[0119] 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 retains the function of the heavy chain variable region similar to that of the first antigen binding region.

[0120] 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 in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 13, as long as the altered amino acid sequence substantially maintains the function of the heavy chain variable region of the similar first antigen binding region.

[0121] 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 with the sequence shown in SEQ ID NO:14.

[0122] 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.

[0123] 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 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 similar function of the light chain variable region of the first antigen binding region.

[0124] 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 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 of the light chain variable region similar to that of the first antigen binding region.

[0125] 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 in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 14, as long as the altered amino acid sequence basically maintains the function of the light chain variable region of the similar first antigen binding region.

[0126] In some embodiments of the first or second aspects, 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 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical 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 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 14.

[0127] 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 shown in SEQ ID NO: 13 and the amino acid sequence of the light chain variable region of the first antigen binding region is shown in SEQ ID NO: 14.

[0128] In some embodiments of 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 with the sequence shown in SEQ ID NO: 15.

[0129] 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.

[0130] 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 similar function of the heavy chain variable region of the second antigen binding region.

[0131] 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 retains similar function of the heavy chain variable region of the second antigen binding region.

[0132] 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 in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 15, as long as the altered amino acid sequence substantially maintains the similar function of the heavy chain variable region of the second antigen binding region.

[0133] 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 with the sequence shown in SEQ ID NO:16.

[0134] 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.

[0135] 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 similar function of the light chain variable region of the second antigen binding region.

[0136] 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 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 the similar function of the light chain variable region of the second antigen binding region.

[0137] 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 in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 16, as long as the altered amino acid sequence basically maintains the similar function of the light chain variable region of the second antigen binding region.

[0138] In some embodiments of 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 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical 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 is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence shown in SEQ ID NO: 16.

[0139] In some embodiments of the first or second aspect, the amino acid sequence of the heavy chain variable region of the second antigen binding region is shown in SEQ ID NO: 15 and the amino acid sequence of the light chain variable region of the second antigen binding region is shown in SEQ ID NO: 16.

[0140] In some embodiments of the first or second aspect, the first antigen-binding region is in the form of a single-chain antibody (scFv).

[0141] In some embodiments of the first or second aspect, the second antigen-binding region is in the form of a single-chain antibody (scFv).

[0142] In some embodiments described in the first or second aspect, the first antigen-binding region and the second antigen-binding region are connected 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, wherein n is an integer from 1 to 10, for example, 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, wherein n is an integer from 1 to 5, for example, 1, 2, 3, 4 or 5. In some specific embodiments, the peptide linker is GGGGS (SEQ ID NO: 19).

[0143] 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 is connected to the light chain variable region 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, wherein n is an integer from 1 to 10, for example, 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, wherein n is an integer from 1 to 5, for example, 1, 2, 3, 4 or 5. In some specific embodiments, the peptide linker is GGGGSGGGGSGGGGS (SEQ ID NO: 20).

[0144] 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 is connected to the light chain variable region 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, wherein n is an integer from 1 to 10, for example, 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, wherein n is an integer from 1 to 5, for example, 1, 2, 3, 4 or 5. In some specific embodiments, the peptide linker is GGSGGSGGSGGSGGS (SEQ ID NO: 21).

[0145] In some embodiments of the first or second aspect, the bispecific antibody has the following connection mode: CD16A VL1-linker 1-CD16AVH1-linker 2-CD70 VL2-linker 3-CD70 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 are GS-type flexible peptide linkers. In some embodiments, the peptide linker 1, the peptide linker 2 or the peptide linker 3 are (G4S)n or (G2S)n, wherein n is an integer from 1 to 10, for example, 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, wherein n is an integer from 1 to 5, for example, 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 (SEQ ID NO: 21).

[0146] In some embodiments of the first or second aspect, the bispecific antibody comprises a first antigen-binding region that binds to a CD70 molecule and a second antigen-binding region that binds to a CD16A molecule, wherein the first antigen-binding region comprises the amino acid sequence shown in SEQ ID NO: 17.

[0147] 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.

[0148] 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.

[0149] 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 similar function of the first antigen binding region.

[0150] 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 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 similar function of the first antigen binding region.

[0151] 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 in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 17, as long as the altered amino acid sequence basically maintains the similar function of the first antigen binding region.

[0152] In some embodiments of the first or second aspect, the bispecific antibody comprises a first antigen-binding region that binds to a CD70 molecule and a second antigen-binding region that binds to a CD16A molecule, wherein the second antigen-binding region comprises the amino acid sequence shown in SEQ ID NO: 18.

[0153] 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.

[0154] 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.

[0155] 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 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 similar function of the second antigen binding region.

[0156] 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 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 a similar function of the second antigen binding region.

[0157] 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 in the region other than the C-terminus or N-terminus of the amino acid sequence shown in SEQ ID NO: 18, as long as the altered amino acid sequence basically maintains the similar function of the second antigen binding region.

