CD16 antibodies and uses thereof

Abstract

The invention discloses a CD16 antibody and application thereof, the CD16 antibody comprises an antibody specifically binding to CD16A and an antibody strongly binding to CD16B but weakly binding to CD16A, the antibody specifically binding to CD16A can bind to CD16A but not binding to CD16B, and selectively activates NK cells and macrophages, so that adverse inflammatory reaction caused by cross reaction of CD16B is avoided; the antibody which is strongly combined with CD16B but weakly combined with CD16A has unique CD16B combining capacity, and can be used for enhancing the function of neutrophil and promoting immune complex removal, so that inflammatory response is regulated; the two antibodies can be developed into a plurality of drugs, have a wide application field, and provide a valuable technical platform for subsequent development of novel antibody drugs.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology and relates to an antibody, specifically a CD16 antibody and its applications. Background Technology

[0002] CD16 molecules, including CD16A (FcγRIIIa) and CD16B (FcγRIIIb), are important members of the Fcγ receptor family. They mainly mediate various immune functions by binding to the Fc fragment of immunoglobulin G (IgG).

[0003] CD16A is expressed on immune effector cells such as NK cells, monocytes, and macrophages, and mainly participates in immune clearance by triggering antibody-dependent cell-mediated cytotoxicity (ADCC) responses. In cancer immunotherapy, CD16A is a key molecule for the efficacy of many antibody drugs. For example, in treatments mediated by certain antibody drugs (such as rituximab and trastuzumab), CD16A activates NK cells, producing a highly efficient killing effect on target cells. CD16B is expressed on the surface of neutrophils and has a higher immune complex binding capacity than CD16A, but lower signal transduction activity; it mainly participates in the clearance of immune complexes and the regulation of inflammation.

[0004] However, CD16A shares approximately 95% amino acid sequence similarity with its homologous isoform CD16B (FcγRIIIb), with the main differences concentrated at a few key sites in the extracellular region. CD16B is primarily expressed on the surface of neutrophils and participates in the clearance of immune complexes. Due to this high homology, most existing antibodies against CD16A also bind to CD16B, leading to nonspecific binding and potential side effects. These side effects may include interactions with non-target cells, excessive immune activation, and unexpected toxic reactions.

[0005] In recent years, antibodies that specifically recognize CD16A but do not bind to CD16B have become a new research hotspot. These antibodies can not only enhance the specific activation of NK cells but also avoid the immune side effects caused by CD16B binding. Such specific antibodies have significant application value in the development of tumor therapeutic antibodies, bispecific antibodies (BsAbs), and in improving the efficacy of antibody drugs. For example, in CAR-NK cell therapy, CD16A-specific antibodies can be used to enhance the efficacy of CAR-NK cells while reducing treatment-related side effects.

[0006] CD16B, a member of the FcγRIII family, is expressed almost exclusively on the surface of neutrophils and is one of the specific markers of human neutrophils. CD16B is anchored to the neutrophil membrane via glycosylphosphatidylinositol (GPI), rather than through a transmembrane domain (like the transmembrane region possessed by CD16A). Neutrophils can recognize IgG-coated target cells (such as pathogens and tumor cells) and exert cytotoxic effects by binding to the Fc fragment of IgG via CD16B. Through binding to IgG, CD16B activates neutrophil signaling pathways and regulates their functions, including: enhancing chemotaxis (towards sites of inflammation), stimulating degranulation, releasing lysosomal enzymes and cytotoxic substances, and regulating neutrophil lifespan (anti-apoptosis). CD16B can also bind to immune complexes on the neutrophil surface, helping to clear them and preventing tissue damage caused by excessive inflammatory activation or immune complex deposition. Abnormal CD16B function may lead to incomplete clearance of immune complexes, triggering inflammation and tissue damage. Changes in CD16B expression and function are associated with the overactivation of neutrophils in arthritis. If antibodies against CD16B can be developed, it will help in the treatment of inflammation-related diseases.

[0007] Therefore, in response to the above-mentioned technical problems, the inventors have developed a novel CD16 antibody, providing a valuable technical platform for the development of novel antibody drugs. Summary of the Invention

[0008] The purpose of this invention is to provide a CD16 antibody and its applications. Two antibodies were screened using a combination of various biotechnologies: an antibody that specifically binds to CD16A and an antibody that strongly binds to CD16B but weakly binds to CD16A. The antibody that specifically binds to CD16A can bind to CD16A but not CD16B, selectively activating NK cells and macrophages, avoiding adverse inflammatory responses caused by CD16B cross-reactivity. The antibody that strongly binds to CD16B but weakly binds to CD16A possesses unique CD16B binding ability and can be used to enhance neutrophil function, promote immune complex clearance, and thus regulate inflammatory responses. Furthermore, both antibodies can be developed into various drugs with broad application areas, providing a valuable technological platform for the subsequent development of novel antibody drugs.

[0009] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0010] On one hand, the present invention provides an antibody or antigenic binding fragment thereof that specifically binds to CD16A, comprising a first heavy chain variable region and a first light chain variable region, wherein the first heavy chain variable region contains HCDR1, HCDR2 and HCDR3, and the sequences of HCDR1, HCDR2 and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; the first light chain variable region contains LCDR1, LCDR2 and LCDR3, and the sequences of LCDR1, LCDR2 and LCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:4, STS and SEQ ID NO:5.

[0011] In one or more embodiments of the present invention, the sequences of HCDR1, HCDR2, and HCDR3 are as shown in positions 26-33, 51-58, and 97-108 of SEQ ID NO:6, respectively, or have 90% or more homology with the sequences shown in positions 26-33, 51-58, and 97-108 of SEQ ID NO:6, respectively; the sequences of LCDR1, LCDR2, and LCDR3 contain positions 27-31, 49-51, and 88-97 of SEQ ID NO:7, respectively, or have 90% or more homology with the sequences shown in positions 27-31, 49-51, and 88-97 of SEQ ID NO:7, respectively.

[0012] In one or more embodiments of the present invention, the antibody that specifically binds to CD16A or its antigenic binding fragment comprises a first heavy chain and a first light chain, wherein the sequence of the first heavy chain is as shown in SEQ ID NO:6 or has 90% or more homology with it, and the sequence of the first light chain is as shown in SEQ ID NO:7 or has 90% or more homology with it.

[0013] On the other hand, the present invention also provides a nucleic acid molecule encoding an antibody or an antigenic binding fragment thereof that specifically binds to CD16A.

[0014] In another aspect, the present invention provides a construct containing the aforementioned nucleic acid molecules.

[0015] In one or more embodiments of the present invention, the construct is a chimeric antigen construct or a vector.

[0016] In another aspect, the present invention also provides a cell that expresses the above-mentioned antibody that specifically binds to CD16A or its antigenic binding fragment, or the above-mentioned chimeric antigen construct, wherein the cell may be an NK cell, T cell, dendritic cell, macrophage, B cell, etc.