[0158] In some embodiments of the first or second aspect, the bispecific antibody comprises a first antigen-binding region with an amino acid sequence as shown in SEQ ID NO: 17 and a second antigen-binding region with an amino acid sequence as shown in SEQ ID NO: 18.

[0159] In some embodiments of the first or second aspect, the amino acid sequence of the bispecific antibody is as shown in SEQ ID NO:23.

[0160] In some embodiments of the first or second aspect, the N-terminus of the bispecific antibody is connected to a purification tag, such as a histidine tag HHHHHH (SEQ ID NO: 22). In some embodiments, the amino acid sequence of the bispecific antibody is as shown in SEQ ID NO: 24.

[0161] 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.

[0162] Where conditions permit, the pharmaceutical composition can be provided directly 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, which can be thawed and revived at the time of use.

[0163] In some embodiments of the third aspect, the pharmaceutical composition comprises sodium chloride and / or human serum albumin.

[0164] In some embodiments of the third aspect, the pharmaceutical composition comprises trehalose, sucrose, dextran, DMSO, or any combination thereof.

[0165] In some embodiments of the third aspect, the pharmaceutical composition is used to prevent or treat a tumor in an individual.

[0166] In some embodiments of the third aspect, the tumor is a tumor cell that highly expresses the CD70 molecule (CD70 + In some embodiments, the tumor cells highly express the CD70 molecule (CD70 + ) refers to a tumor in which at least 60% of the tumor cells in the tumor cell population express CD70 molecules. In some embodiments, the tumor cells highly express CD70 molecules (CD70 + ) refers to a tumor in which at least 70% of the tumor cells in the tumor cell population express CD70 molecules. In some embodiments, the tumor cells highly express CD70 molecules (CD70 + ) refers to a tumor in which at least 80% of the tumor cells in the tumor cell population express CD70 molecules. In some embodiments, the tumor cells highly express CD70 molecules (CD70 + ) refers to a tumor in which at least 90% of the tumor cells in the tumor cell population express CD70 molecules. In some embodiments, the tumor cells highly express CD70 molecules (CD70 + ) refers to a tumor in which at least 95% of the tumor cells in the tumor cell population express CD70 molecules. In some embodiments, the tumor cells highly express CD70 molecules (CD70 + ) refers to a tumor in which at least 98% of the tumor cells in the tumor cell population express CD70 molecules. In some embodiments, the tumor cells highly express CD70 molecules (CD70 + ) refers to a tumor in which at least 99% of the tumor cells in the tumor cell population express the CD70 molecule.

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

[0168] In some embodiments of the third aspect, the tumor is a hematological tumor.

[0169] In some embodiments of the third aspect, the tumor is a malignant tumor.

[0170] In some embodiments of the third aspect, the tumor is cancer.

[0171] In some embodiments of the third aspect, the tumor is selected from: renal cell carcinoma (e.g., renal clear cell carcinoma), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma or lung squamous cell carcinoma), nasopharyngeal carcinoma, thymoma, glioblastoma, lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma or peripheral T-cell lymphoma), leukemia (e.g., acute myeloid leukemia), and metastases of the above cancers.

[0172] In a fourth aspect, the present application provides 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 medicament for preventing or treating tumors in an individual.

[0173] In some embodiments of the fourth aspect, the tumor is a tumor cell that highly expresses the CD70 molecule (CD70 + In some embodiments, the tumor cells highly express the CD70 molecule (CD70 + ) refers to a tumor in which at least 60% of the tumor cells in the tumor cell population express CD70 molecules. In some embodiments, the tumor cells highly express CD70 molecules (CD70 + ) refers to a tumor in which at least 70% of the tumor cells in the tumor cell population express CD70 molecules. In some embodiments, the tumor cells highly express CD70 molecules (CD70 + ) refers to a tumor in which at least 80% of the tumor cells in the tumor cell population express CD70 molecules. In some embodiments, the tumor cells highly express CD70 molecules (CD70 + ) refers to a tumor in which at least 90% of the tumor cells in the tumor cell population express CD70 molecules. In some embodiments, the tumor cells highly express CD70 molecules (CD70 + ) refers to a tumor in which at least 95% of the tumor cells in the tumor cell population express CD70 molecules. In some embodiments, the tumor cells highly express CD70 molecules (CD70 + ) refers to a tumor in which at least 98% of the tumor cells in the tumor cell population express CD70 molecules. In some embodiments, the tumor cells highly express CD70 molecules (CD70 + ) refers to a tumor in which at least 99% of the tumor cells in the tumor cell population express the CD70 molecule.

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

[0175] In some embodiments of the fourth aspect, the tumor is a hematological tumor.

[0176] In some embodiments of the fourth aspect, the tumor is a malignant tumor.