[0017] In another aspect, the present invention also provides a bispecific antibody, comprising the above-mentioned antibody specifically binding to CD16A or its antigenic binding fragment and an antibody or antigenic fragment binding to a tumor antigen, wherein the tumor antigen is preferably HER2, PD-L1, CD33, PSMA, FAP, ROR1, HER3, CD19, CD20, BCMA, CD22, CD38, CD123, 5T4 or EGFR, which can target CD16A and specific tumor antigens (such as HER2, EGFR, PD-L1, CD33, PSMA, FAP, ROR1, HER3, CD19, CD20, BCMA, CD22, CD38 or CD123), thereby significantly enhancing the targeted killing power of NK cells against tumor cells, while reducing the off-target effects of traditional therapies and avoiding side effects on normal tissues.

[0018] In one or more embodiments of the present invention, the bispecific antibody includes the antibody that specifically binds to CD16A as described in claim 1 or 2, or its antigenic binding fragment, and the antibody or antigenic fragment that binds to tumor antigen 5T4.

[0019] In one or more embodiments of the present invention, the bispecific antibody is 12H7-5T4-VHH(VL-N), 12H7-5T4-VHH(VL-C), or 12H7-5T4-VHH(Fc-C). The heavy chain sequences of 12H7-5T4-VHH(VL-N) and 12H7-5T4-VHH(VL-C) are shown in SEQ ID NO:23. The heavy chain sequence of 12H7-5T4-VHH(VL-N) is shown in SEQ ID NO:24. The light chain sequence of 12H7-5T4-VHH(VL-C) is shown in SEQ ID NO:25. The heavy chain sequence of 12H7-5T4-VHH(Fc-C) is shown in SEQ ID NO:26. The light chain sequence of 12H7-5T4-VHH(Fc-C) is shown in SEQ ID NO:27.

[0020] In another aspect, the present invention also provides a trispecific antibody, including the above-mentioned antibody that specifically binds to CD16A or its antigenic binding fragment, antibody or antigenic binding fragment that targets immunomodulatory factors, or antibody or antigenic fragment that binds to tumor antigens, thereby simultaneously activating NK cells and T cells to achieve a combined anti-tumor effect.

[0021] In one or more embodiments of the present invention, the immunomodulatory factor includes CD3, IL-15, or PD-1.

[0022] In another aspect, the present invention also provides a pharmaceutical composition comprising the above-described antibody that specifically binds to CD16A or its antigenic binding fragment, or the above-described cell-expressed antibody.

[0023] In one or more embodiments of the present invention, the pharmaceutical composition further includes nanoparticles, small molecule compound drugs, ADC drugs, or immune cells; wherein, when the pharmaceutical composition includes nanoparticles, the pharmaceutical composition is a drug delivery carrier; preferably, the immune cells are CAR-NK or CAR-T, and by binding with nanoparticles or immune cells (such as CAR-NK or CAR-T), the targeting and therapeutic efficacy of the drug can be further enhanced.

[0024] In another aspect, the present invention also provides the use of the above-mentioned antibody that specifically binds to CD16A or its antigenic binding fragment, nucleic acid molecule, construct, cell, bispecific antibody, trispecific antibody or pharmaceutical composition in the preparation of anticancer drugs, anti-infective disease drugs or autoimmune-related disease drugs.

[0025] In one or more embodiments of the present invention, the infectious disease is HIV, HBV, or bacterial sepsis; or

[0026] The autoimmune-related diseases mentioned are rheumatoid arthritis or systemic lupus erythematosus.

[0027] On the other hand, the present invention also provides a method for treating cancer, infectious diseases and autoimmune-related diseases, comprising administering the above-mentioned antibody that specifically binds to CD16A or its antigenic binding fragment, nucleic acid molecule, construct, cell, bispecific antibody, trispecific antibody or pharmaceutical composition to a diseased host.

[0028] In another aspect, the present invention also provides the use of the above-mentioned antibody that specifically binds to CD16A or its antigenic binding fragment in expressing cells for detecting CD16A or in preparing reagents for detecting cells expressing CD16A.

[0029] On the other hand, the present invention also provides a method for detecting CD16A, comprising labeling an antibody or antigenic binding fragment thereof that specifically binds to CD16A to detect CD16A.

[0030] In another aspect, the present invention also provides the use of the above-mentioned antibody that specifically binds to CD16A or its antigenic binding fragment in activating ADCC of NK cells or in preparing ADCC reagents for activating NK cells.

[0031] In another aspect, the present invention also provides a method for activating ADCC in NK cells, comprising adding the above-mentioned antibody that specifically binds to CD16A or its antigenic binding fragment to NK cells.

[0032] On the other hand, the present invention also provides an antibody or antigenic binding fragment thereof that strongly binds to CD16B but weakly binds to CD16A, comprising a second heavy chain variable region and a second light chain variable region, wherein the second heavy chain variable region contains HCDR4, HCDR5, and HCDR6, and the sequences of HCDR4, HCDR5, and HCDR6 respectively contain the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; and the second light chain variable region contains LCDR4, LCDR5, and LCDR6, and the sequences of LCDR4, LCDR5, and LCDR6 respectively contain the amino acid sequences shown in SEQ ID NO:11, WAS, and SEQ ID NO:12.

[0033] In one or more embodiments of the present invention, the sequences of HCDR4, HCDR5, and HCDR6 are as shown in positions 26-33, 51-58, and 97-106 of SEQ ID NO:13, respectively, or have 90% or more homology with the sequences shown in positions 26-33, 51-58, and 97-106 of SEQ ID NO:13, respectively; the sequences of LCDR4, LCDR5, and LCDR6 contain positions 27-32, 50-52, and 89-97 of SEQ ID NO:14, respectively, or have 90% or more homology with the sequences shown in positions 27-32, 50-52, and 89-97 of SEQ ID NO:14, respectively.

[0034] In one or more embodiments of the present invention, the antibody or its antigenic binding fragment that strongly binds CD16B but weakly binds CD16A includes a second heavy chain and a second light chain, wherein the sequence of the second heavy chain is as shown in SEQ ID NO:13 or has 90% or more homology with it, and the sequence of the second light chain is as shown in SEQ ID NO:14 or has 90% or more homology with it.

[0035] In another aspect, the present invention also provides a nucleic acid molecule encoding an antibody that strongly binds to CD16B but weakly binds to CD16A, or an antigenic binding fragment thereof.

[0036] In another aspect, the present invention also provides a construct containing the above-mentioned nucleic acid molecules, preferably, the construct being a chimeric antigen construct or a vector.

[0037] On the other hand, the present invention also provides a cell that expresses the antibody that strongly binds to CD16B but weakly binds to CD16A or its antigenic binding fragment, or the chimeric antigen construct described above. The cell may be an NK cell, T cell, dendritic cell, macrophage, B cell, etc.

[0038] In another aspect, the present invention also provides a bispecific antibody, including the antibody that strongly binds to CD16B but weakly binds to CD16A or its antigenic binding fragment, or an antibody or antigenic fragment that binds to inflammatory factors.

[0039] In another aspect, the present invention also provides a pharmaceutical composition comprising the above-described antibody that strongly binds to CD16B but weakly binds to CD16A, or an antigenic binding fragment thereof, or a cell-expressed antibody.

[0040] In one or more embodiments of the present invention, the pharmaceutical composition further includes nanoparticles, small molecule compound drugs, ADC drugs, or immune cells. When the pharmaceutical composition includes nanoparticles, the pharmaceutical composition is a drug delivery carrier. Preferably, the immune cells are CAR-NK or CAR-T cells.

[0041] In another aspect, the present invention also provides the use of the above-mentioned antibody that strongly binds to CD16B but weakly binds to CD16A, or its antigenic binding fragment, nucleic acid molecule, construct, cell, bispecific antibody, or pharmaceutical composition in the preparation of drugs for inflammation-related diseases, drugs for immune complex-related diseases, or drugs for blood diseases.

[0042] In one or more embodiments of the present invention, the inflammation-related disease includes inflammatory diseases and inflammatory diseases caused by systemic immune diseases, such as allergic purpura, systemic lupus erythematosus, rheumatoid arthritis, acute respiratory distress syndrome (ARDS), or inflammatory bowel disease (such as Crohn's disease).

[0043] In one or more embodiments of the present invention, the blood disease is immune thrombocytopenic purpura (ITP) or neutrophil-related disease.

[0044] In another aspect, the present invention also provides a method for treating inflammatory-related diseases, immune complex-related diseases, or blood diseases, comprising administering a therapeutically effective amount of the aforementioned antibody that strongly binds CD16B but weakly binds CD16A, or its antigenic binding fragment, nucleic acid molecule, construct, cell, bispecific antibody, or pharmaceutical composition to a diseased host.

[0045] In another aspect, the present invention also provides a combination target polyclonal antibody, including the above-mentioned antibody that specifically binds to CD16A or its antigenic binding fragment, antibody that strongly binds to CD16B but weakly binds to CD16A or its antigenic binding fragment, and antibody or antigenic binding fragment that binds to immunomodulatory factors, thereby enabling the combined activation of NK cells and other immune effector cells.

[0046] In another aspect, the present invention also provides the application of the above-mentioned combined target polyclonal antibody in the preparation of drugs for immune-related diseases.

[0047] In one or more embodiments of the present invention, the immune-related disease is multiple sclerosis and systemic lupus erythematosus.

[0048] On the other hand, the present invention also provides a method for treating immune-related diseases, comprising administering a therapeutically effective amount of the above-mentioned combined target polyclonal antibody to a diseased host.

[0049] In another aspect, the present invention also provides the use of the above-mentioned antibody that strongly binds to CD16B but weakly binds to CD16A, or its antigenic binding fragment, in detecting cells expressing CD16B or in preparing reagents for detecting cells expressing CD16B.

[0050] In another aspect, the present invention also provides a method for detecting CD16B, comprising labeling an antibody or its antigenic binding fragment that strongly binds CD16B but weakly binds CD16A, thereby detecting CD16B.

[0051] Compared with existing technologies, the CD16 antibody and its application of the present invention, through a variety of techniques such as mouse immunization, hybridoma cell screening, ELISA verification, and flow cytometry, and through refined antibody screening and optimization techniques, successfully obtained the following two types of antibodies:

[0052] 1. Antibodies that specifically recognize CD16A but not CD16B exhibit high specificity, binding to CD16A but not CD16B. These antibodies can selectively activate NK cells and macrophages, avoiding adverse inflammatory responses caused by CD16B cross-reactivity. These antibodies have significant application value in tumor immunotherapy, ADCC enhancement therapy, and the treatment of related immune diseases.

[0053] 2. Antibodies that weakly bind to CD16A but strongly bind to CD16B exhibit the characteristic of weak binding to CD16A but strong binding to CD16B. These antibodies have a unique CD16B binding ability and can be used to enhance neutrophil function, promote the clearance of immune complexes, thereby regulating the inflammatory response and contributing to the treatment of inflammation-related diseases.

[0054] Furthermore, these two antibodies can form bispecific antibodies against tumor or inflammatory factors, which is more conducive to the treatment of diseases. Experiments have shown that the bispecific antibodies formed have significant advantages in terms of structural rationality, functional synergy, and cell killing efficiency, providing a more promising antibody engineering design strategy for clinical applications. Among them, the antibody that specifically recognizes CD16A but does not recognize CD16B can also form trispecific antibodies with other antigens, while activating NK cells and T cells to achieve a combined anti-tumor effect. Moreover, these two antibodies can also be combined with other immunomodulatory factors to form multispecific antibodies for the treatment of complex immune-related diseases, such as multiple sclerosis and systemic lupus erythematosus. In addition, these two antibodies can also be combined with nanoparticles or cell therapy platforms (such as CAR-NK or CAR-T) to further enhance the targeting and therapeutic efficacy of drugs.

[0055] In summary, the two antibodies developed in this invention are comprehensive in application and have a wide range of applications, providing clear guidance for subsequent drug development, fully demonstrating the clinical and commercial value of the two types of antibodies, and providing a valuable technical platform for the development of novel antibody drugs. Attached Figure Description

[0056] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0057] Figure 1 This is a graph showing the SDS-PAGE reduction electrophoresis results of the recombinant CD16A and CD16B proteins in Example 1 of the present invention.

[0058] Figure 2 This is a flow cytometry result of CD16A / 293F cells and CD16B / 293F cells in Example 2 of the present invention;

[0059] Figure 3 The results of SDS-PAGE reduction electrophoresis detection of four recombinant antibodies against CD16 in Example 5 of the present invention are shown. Among them, 1 is recombinant 12A1 antibody, 2 is recombinant BMK antibody, 3 is recombinant 12H7 antibody, and 4 is recombinant ISO antibody.

[0060] Figure 4 The results of ELISA detection of the binding of the recombinant antibody to the CD16A-his antigen in Example 6 of this invention;

[0061] Figure 5The results of ELISA detection of the binding of the recombinant antibody to the CD16B-his antigen in Example 6 of this invention;

[0062] Figure 6 The flow cytometry results are as follows: The recombinant antibody in Example 7 of this invention binds to CD16A-293F cells.

[0063] Figure 7 The flow cytometry results are as follows: The recombinant antibody in Example 7 of this invention binds to CD16B-293F cells.