[0177] In some embodiments of the fourth aspect, the tumor is cancer.

[0178] In some embodiments of the fourth aspect, the tumor is selected from: renal cell carcinoma (e.g., renal clear cell carcinoma), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma or lung squamous cell carcinoma), nasopharyngeal carcinoma, thymoma, glioblastoma, lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma or peripheral T-cell lymphoma), leukemia (e.g., acute myeloid leukemia), and metastases of the above cancers.

[0179] In a fifth aspect, the present application provides a method for preventing or treating tumors in an individual, comprising administering to an individual in need thereof 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.

[0180] In some embodiments of the fifth aspect, the tumor is a tumor cell that highly expresses the CD70 molecule (CD70 + In some embodiments, the tumor cells highly express the CD70 molecule (CD70 + ) refers to a tumor in which at least 60% of the tumor cells in the tumor cell population express CD70 molecules. In some embodiments, the tumor cells highly express CD70 molecules (CD70 + ) refers to a tumor in which at least 70% of the tumor cells in the tumor cell population express CD70 molecules. In some embodiments, the tumor cells highly express CD70 molecules (CD70 + ) refers to a tumor in which at least 80% of the tumor cells in the tumor cell population express CD70 molecules. In some embodiments, the tumor cells highly express CD70 molecules (CD70 + ) refers to a tumor in which at least 90% of the tumor cells in the tumor cell population express CD70 molecules. In some embodiments, the tumor cells highly express CD70 molecules (CD70 + ) refers to a tumor in which at least 95% of the tumor cells in the tumor cell population express CD70 molecules. In some embodiments, the tumor cells highly express CD70 molecules (CD70 +) refers to a tumor in which at least 98% of the tumor cells in the tumor cell population express CD70 molecules. In some embodiments, the tumor cells highly express CD70 molecules (CD70 + ) refers to a tumor in which at least 99% of the tumor cells in the tumor cell population express the CD70 molecule.

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

[0182] In some embodiments of the fifth aspect, the tumor is a hematological tumor.

[0183] In some embodiments of the fifth aspect, the tumor is a malignant tumor.

[0184] In some embodiments of the fifth aspect, the tumor is cancer.

[0185] In some embodiments of the fifth aspect, the tumor is selected from the group consisting of renal cell carcinoma (e.g., renal clear cell carcinoma), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, lung adenocarcinoma, or lung squamous cell carcinoma), nasopharyngeal carcinoma, thymoma, glioblastoma, lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, or peripheral T-cell lymphoma), leukemia (e.g., acute myeloid leukemia), and metastases of the above cancers.

[0186] The present application also provides nucleic acid molecules encoding the bispecific antibodies described in the second aspect, vectors comprising the nucleic acid molecules, and host cells comprising the nucleic acid molecules or vectors. In other aspects, the present application also provides methods for producing the bispecific antibodies described in the second aspect. In some embodiments, the method for producing the bispecific antibodies described in the second aspect comprises culturing host cells to express the nucleic acid molecules. In some embodiments, the method for producing the bispecific antibodies described in the second aspect further comprises recovering the bispecific antibodies from the host cell culture medium.

[0187] It should be understood that the above detailed description is only for the purpose of enabling those skilled in the art to more clearly understand the content of the present application and is not intended to limit the present invention in any respect. Those skilled in the art can make various modifications and variations to the embodiments described.

[0188] The following examples are for illustration purposes only and are not intended to limit the scope of this application. Example

[0189] Example 1: Preparation of bispecific antibodies

[0190] 1.1 Gene design and cloning

[0191] Gene synthesis: Based on the determined antibody fragment sequence, the corresponding gene was synthesized, where the amino acid sequence of the anti-CD70 and CD16A bispecific antibody (CD16A VL1-Linker 1-CD16AVH1-Linker 2-CD70 VL2-Linker 3-CD70 VH2-His) is shown below: (SEQ ID NO: 24).

[0192] Construction of expression vector: The synthesized gene was inserted into the pET22b plasmid.

[0193] 1.2 Bacterial expansion culture

[0194] (1) Take 100 μL of BL21(DE3) / pET22b-CD70-CD16 and add it to 10 mL of LB medium (containing kanamycin). Incubate at 37°C and 150 rpm for 6-7 h.

[0195] (2) Add the bacterial solution obtained in step (1) to 100 mL of LB medium (containing kanamycin) and culture overnight at 37°C and 150 rpm.

[0196] (3) The bacterial solution obtained in step (2) was added to 2 L of LB medium (containing kanamycin), cultured at 37°C and 150 rpm for 6-7 h, then cooled to 32°C and IPTG was added to a final concentration of 0.2 mmol / L. The culture was continued for 15 h, and the supernatant was collected by centrifugation.

[0197] 1.3 After centrifugation, the collected supernatant was concentrated to 250 mL using a membrane with a molecular weight cutoff of 10 kDa.

[0198] 1.4 Purification

[0199] 1.4.1 NI column purification

[0200] Column volume: 10 mL

[0201] Flow rate: 3 mL / min

[0202] Equilibration buffer: 20 mM PB, pH 7.0

[0203] Elution buffer: 20 mM PB, 0.5 M NaCl, 0.5 M imidazole, pH 7.0

[0204] Purification method: After loading, rinse with equilibrium buffer until UV280 reaches the baseline, then elute in one step with elution buffer, and adjust the eluent to pH 5.0.

[0205] 1.4.2 SPFF purification

[0206] Column volume: 15 mL

[0207] Flow rate: 3 mL / min

[0208] Equilibration buffer: 50 mM PB, pH 5.0

[0209] Elution buffer: 50 mM PB, 1 M NaCl, pH 5.0

[0210] Purification method: The eluate from the NI column was diluted 10 times with equilibration buffer and loaded onto the sample. The sample was washed with equilibration buffer until UV280 reached the baseline, and then eluted in one step with elution buffer.

[0211] The solution was replaced with an ultrafiltration tube with a molecular weight cut-off of 10 kDa and concentrated to 1 / 10 of the initial volume.

[0212] Example 2: Preparation of bispecific antibody-NK cell conjugates

[0213] 2.1 NK cell culture

[0214] 2.1.1 Isolation of human peripheral blood mononuclear cells (PBMCs)

[0215] Blood (50 mL each) was collected from healthy volunteers, and all volunteers signed informed consent. The volunteer selection criteria are as follows:

[0216] 1. Basic Standards:

[0217] 1. Age range: 18-45 years old, gender not limited.

[0218] 2. No serious chronic disease or active infection.

[0219] 3. No history of malignant tumor.

[0220] 4. No history of long-term or recent use of immunosuppressants.

[0221] 5. No history of drug, alcohol or other substance abuse.

[0222] 6. Non-pregnant or breastfeeding women.

[0223] 2. Hematological criteria:

[0224] 1. Hemoglobin, white blood cell count, and platelet count are within normal range.

[0225] 2. Liver function and kidney function tests are normal.

[0226] 3. Immunological standards:

[0227] 1. NK cell activity is normal.

[0228] 2. No known immunodeficiency or autoimmune disease.

[0229] 4. Infectious Disease Screening:

[0230] 1. Negative results for HIV, HBV, HCV, HTLV-1 / 2, CMV, etc. virological screening.

[0231] 2. Screening results for infectious diseases such as syphilis and tuberculosis are negative.

[0232] 5. Family history / genetic disease history:

[0233] 1. Genetic diseases without familial clustering, such as hemophilia, cystic fibrosis, etc.

[0234] 2. No history of immediate family members (parents, siblings) suffering from malignant tumors in early years.

[0235] 3. No clear genetic diseases or gene mutations related to the research.

[0236] (1) Transfer peripheral blood: Use a pipette to transfer the donor's peripheral blood into a 50 mL centrifuge tube.

[0237] (2) Centrifugation: Balance the centrifuge tube, centrifuge at 800g, speed up 6, speed down 6, and centrifuge for 15 minutes.

[0238] (3) Plasma transfer: Transfer the upper plasma layer obtained by centrifugation to a 50 mL centrifuge tube and store at 4°C.

[0239] (4) Dilution: Dilute the peripheral blood with sodium chloride injection at a volume ratio of 2:1 and mix well.

[0240] (5) Layering and sample addition: Add 15 mL of separation solution to each 50 mL centrifuge tube, with the volume ratio of diluted blood to separation solution being 2:1. Centrifuge at 800 g, speed 6, speed 3, for 20 min.

[0241] (6) Extraction of mononuclear cells: After centrifugation, obvious stratification appears in the centrifuge tube. Aspirate the cell suspension of the mononuclear cell layer and transfer it into a 50 mL centrifuge tube.

[0242] (7) Washing: Add sodium chloride injection and resuspend in a 50 mL centrifuge tube. Centrifuge at 300 g, speed 9, speed 7, for 10 min. Discard the supernatant after centrifugation.

[0243] (8) Wash again: Add sodium chloride injection and resuspend in a 50 mL centrifuge tube, mix well and count (retain 1 mL for counting before incubation), centrifuge at 400 g, speed 6, speed 6, for 10 min. After centrifugation, discard the supernatant. The precipitate is the mononuclear cells, which is collected for later use.

[0244] 2.1.2 Magnetic bead purification of NK cells from PBMC:

[0245] (1) Resuspend the mononuclear cell pellet obtained in Section 2.1.1 in separation buffer to a concentration of 1×10 8 cells / mL.

[0246] (2) Add 100 μL CD56 per ml of resuspension + The magnetic beads were sorted and incubated in a horizontal shaker at 2-8°C for 15 min.