[0064] Figure 8 The flow cytometry results of the recombinant antibody binding to NK92 cells in Example 7 of this invention;

[0065] Figure 9 The flow cytometry results of the binding of the recombinant antibody to NK cells derived from umbilical cord blood in Example 7 of this invention;

[0066] Figure 10 This is a schematic diagram of the structure of the 12H7-5T4-VHH (VL-N) antibody in Example 8 of the present invention;

[0067] Figure 11 This is a schematic diagram of the structure of the 12H7-5T4-VHH(VL-C) antibody in Example 8 of the present invention;

[0068] Figure 12 This is a schematic diagram of the results of the 12H7-5T4-VHH (Fc-C) antibody in Example 8 of the present invention;

[0069] Figure 13 The results of the ELISA method in Example 8 of this invention for detecting the binding ability of different forms of antibodies to the 5T4 antigen;

[0070] Figure 14 The results of the ELISA method in Example 8 of this invention for detecting the binding ability of different forms of antibodies to CD16 antigen;

[0071] Figure 15 The results of flow cytometry detection of the target binding ability of anti-5T4 antibody and its derivative constructs in Example 8 of this invention;

[0072] Figure 16 This is a graph showing the results of testing the killing activity of the bispecific antibody in Example 8 of the present invention;

[0073] In the above figures, MFI refers to average fluorescence intensity. Detailed Implementation

[0074] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention. Invention Details

[0076] definition

[0077] "Antibody" in a broad sense refers to and includes immunoglobulin molecules, including monoclonal antibodies (including mouse, human, humanized and chimeric monoclonal antibodies), antibody fragments, multispecific antibodies, dimer, tetramer or multimer antibodies, single-chain antibodies, domain antibodies and any other modified configuration of immunoglobulin molecules containing antigen-binding sites with desired specificity.

[0078] A full-length antibody molecule consists of two heavy chains (HC) and two light chains (LC) linked by disulfide bonds, and their polymers (e.g., IgM). Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (composed of domains CH1, hinge, CH2, and CH3). Each light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), interspersed with framework regions (FRs). Each VH and VL consists of three CDRs and four FR segments, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0079] The “complementarity-determining region (CDR)” is the “antigen-binding site” in an antibody. CDRs can be defined using various terms: (i) the complementarity-determining region (CDR), the three in VH (HCDR1, HCDR2, HCDR3) and the three in VL (LCDR1, LCDR2, LCDR3) based on sequence variability.

[0080] "Antibody fragment," "antigen-binding fragment," or "antigen-binding portion" refers to a portion of an immunoglobulin molecule that retains the heavy chain and / or light chain antigen-binding sites, such as heavy chain complementarity-determining regions (HCDRs) 1, 2, and 3, light chain complementarity-determining regions (LCDRs) 1, 2, and 3, heavy chain variable regions (VH), or light chain variable regions (VL). Antibody fragments include the well-known Fab, F(ab')2, Fd, and Fv fragments, as well as domain antibodies (dAbs) consisting of a single VH domain. VH and VL domains can be linked together via synthetic linkers to form various types of single-chain antibody designs, where the VH / VL domains can pair intramolecularly or intermolecularly, in the case that the VH and VL domains are expressed by separate single-chain antibody constructs, to form monovalent antigen-binding sites, such as single-chain Fv (scFv) or biantibodies.

[0081] The terms “polynucleotide,” “oligonucleotide,” “nucleic acid,” and “nucleic acid molecule” are used interchangeably in this document to include the polymeric form of nucleotides (ribonucleotides or deoxyribonucleotides). This term refers only to the primary structure of the molecule.

[0082] A "vector" is a polynucleotide capable of replicating within or moving between biological systems. Vector polynucleotides typically contain elements such as origins of replication, polyadenylation signals, or selection markers that function to facilitate or maintain the replication of these polynucleotides within biological systems, such as cells, viruses, animals, plants, and biological systems reconstructed using biological components capable of replication. Vector polynucleotides can be DNA or RNA molecules, cDNA, or hybrids thereof, and can be single-stranded or double-stranded.

[0083] "Expression vector" refers to a vector that can be used in a biological system or a reconstructed biological system to guide the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector, as used herein. "Host cell" means an in vivo or in vitro eukaryotic cell or a cell (e.g., a cell line) from a multicellular organism cultured as a single-cell entity. Eukaryotic cells can or have been used as recipients of nucleic acids (e.g., expression vectors containing nucleotide sequences encoding the multimeric polypeptides disclosed herein) and include the progeny of the original cell that has been genetically modified with nucleic acid. It should be understood that the progeny of a single cell is not necessarily identical to the original parent in morphology or genome or total DNA complementarity due to natural, accidental, or intentional mutations. "Recombinant host cell" (also called "genetically modified host cell") is a host cell in which a heterologous nucleic acid, such as an expression vector, has been introduced. For example, genetic modification of a eukaryotic host cell is achieved by introducing a heterologous nucleic acid, such as a foreign nucleic acid, into a suitable eukaryotic host cell, or a recombinant nucleic acid that is not normally present in eukaryotic host cells.

[0084] "Specific binding," "specifically binding," or simply "binding" refers to an antibody binding to a specific antigen with a greater affinity than to other antigens. Typically, the equilibrium dissociation constant (KD) for binding is approximately 1 x 10⁻⁶. -8 M or smaller, for example, about 1x10 -9 M or smaller, approximately 1x10 -10 M or smaller, approximately 1x10 -11 M or smaller, or approximately 1x10 -12 When M is smaller, the KD of an antibody that binds specifically is typically at least one hundred times smaller than the KD of its binding to non-specific antigens (such as BSA or casein). KD can be measured using standard procedures.

[0085] "Pharmaceutical composition" refers to a substance comprising the antibody described in this invention (such as antibody 12H7 that specifically binds to CD16A or antibody 12A1 that strongly binds to CD16B) or its antigenic binding fragment, nucleic acid molecule, construct or cell expressing the above antibody / construct as an active ingredient, and optionally containing at least one pharmaceutical excipient (such as a carrier, diluent or excipient).

[0086] In this invention, pharmaceutical compositions not only comprise mixtures of active ingredients with conventionally pharmacologically acceptable buffers or stabilizers, but also specifically encompass formulations combining the aforementioned active ingredients with nanoparticles, small molecule compound drugs, ADC drugs, or immune cells (such as CAR-NK or CAR-T). When the composition contains nanoparticles, it can serve as a drug delivery carrier to further enhance the drug's targeting and therapeutic efficacy in vivo. Such compositions are intended for administration to subjects to achieve desired pharmacological effects such as prevention, inhibition, or relief of cancer, infectious diseases, inflammation-related diseases, and autoimmune-related diseases.