[0247] (3) Add sodium chloride injection and resuspend in a 50 mL centrifuge tube. Centrifuge at 300 g, speed 9, speed 7, for 10 min. Discard the supernatant.

[0248] (4) Every 1×10 8 Add 500 μL of separation buffer to each cell and resuspend the cells.

[0249] (5) Add the cell suspension to a separation column placed on a magnetic stand and discard the liquid that flows through the separation column.

[0250] (6) Wash the separation column three times with 30 mL of separation solution.

[0251] (7) Add 20 mL of eluent to the separation column and separate the purified CD56 + NK cells were eluted.

[0252] 2.1.3 NK cell inoculation and culture:

[0253] (1) Cell inoculation: Purified NK cells were inoculated in a 75cm 2 In the culture flask, the inoculation concentration is about 1×10 6 Aspirate the PBS with a pipette and discard it. Add 50 mL of complete culture medium (GT551 culture medium containing IL-2 500 U / mL, IL-15 800 U / mL, IL-21400 U / mL and 5% autologous plasma) and culture in a 37°C, saturated humidity, 5% CO2 incubator.

[0254] (2) Cell transfer: Cells can be passaged after 3-5 days of culture. Observe whether the culture medium turns yellow. Under the microscope, observe whether the cells are growing well and the cell concentration is ≥3×10 6 Cells were passaged at 400 cells / mL. Transfer the cells, culture medium, and plasma from the original culture flask to a cell culture bag and add 250 mL of complete culture medium. Simultaneously, 50 mL of complete culture medium was added to the original culture flask for continued culture. The next day, cells were transferred back to the culture bag. Label the culture bag with information such as the cell number and culture time, and culture in a 37°C, saturated humidity, and 5% CO2 incubator.

[0255] (3) Adding culture medium: After cell transfer, observe the color change of the cell suspension. If the cell suspension turns obviously yellow, it is necessary to add complete culture medium to a final volume of ≤1800 mL and culture in a 37°C, saturated humidity, 5% CO2 incubator.

[0256] (4) Cell counting: After the cells are fully replenished, 20 mL of culture medium is extracted from the cell culture bag using a disposable syringe 3 days before release and sent to the quality control center for blood culture, cell counting, and bacterial, endotoxin, mycoplasma, Gram staining, and cell surface antibody testing. After the test is qualified, the technician will notify the cell release planner to arrange the cell release time.

[0257] 2.2 NK cells coupled with bispecific antibodies:

[0258] (1) Cell collection: Transfer the cell suspension in the culture bag into a 250 mL centrifuge tube, centrifuge at 300 g, increase the speed by 9, decrease the speed by 7, centrifuge for 10 min, and discard the supernatant; collect the cell suspension into a 50 mL centrifuge tube, resuspend it in sodium chloride injection to 45 mL, centrifuge at 300 g, increase the speed by 9, decrease the speed by 7, centrifuge for 10 min, and discard the supernatant.

[0259] (2) Bispecific Antibody Coupling: Resuspend the mixture to 45 mL in sodium chloride injection in a 50 mL centrifuge tube, add 500 μL of recombinant anti-CD70 and CD16A bispecific antibodies, and incubate at 4°C for 30 min on a horizontal shaker. Centrifuge at 300 g, speed 9°C, speed 7°C, for 10 min, and discard the supernatant. Repeat once. Transfer the supernatant to a 50 mL centrifuge tube before packaging and store at 4°C for 48 h for future reference.

[0260] (3) Cell resuspension: Add the sodium chloride injection solution obtained in the previous step to the cells, pipette the suspended cells, and filter with a 100 μm sterile cell strainer. According to the cell count before distribution, the cell suspension was 5×10 9 Each 100mL bag is divided into 100mL transfer bags, and 2mL is reserved for partial testing before dividing.

[0261] 2.3 Identification of NK cell phenotype and coupling efficiency

[0262] Take 4 flow cytometry tubes and number them sequentially ①, ②, ③, and ④. 100-200 μL of well-mixed cell suspension (cell count, 0.5-1×10 6 10 μL of isotype IgG1-FITC and IgG1-PE antibodies were added to tube ①. 10 μL of CD3-FITC antibody was added to tubes ②, ③, and ④, respectively. 10 μL of CD16-PE antibody was added to tube ②. 10 μL of CD56-APC antibody was added to tube ③. 10 μL of CD70-Cy5.5 recombinant protein was added to tube ④. Gently shake to mix thoroughly and incubate at room temperature in the dark for 30 minutes. After incubation, centrifuge at 300g for 5 minutes and discard the supernatant. 1 mL of PBS buffer was added to each tube, vortexed to mix thoroughly, and centrifuged at 300g for 5 minutes. The supernatant was discarded. 1 mL of PBS buffer was added to each tube. Incubate at 4°C in the dark for 1 hour before analysis.