[0087] In this invention, the term "treatment" and the like refers to achieving a desired pharmacological and / or physiological effect. Such effect may be preventative, i.e., complete or partial prevention of the disease or its symptoms, and / or therapeutic, i.e., partial or complete cure of the disease and / or side effects attributable to the disease. As used herein, "treatment" encompasses any treatment of a disease in mammals, such as humans, and includes: (a) preventing the occurrence of the disease in subjects who may be susceptible to it but have not yet been diagnosed with it; (b) inhibiting the disease, i.e., preventing its development; and (c) alleviating the disease, i.e., causing the disease to regress.

[0088] The terms “individual,” “subject,” “host,” and “patient,” which may be used interchangeably in this document, refer to mammals, including but not limited to rodents (e.g., rats, mice), rabbits (e.g., rabbits), non-human primates, humans, canines, felines, and ungulates (e.g., equines, bovines, ovines, suidae, capitols).

[0089] "Therapeutic effective dose" or "effective dose" refers to the amount of a drug or combination of two drugs that, when administered to mammals or other subjects to treat a disease, is sufficient to affect the treatment of the disease. Therapeutic effective doses vary depending on the drug, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0090] Unless otherwise specified, all reagents and materials mentioned in this article are available from conventional sources.

[0091] Unless otherwise specified, "12H7 antibody" in this article refers to "antibody that specifically recognizes CD16A and does not recognize CD16B", also known as "CD16A antibody".

[0092] Unless otherwise specified, "12A1 antibody" in this article refers to "antibody that weakly binds to CD16A but strongly binds to CD16B", also known as "CD16B antibody".

[0093] Example 1. Preparation of CD16A and CD16B proteins

[0094] 1. Construction of protein expression vectors

[0095] Using genetic engineering techniques, the genes encoding the extracellular domains of human CD16A (GenBank: AAH17865.1) *Gly17-Gln208 and CD16B (UniProtKB / Swiss-Prot: O75015.3) (Thr20-Gln208) proteins were cloned into eukaryotic expression vectors (pCDNA3.4). To facilitate subsequent purification, 6×His tags were fused to the C-terminus of the CD16A and CD16B genes, respectively, and designated as CD16A-His and CD16B-His. The recombinant plasmids were validated by sequencing and then used for protein expression.

[0096] The amino acid sequence of CD16A-His (SEQ ID NO:15) is as follows:

[0097] GMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQHHHHHH*

[0098] The amino acid sequence of CD16B-His (SEQ ID NO:16) is as follows:

[0099] GMRTEDLPKAVVFLEPQWYSVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVNDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKDRKYFHHNSDFHIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFHHHHHHHH*.

[0100] 2. Cell Culture and Transfection

[0101] Protein expression was performed using 293F cells (a suspension culture line of human embryonic kidney cells, purchased from Zhuhai Kairui Biotechnology). 293F cells were cultured in serum-free medium (Zhuhai Kairui Biotechnology). KOP293 Cells were cultured in suspension at 37°C with a carbon dioxide concentration of 5%, using a rotary shaker (120 rpm) to maintain cell suspension. When the cell density reached approximately 1 × 10⁻⁶ cells / year... 6 When the cells / mL is used, use the transfection reagent (Zhuhai Kairui Biotechnology). TA-293 Transfection was mediated by a medium containing plasmid DNA (catalog number: K20001). 1 μg plasmid DNA was added to 5 μL TA-293 per mL of cell suspension, and the mixture was incubated at room temperature for 10 minutes before being added to the cell suspension. The cells were cultured for 5-7 days post-transfection. The cell supernatant contained secreted CD16A-His and CD16B-His proteins. The cell culture supernatant was collected and centrifuged (3000×g, 10 min) to remove cell debris. The supernatant was then filtered through a 0.22 μm filter to remove residual impurities, ensuring successful subsequent purification.

[0102] 3. Protein purification

[0103] CD16A-His and CD16B-His proteins were purified using Ni-NTA (nickel affinity chromatography column, purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.). The Ni-NTA affinity column was equilibrated with 10 column volumes of equilibration buffer (50 mM Tris, 300 mM NaCl, 10 mM imidazole, pH 8.0). The filtered cell supernatant was slowly loaded into the affinity column to ensure sufficient binding of the target protein to the nickel ligand. The column was washed with elution buffer (50 mM Tris, 300 mM NaCl, 20 mM imidazole, pH 8.0) to remove non-specifically bound proteins. The target proteins were eluted with high-concentration imidazole elution buffer (50 mM Tris, 300 mM NaCl, 250 mM imidazole, pH 8.0). The eluted fraction was collected and protein purity was confirmed by SDS-PAGE. The results are shown below. Figure 1 As shown, from Figure 1 As can be seen from the SDS-PAGE results, CD16A-His and CD16B-His were successfully prepared with a purity greater than 95%, and can be used for subsequent experiments. The protein concentration was determined using Nanodrop. The purified CD16A-His and CD16B-His proteins were replaced with PBS buffer (pH 7.4), aliquoted, and stored at -80°C for later use.

[0104] Example 2. Preparation of CD16A and CD16B overexpression cell lines

[0105] 1. Construction of expression carriers

[0106] Plasmids stably expressing CD16A and CD16B were constructed using the PiggyBac transposon system. The gene fragments encoding human CD16A and CD16B were amplified by PCR and cloned into the multiple cloning site XbaI / NotI of the PiggyBac expression vector (PiggyBac Dual promoter (PB513B-1) mammalian transposon plasmid, purchased from Beijing Huayueyang Biotechnology). For subsequent analysis, the constructed recombinant plasmids were verified by restriction enzyme digestion and sequencing to confirm the correct insertion fragments and the absence of mutations. 293F cells were cultured in serum-free suspension medium to the logarithmic growth phase, and the density was adjusted to 1 × 10⁻⁶ cells / year. 6 The constructed PiggyBac expression vector and PiggyBac transposase expression vector (Super PiggyBac Transposase (PB200PA-1), purchased from Beijing Huayueyang Biotechnology) were mixed at a mass ratio of 3:1 (total DNA concentration of 1 μg / mL). Transfection was performed using transfection reagent (Zhuhai Kairui Biotechnology). TA-293(Catalog No.: K20001) The DNA:PEI mixture was prepared at a 1:3 ratio and incubated at room temperature for 10 minutes before being added to the cell culture medium. The cells were cultured for another 48 hours post-transfection. 48 hours after transfection, puromycin (5 μg / mL) was added for selection. During selection, the culture medium was changed every 2-3 days, gradually eliminating untransfected cells. After approximately 7-10 days, the surviving cell population constituted the stable 293F cell line successfully integrating the target gene. The selected cells were further diluted to single-cell levels and seeded into 96-well plates. After single-clone cell expansion, single clones were picked for expression analysis. The selected cell lines were observed using a fluorescence microscope to confirm the expression of the target protein (CD16A or CD16B) and GFP. Cells exhibiting green fluorescence were considered successfully expressed clones. Cells were collected, washed with PBS, resuspended, and the cell density was adjusted to 1×10⁶ cells / mL. 6 The cells / mL were analyzed using flow cytometry to detect GFP fluorescence signal and CD16A and CD16B gene expression. Results showed that the successfully constructed CD16A and CD16B overexpression cell lines exhibited high levels of GFP fluorescence signal, with a positive rate exceeding 95%. The successfully expressed CD16A and CD16B overexpression 293F cell lines were aliquoted into cryovials, infused with 10% DMSO cryopreservation solution, and gradually cooled before short-term storage at -80°C or long-term storage in liquid nitrogen.

[0107] Experimental results are as follows Figure 2 As shown, from Figure 2 It can be seen that both CD16A-293F and CD16B-293F cells express GFP, and antibody staining confirms that CD16A molecules are successfully expressed in CD16A-293F cells and CD16B molecules are successfully expressed in CD16B-293F cells. The positive rate of CD16 molecule gene expression in both cells is greater than 95%, indicating that the cell construction was successful.

[0108] The amino acid and nucleotide sequences of CD16A and CD16B in this embodiment are as follows:

[0109] >Amino acid sequence of CD16A (SEQ ID NO:17):

[0110] MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDKGS*

[0111] >Amino acid sequence of CD16B (SEQ ID NO:18):

[0112] MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYSVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVNDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGKDRKYFHHNSDFHIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFSPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIGS*

[0113] >Nucleotide sequence of CD16A (SEQ ID NO:19):

[0114] ATGTGGCAGCTGCTCCTCCCAACTGCTCTGCTACTTCTAGTTTCAGCTGGCATGCGGACTGAAGATCTCCCAAAGGCTGTGGTGTTCCTGGAGCCTCAATGGTACAGGGTGCTCGAGAAGGACAGTGTGACTCTGAAGTGCCAGGGAGCCTACTCCCCTGAGGACAATTCCACACAGTGGTTTCACAATGAGAGCCTCATCTCAAGCCAGGCCTCGAGCTACTTCATTGACGCTGCCACAGTCGACGACAGTGGAGAGTACAGGTGCCAGACAAACCTCTCCACCCTCAGTGACCCGGTGCAGCTAGAAGTCCATATCGGCTGGCTGTTGCTCCAGGCCCCTCGGTGGGTGTTCAAGGAGGAAGACCCTATTCACCTGAGGTGTCACAGCTGGAAGAACACTGCTCTGCATAAGGTCACATATTTACAGAATGGCAAAGGCAGGAAGTATTTTCATCATAATTCTGACTTCTACATTCCAAAAGCCACACTCAAAGACAGCGGCTCCTACTTCTGCAGGGGGCTTTTTGGGAGTAAAAATGTGTCTTCAGAGACTGTGAACATCACCATCACTCAAGGTTTGGCAGTGTCAACCATCTCATCATTCTTTCCACCTGGGTACCAAGTCTCTTTCTGCTTGGTGATGGTACTCCTTTTTGCAGTGGACACAGGACTATATTTCTCTGTGAAGACAAACATTCGAAGCTCAACAAGAGACTGGAAGGACCATAAATTTAAATGGAGAAAGGACCCTCAAGACAAAGGATCC

[0115] > Nucleotide sequence of CD1\6B (SEQ ID NO:20):

[0116] .

[0117] Example 3. Hybridoma screening for monoclonal antibodies against CD16A and CD16B

[0118] This invention employs hybridoma technology. Mice are immunized with CD16A protein obtained in Example 1 (methods and procedures are shown in Table 1). Then, mouse spleen cells are fused with myeloma cells to obtain hybridoma cells capable of secreting CD16A antibodies.

[0119] Table 1: Laboratory Animals and Immunological Information

[0120]

[0121] Hybridoma Fusion: Mice were euthanized, and the spleen was removed from the abdominal cavity aseptically, with surrounding connective tissue removed. Splenic cells were fully released by squeezing with a needle to prepare a spleen cell suspension. The cell suspension was filtered through a 70 μM cell sieve and washed once with RPMI-1640 medium, then centrifuged at 1200 rpm for 6 min. The supernatant was removed, and the cells were resuspended in RBC lysis buffer (purchased from Shanghai Sangon Biotech, catalog number: B541001). Red blood cells were lysed, centrifuged to remove the supernatant, and then resuspended in RPMI-1640 medium and counted. SP2 / 0 and lysed red blood cells were mixed at a ratio of 1:2 to 1:1 and centrifuged at 1000 rpm for 6 min. The supernatant was removed, and the mixed cells were resuspended in fusion buffer (brand: BTX, name: BTXpress Cytofusion Medium C, catalog number: 47-0001). 15 mL of fusion buffer was added, and the mixture was centrifuged at 1000 rpm for 5 min, then the supernatant was removed. After repeating the above steps once, add an appropriate volume of fusion buffer to resuspend the cells and adjust the mixed cell density to 1×10⁻⁶. 7 Cells / mL. After fusion using an electrofusion apparatus, cells were incubated at room temperature for 5 min in an electrofusion dish. Cells were then transferred to centrifuge tubes and diluted to 1–2 × 10⁶ cells / mL. 4 Cells / mL. Add 100 μL of cell suspension to each well of a 96-well plate. Change the culture medium on day 5 after confluence. After day 10 (or longer, depending on cell growth status), collect the supernatant for ELISA and flow cytometry (FACS) analysis to screen for positive clones.

[0122] Hybridoma cells that specifically bind to CD16A molecules were screened using ELISA and flow cytometry (FACS).

[0123] 1. ELISA screening for hybridomas that bind to CD16A or CD16B molecules.

[0124] Recombinant CD16A-His protein and CD16B-His protein (2 μg / mL) were coated onto 96-well ELISA plates, 100 μL / well, and incubated overnight at 4°C. The next day, unbound proteins were discarded, the plates were washed three times with PBS, and blocked with 5% BSA for 1 hour. Hybridoma cell supernatant was collected and added to wells coated with CD16A and CD16B, respectively, and incubated at 37°C for 1 hour. After washing the wells three times with PBS, HRP-labeled secondary antibody was added, and the plates were incubated at 37°C for 30 minutes. The plates were then developed with TMB substrate, and the reaction was stopped with 2 M H2SO4. OD values ​​were measured at 450 nm. Screening criteria: antibodies with high OD450 values ​​in CD16A wells and near-background levels in CD16B wells, or antibodies with high OD450 values ​​in CD16B wells and near-background levels in CD16A wells.