[0263] Figure 1 The results showed that the phenotype of NK cells was CD3- / CD16 + CD56 + , the NK cell purity was 99.18%. Figure 2 The results showed that the coupling efficiency of the isotype control antibody to NK cells was 0.34%, and the coupling efficiency of the anti-CD16A and CD70 bispecific antibody to NK cells was 89.55%.

[0264] Example 3: In vitro killing verification of bispecific antibody-NK cell conjugates

[0265] 3.1 Cell plating

[0266] In a 96-well plate, add the appropriate number of bispecific antibody-NK cell conjugates or NK cells and 786-O cells at the preset effector-target ratio. Set up at least three replicates for each effector-target ratio to minimize variability. Also, set up control wells containing NK cells alone, 786-O cells alone, and blank medium.

[0267] Gently shake the 96-well plate to evenly distribute the cells.

[0268] 3.2 Co-culture

[0269] The 96-well plate was placed in a 37° C., 5% CO 2 incubator, and the NK cells and 786-O cells were co-cultured for 4 h.

[0270] 3.3 CCK8 detection

[0271] After co-culture, 10 μL of CCK8 reagent was added to each well.

[0272] The 96-well plate was placed in the incubator and incubated for 2 h to allow the CCK8 reagent to fully react with the cells.

[0273] Use a microplate reader to measure the absorbance (OD value) at a wavelength of 450 nm and record the OD value of each well.

[0274] Figure 3 The results showed that compared with NK cells, the bispecific antibody-NK cell conjugate has a stronger cell killing efficiency.

[0275] Example 4: ADCC effect mediated by bispecific antibodies

[0276] 4.1 Cell culture and preparation

[0277] 786-O target cells were cultured to the logarithmic growth phase and seeded into 96-well plates at a seeding concentration of approximately 1×10 6 pieces / mL.

[0278] At the same time, Jurkat effector cells stably expressing NFAT luciferase reporter gene and FcγRIIIa were prepared and cultured to an appropriate density.

[0279] 4.2 Luciferase substrate preparation

[0280] Prepare the luciferase substrate solution D-luciferin at a concentration of 1 mM according to the instructions of the luciferase assay kit.

[0281] 4.3 Bispecific Antibody Binding to Target Cells

[0282] In a 96-well plate containing 786-O target cells, the bispecific antibody (final concentration of 10 μg / mL) was added, and a vehicle control and an isotype IgG antibody control were set up. Three replicate wells were set up for each treatment to reduce experimental error. The cells were incubated at 37°C, 5% CO2 for 60 min.

[0283] 4.4 Co-culture of effector cells and target cells

[0284] The target cells bound to the bispecific antibody were mixed with Jurkat effector cells at a certain ratio of 5:1, 1:1, and 1:5, respectively, and co-cultured at 37°C and 5% CO2 for 6 hours.

[0285] 4.5 Luciferase activity assay

[0286] After the co-culture, 100 μL of luciferase substrate D-luciferin solution was added.

[0287] The fluorescence signal was detected using a multifunctional microplate reader and the relative light units (RLU) were recorded.

[0288] Figure 4 The results showed that the bispecific antibody against CD16A and CD70 can effectively mediate ADCC effect.

[0289] Example 5: Validation of tumor inhibition in animal models of bispecific antibody-NK cell conjugates

[0290] 5.1 Experimental Preparation:

[0291] Experimental animals: NOD / SCID mice were selected because they have immunodeficiency characteristics and are suitable for xenograft tumor model experiments.

[0292] Cell preparation: Human renal clear cell adenocarcinoma cells 786-O are used for tumor bearing; NK cells and bispecific antibody-NK cell conjugates are expanded and activated in vitro for tail vein injection.

[0293] Experimental groups: The experiment was divided into three groups, with 10 mice in each group, namely PBS injection group, NK cell injection group, and bispecific antibody-NK cell conjugate injection group.

[0294] 5.2 Establishment of tumor-bearing model

[0295] 786-O cell passaging and expansion: 786-O cells were cultured and expanded in vitro to ensure that the cells were in good condition.

[0296] Cell inoculation: 786-O cells in the logarithmic growth phase were collected and the cell density was adjusted to 2.5×10 8 The cells were inoculated subcutaneously in the groin of NOD / SCID mice with an injection volume of 1 ml.

[0297] Observe tumor formation: After inoculation, regularly observe the status of the mice and tumor growth, and record changes in tumor volume. After 7 days, the long diameter of the tumor is about 1 cm.

[0298] 5.3 Administration and Evaluation of Bispecific Antibody-NK Cell Conjugates

[0299] Tail vein injection: On the 8th day after tumor bearing, mice were given PBS, NK cells and bispecific antibody-NK cell conjugates according to their grouping. The cell dosage was 1×10 9 / kg body weight.