[0125] 2. FACS screening for hybridomas binding to CD16A or CD16B molecules

[0126] Using 293F cells stably expressing CD16A and CD16B constructed in Example 2, cells were collected, washed with PBS, and the concentration was adjusted to 1×10⁻⁶. 6 cells / mL. The hybridoma supernatant obtained from the initial screening was diluted (1:10 or 1:100), and 100 μL was added to the cell suspension. The cells were then incubated with CD16A-GFP and CD16B-GFP cells at 4°C for 30 minutes, respectively. After washing, APC-labeled anti-mouse IgG secondary antibody was added, and the cells were incubated at 4°C for 30 minutes, followed by another wash. The PE fluorescence signal of the cells was detected using flow cytometry. Screening criteria: Antibodies showing high APC signal in CD16A-293F cells and near-background signal in CD16B-293F cells, or antibodies showing high APC signal in CD16B-293F cells and near-background signal in CD16A-293F cells.

[0127] After the above ELISA and FACS screening, a hybridoma that binds to CD16A but not CD16B was obtained, clone number: 12H7; and a hybridoma that strongly binds to CD16B and weakly binds to CD16A was obtained, clone number: 12A1.

[0128] Example 4. Obtaining the antibody sequence

[0129] RNA was extracted from hybridoma cells screened in Example 3 using the Trizol method; cDNA was reverse transcribed using the 5'RACE method, and amplified to obtain the full-length sequences of the heavy and light chain variable regions; PCR products were used for library construction according to the next-generation sequencing library construction procedure, and library quality QC was performed; high-throughput sequencing was performed using ILLUMINA Miseq / NextSeq2000 PE300bp mode, and the sequencing data were subjected to quality QC; the sequencing results were compared with the IMGT database using bioinformatics analysis for further analysis; the analysis results were summarized to generate antibody sequencing sequences. The sequencing data were quality-controlled and assembled, and the sequence with the highest abundance in each sample was selected as the target antibody sequence. Then, the antibody reference sequences for this species in the IMGT database were compared using NCBI igblast software to filter out sequences without biological function (containing frameshift mutations or stop codons) and non-full-length antibody sequences. Finally, the obtained sequence annotation information was summarized. This experiment was commissioned to Suzhou Baida Biotechnology Co., Ltd.

[0130] Antibodies obtained through hybridoma 12H7 selection that bind CD16A but not CD16B are designated as 12H7 antibodies (CD16A antibodies), with the heavy chain denoted as 12H7-VH and the light chain as 12H7-VL. Antibodies obtained through hybridoma 12A1 selection that strongly bind CD16B and weakly bind CD16A are designated as 12A1 antibodies (CD16B antibodies), with the heavy chain denoted as 12A1-VH and the light chain as 12A1-VL. The specific sequences are as follows:

[0131] >12H7-VH (SEQ ID NO:6)

[0132] QVQLQQSGAELVRPGTSVKVSCKAS GYAFALFL IEWIKQGPGQGLEWIGV INPGSGVT NYNEKFKAKATLTADTSSSTAYMQLSSLTSDDSAVYFC ARSGSYRYGFLY WGQGTLVTVSA, where the three segments marked with bold underline are its three CDR regions, namely HCDR1 (SEQ ID NO:1, GYAFALFL), HCDR2 (SEQ ID NO:2, INPGSGVT) and HCDR3 (SEQ ID NO:3, ARSGSYRYGFLY).

[0133] >12H7-VL (SEQ ID NO:7)

[0134] QIVLTQSPAIMSASLGEEITLTCSAS STLSY MHWYQQKSGTSPKLLIY STS NLASGVPSRFSGSGSGTFYSLTISSVEAEDAADYYC HQWSSYPWT FGGGTKLEIK, where the three segments marked with black underline are its three CDR regions, namely LCDR1 (SEQ ID NO:4, STLSY), LCDR2 (STS) and LCDR3 (SEQ ID NO:5, HQWSSYPWT).

[0135] >12A1-VH (SEQ ID NO:13)

[0136] QVQLQQPGAELVRPGASVRLSCKAS DYTFTNYW IHWVKQRPGQGLEWIGE INPSTGRT NYNERFKYKATLTVDKSSSTTYMQLSGLTSEDSAVYYC VWTTDYPMDYWGQGTSVTVSS, where the three segments marked with black underline are its three CDR regions, namely HCDR4 (SEQ ID NO:8, DYTFTNYW), HCDR5 (SEQ ID NO:9, INPSTGRT) and HCDR6 (SEQ ID NO:10, VWTTDYPMDY).

[0137] >12A1-VL (SEQ ID NO:14)

[0138] DIVMTQSHKLMSTSVGDRVSITCKAS QDVGSA VAWYHQKPGQSPKILIY WAS SRHTGLPDRFTGSGSGTDFTLTISNVQSEDLADYFC QQYRSYPYT FGGGTKLEIK, where the three segments marked with black underline are its three CDR regions, namely LCDR4 (SEQ ID NO:11, QDVGSA), LCDR5 (WAS) and LCDR6 (SEQ ID NO:12).

[0139] Example 5. Preparation of recombinant 12H7 and 12A1 antibodies and expression of control antibody

[0140] The variable regions of the heavy and light chains of 12H7 and 12A1 were spliced ​​with the constant regions of the heavy and κ chains of the human IgG1 subtype, respectively, to construct human-mouse chimeric antibodies. L234A, L235A, and P329G (LALAPG) mutations were introduced into the constant region of the heavy chain of 12H7 to eliminate the binding ability of the antibody's Fc fragment to the CD16A molecule, thus reducing Fc-mediated effector function. An ISO negative control antibody (specifically, an antibody targeting KLH) and the known CD16A positive antibody BMK were simultaneously expressed as experimental controls. The binding ability of the antibody's Fc fragment to the CD16A molecule was also eliminated using the same method as with 12H7, reducing Fc-mediated effector function.

[0141] Based on the antibody sequences designed using the above mutations, the heavy chain (VH+CH) and light chain (VL+CL) gene sequences of recombinant 12H7, 12A1, ISO, and BMK were synthesized (contracted to Wuxi Cysof Biotechnology Co., Ltd.). The heavy chain and light chain genes were cloned into the pCDNA3.1 expression vector, respectively, and named as follows:

[0142] opCDNA3.1-12H7-HC, pCDNA3.1-12H7-LC

[0143] opCDNA3.1-12A1-HC, pCDNA3.1-12A1-LC

[0144] opCDNA3.1-ISO-HC, pCDNA3.1-ISO-LC

[0145] opCDNA3.1-BMK-HC, pCDNA3.1-BMK-LC

[0146] The amino acid sequences of the heavy chain (BMK-VH) and light chain (BMK-VL) of BMK are as follows:

[0147] >BMK-VL (SEQ ID NO:21)

[0148] SYVLTQPSSVSVAPGQTATISCGGHNIGSKNVHWYQQRPGQSPVLVIYQDNKRPSGIPERFSGSNSGNTATLTISGTQAMDEADYYCQVWDNYSVLFGGGTKLTVL

[0149] >BMK-VH (SEQ ID NO:22)