[0300] Observation and evaluation: Regularly observe the mental state, activity, diet, body weight, tumor size and survival of mice.

[0301] Figure 5 The results showed that compared with NK cells, the bispecific antibody-NK cell conjugate can effectively inhibit tumor growth in mice and improve the survival rate of mice.

[0302] All publications and patent documents cited in this specification are incorporated herein by reference, as if each publication or patent was expressly indicated to be incorporated herein by reference. Without departing from the true idea and scope of the present disclosure, various changes and equivalents may be made to each embodiment disclosed in the present application. Unless otherwise indicated in the context, any feature, step or embodiment of the embodiment of the present disclosure may be used in combination with any other feature, step or embodiment.

[0303] Sequence information

[0304] SEQ ID NO: 1

[0305] DTYMH

[0306] SEQ ID NO:2

[0307] RIAPAIGDTKYDPKFQG

[0308] SEQ ID NO:3

[0309] DYGGYFDV

[0310] SEQ ID NO:4

[0311] KASDHINNWLA

[0312] SEQ ID NO:5

[0313] GASILET

[0314] SEQ ID NO:6

[0315] QQYWSTPLT

[0316] SEQ ID NO:7

[0317] SYYMH

[0318] SEQ ID NO:8

[0319] AIEPMYGSTSYAQKFQG

[0320] SEQ ID NO:9

[0321] GSAYYYDFADY

[0322] SEQ ID NO:10

[0323] GGHNIGSKNVH

[0324] SEQ ID NO:11

[0325] QDNKRPS

[0326] SEQ ID NO:12

[0327] QVWDNYSVL

[0328] SEQ ID NO:13

[0329] MKCSWVIFFLMAVVTGVNSEVQLQQSGAELVKPGASVKLSCTASGFDIIDTYMHWVKQRPEQGLEWIGRIAPAIGDTKYDPKFQGKATITSDTSSNTVYLHLSSLTSEDTALYYCSRDYGGYFDVWGAGTAVTVSS

[0330] SEQ ID NO:14

[0331] MKFPSQLLLFLLFRITGIICDIQMTQSSSYLSVSLGGRVNIACKASDHINNWLAWYQQKPGDVPRLLISGASILETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPLTFGAGTKLELK

[0332] SEQ ID NO:15

[0333] EVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGAIEPMYGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSS

[0334] SEQ ID NO:16

[0335] SYVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVL

[0336] SEQ ID NO:17

[0337] MKFPSQLLLFLLFRITGIICDIQMTQSSSYLSVSLGGRVNIACKASDHINNWLAWYQQKPGDVPRLLISGASILETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPLTFGAGTKLELKGGSGGSGGSGGSGGSMKCSWVIFFLMAVVTGVNSEVQLQQSGAELVKPGASVKLSCTASGFDIIDTYMHWVKQRPEQGLEWIGRIAPAIGDTKYDPKFQGKATITSDTSSNTVYLHLSSLTSEDTALYYCSRDYGGYFDVWGAGTAVTVSS

[0338] SEQ ID NO:18

[0339] SYVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGAIEPMYGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSS

[0340] SEQ ID NO:19

[0341] GGGGS

[0342] SEQ ID NO:20

[0343] GGGGSGGGGSGGGGS

[0344] SEQ ID NO:21

[0345] GGSGGSGGSGGSGGS

[0346] SEQ ID NO:22

[0347] HHHHHH

[0348] SEQ ID NO:23

[0349] SYVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGAIEPMYGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSSGGGGSMKFPSQLLLFLLFRITGIICDIQMTQSSSYLSVSLGGRVNIACKASDHINNWLAWYQQKPGDVPRLLISGASILETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPLTFGAGTKLELKGGSGGSGGSGGSGGSMKCSWVIFFLMAVVTGVNSEVQLQQSGAELVKPGASVKLSCTASGFDIIDTYMHWVKQRPEQGLEWIGRIAPAIGDTKYDPKFQGKATITSDTSSNTVYLHLSSLTSEDTALYYCSRDYGGYFDVWGAGTAVTVSS

[0350] SEQ ID NO:24

[0351] SYVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVLGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGAIEPMYGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSSGGGGSMKFPSQLLLFLLFRITGIICDIQMTQSSSYLSVSLGGRVNIACKASDHINNWLAWYQQKPGDVPRLLISGASILETGVPSRFSGSGSGKDYTLSITSLQTEDVATYYCQQYWSTPLTFGAGTKLELKGGSGGSGGSGGSGGSMKCSWVIFFLMAVVTGVNSEVQLQQSGAELVKPGASVKLSCTASGFDIIDTYMHWVKQRPEQGLEWIGRIAPAIGDTKYDPKFQGKATITSDTSSNTVYLHLSSLTSEDTALYYCSRDYGGYFDVWGAGTAVTVSSHHHHHH。

Claims

1. A bispecific antibody-natural killer cell (NK cell) conjugate, wherein The bispecific antibody comprises a first antigen-binding region that binds to a CD70 molecule and a second antigen-binding region that binds to a CD16A molecule; The bispecific antibody is coupled to the NK cell through antigen-antibody binding between its second antigen-binding region and the CD16A molecule on the NK cell; Wherein the first antigen binding region comprises: HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, HCDR3 as shown in SEQ ID NO: 3, LCDR1 as shown in SEQ ID NO: 4, LCDR2 as shown in SEQ ID NO: 5, and LCDR3 as shown in SEQ ID NO:6; and The second antigen binding region comprises: HCDR1 as shown in SEQ ID NO: 7, HCDR2 as shown in SEQ ID NO: 8, HCDR3 as shown in SEQ ID NO:9, 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; in, The amino acid sequences of HCDRs and LCDRs are according to the Kabat definition.

2. The bispecific antibody-NK cell conjugate according to claim 1, wherein 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 at least 80% identical 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 at least 80% identical to the sequence shown in SEQ ID NO: 14; and 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 at least 80% identical 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 at least 80% identical to the sequence shown in SEQ ID NO:

16.

3. The bispecific antibody-NK cell conjugate of claim 1, wherein 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.

4. The bispecific antibody-NK cell conjugate of claim 1, wherein 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.

5. The bispecific antibody-NK cell conjugate according to any one of claims 1 to 4, wherein 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 scFv.

6. The bispecific antibody-NK cell conjugate according to any one of claims 1 to 4, wherein The first antigen-binding region and the second antigen-binding region are connected via a peptide linker.

7. The bispecific antibody-NK cell conjugate according to claim 6, wherein the peptide linker is a GS-type flexible peptide linker. The bispecific antibody-NK cell conjugate according to claim 6 , wherein the peptide linker is (G4S)n or (G2S)n, wherein n is an integer from 1 to 10.

9. The bispecific antibody-NK cell conjugate of claim 6, wherein the peptide linker is (G4S)n or (G2S)n, wherein n is an integer from 1 to 5.

10. The bispecific antibody-NK cell conjugate according to any one of claims 1 to 4, wherein 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 connected via a peptide linker. The bispecific antibody-NK cell conjugate according to claim 10 , wherein the peptide linker is a GS-type flexible peptide linker. The bispecific antibody-NK cell conjugate according to claim 10 , wherein the peptide linker is (G4S)n or (G2S)n, wherein n is an integer from 1 to 10.

13. The bispecific antibody-NK cell conjugate of claim 10, wherein the peptide linker is (G4S)n or (G2S)n, wherein n is an integer from 1 to 5.

14. The bispecific antibody-NK cell conjugate according to any one of claims 1 to 4, wherein 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 connected via a peptide linker. The bispecific antibody-NK cell conjugate according to claim 14 , wherein the peptide linker is a GS-type flexible peptide linker. The bispecific antibody-NK cell conjugate according to claim 14 , wherein the peptide linker is (G4S)n or (G2S)n, wherein n is an integer from 1 to 10. The bispecific antibody-NK cell conjugate according to claim 14 , wherein the peptide linker is (G4S)n or (G2S)n, wherein n is an integer from 1 to 5.

18. The bispecific antibody-NK cell conjugate according to any one of claims 1 to 4, wherein 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.

19. The bispecific antibody-NK cell conjugate according to any one of claims 1 to 4, wherein the amino acid sequence of the bispecific antibody is as shown in SEQ ID NO: 23 or 24.

20. The bispecific antibody-NK cell conjugate according to any one of claims 1 to 4, wherein The NK cells are obtained from the in vitro culture and expansion of NK cells derived from peripheral blood mononuclear cells (PBMC); The NK cells are obtained from the in vitro culture and expansion of NK cells derived from umbilical cord blood; The NK cells are obtained from the in vitro culture and expansion of NK cell lines; or The NK cells are obtained from in vitro induction, culture and expansion of induced pluripotent stem cells (iPSCs) or mesenchymal stem cells (ESCs).

21. The bispecific antibody-NK cell conjugate according to any one of claims 1 to 4, wherein the phenotype of the NK cell is CD3 - / CD16 + CD56 + .

22. A pharmaceutical composition comprising the bispecific antibody-NK cell conjugate according to any one of claims 1 to 21, and a pharmaceutically acceptable carrier.

23. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition is provided in the form of a sterile fresh preparation or a sterile frozen preparation.

24. Use of the bispecific antibody-NK cell conjugate of any one of claims 1-21 or the pharmaceutical composition of any one of claims 22-23 in the preparation of a medicament for treating a tumor in an individual; wherein the tumor is renal cell carcinoma or a metastasis thereof.

25. The use according to claim 24, wherein the tumor is renal clear cell carcinoma.

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