[0150] QVQLVQSGAEVKKPGESLKVSCKASGYTFTSYYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGSAYYYDFADYWGQGTLVTVSS

[0151] The correctness of the inserted gene was verified by restriction enzyme digestion and Sanger sequencing to ensure no missense mutations. Heavy and light chain expression plasmids were mixed at a 1:1 molar ratio and transfected into 293F cells in logarithmic growth phase using TA293 reagent at a DNA concentration of 1 μg / mL. Transfected cells contained the gene sequences of recombinant 12H7 antibody, 12A1 antibody, ISO antibody, and BMK antibody. After transfection, cells were placed in serum-free medium and cultured at 37°C with shaking at 5% CO2 for 7 days. The culture supernatant was collected, filtered through a 0.22 μm filter, and used directly for antibody purification. The supernatant was loaded onto a Protein A affinity column (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd.) to capture the target antibody. Unbound impurities were washed with PBS (pH 7.4), followed by antibody elution with 0.1 M citric acid (pH 3.0). The eluent was collected and immediately neutralized with 1 M Tris (pH 9.0). The purity of recombinant 12H7, 12A1, ISO and BMK antibodies was analyzed by SDS-PAGE;

[0152] The results are as follows Figure 3 As shown, from Figure 3 As can be seen, four recombinant antibodies were prepared through recombinant expression: recombinant 12A1 antibody, recombinant 12H7 antibody, recombinant BMK antibody, and recombinant ISO antibody. The purity of the antibodies was greater than 95% as determined by SDS-PAGE.

[0153] Example 6. Detection of antibody binding to CD16A and CD16B proteins using ELISA.

[0154] Recombinant CD16A-His and CD16B-His proteins (prepared in Example 1) were coated onto 96-well ELISA plates. The coating concentration was 1 μg / mL, using 100 μL / well of protein solution, and incubated overnight at 4°C. The next day, unbound protein was discarded, and the wells were washed three times with PBS and blocked with 5% bovine serum albumin (BSA), then incubated at room temperature for 1 hour. Different concentrations of diluted recombinant antibody were added to the wells coated with CD16A or CD16B protein, 100 μL / well, and incubated at 37°C for 1 hour. After incubation, unbound antibody was discarded, and the wells were washed three times with PBS. HRP-labeled anti-human IgG secondary antibody, diluted according to the manufacturer's instructions, was added to each well, and incubated at 37°C for 30 minutes. After washing again, TMB substrate was added for color development, and incubated at 37°C for 10 minutes. The reaction was terminated with 2 M H2SO4, and the absorbance value (OD450) was measured at 450 nm using an ELISA reader.

[0155] ELISA assay for binding recombinant antibody to CD16A-his antigen as follows: Figure 4 And as shown in Table 2 below.

[0156] Table 2. EC50 of recombinant antibody binding to CD16A-his antigen

[0157]

[0158] ELISA assay for binding recombinant antibody to CD16B-his antigen as follows: Figure 5 And as shown in Table 3 below.

[0159] Table 3. EC50 of recombinant antibody binding to CD16B-his antigen

[0160]

[0161] from Figure 4-5 As can be seen from Tables 2-3 above, the ELISA results demonstrate that the recombinant 12H7 antibody binds to CD16A molecules but not to CD16B molecules, while the recombinant 12A1 antibody weakly binds to CD16A molecules but strongly binds to CD16B molecules.

[0162] Example 7. Detection of the binding of CD16A and CD16B antibodies to CD16A and CD16B using FACS method.

[0163] CD16A-293F cells and CD16B-293F cells, which stably express CD16A and CD16B respectively, constructed in Example 2, were used. Cells were collected, washed with PBS, and the cell concentration was adjusted to 1×10⁻⁶. 6 cells / mL. Dilute the recombinant antibody prepared in Example 5 to 10 μg / mL (or other appropriate concentration) and add 100 μL to the cell suspension (each group of cells is CD16A-293F and CD16B-293F cells prepared in Example 2, respectively). Incubate at 4°C for 30 minutes, gently mixing during this time to ensure that each recombinant antibody binds fully to the cells. Wash the cells twice with PBS to remove unbound antibodies. Add appropriately diluted APC-labeled anti-human IgG secondary antibody, incubate at 4°C for 30 minutes, and wash twice more. Resuspend the cells in 500 μL PBS for flow cytometry analysis. Detect the APC fluorescence signal of the cells using a flow cytometer (CytoFLEX).

[0164] Following the same method described above, flow cytometry was used to detect the binding ability of different antibodies to NK92 cells (NK-92 cells are a natural killer NK cell line derived from lymphoma patients, purchased from ATCC) and native NK cells (obtained by isolating and culturing umbilical cord blood NK cells).

[0165] ELISA assay of recombinant antibody binding to CD16A-293F cells as follows Figure 6 As shown in Table 4 below.

[0166] Table 4. EC50 of recombinant antibody binding to CD16A-293F cells

[0167]

[0168] ELISA assay of recombinant antibody binding to CD16B-293F cells as follows Figure 7 As shown in Table 5 below.

[0169] Table 5. EC50 of recombinant antibody binding to CD16B-293F cells

[0170]

[0171] ELISA assay of recombinant antibody binding to NK92 cells as follows Figure 8 As shown in Table 6 below.

[0172] Table 6. EC50 of recombinant antibody binding to NK92 cells

[0173]

[0174] ELISA assay of recombinant antibody binding to NK92 cells as follows Figure 9 And as shown in Table 7 below.

[0175] Table 7. EC50 of recombinant antibodies and NK cells derived from umbilical cord blood.

[0176]

[0177] From the above Figure 6-9 As shown in Table 4-7, the FACS results demonstrate that the recombinant 12H7 antibody binds to CD16A-overexpressing cells, including CD16A-293F cells, NK92 cells, and NK cells derived from umbilical cord blood, but does not bind to CD16B-293F cells; while the recombinant 12A1 antibody weakly binds to CD16A-293F cells, but strongly binds to CD16B-293F cells.

[0178] Example 8. CD16A antibody-mediated killing assay

[0179] This embodiment aims to demonstrate the mediating ability of antibody 12H7, which targets CD16A, as a universal effector cell activation cytoskeleton by constructing three different bispecific antibody structures. We used the anti-tumor-specific antigen 5T4 antibody VHH as a representative tumor-associated antigen (TAA) targeting element for systematic evaluation:

[0180] a) After the 12H7 backbone is fused with the TAA targeting element, it can effectively mediate CD16A activation and produce killing ability against TAA-positive tumor cells.

[0181] b) The different fusion sites of the TAA targeting element (VHH) on the 12H7 backbone are key structural elements that affect the final bispecific antibody-mediated cell killing activity and efficiency.

[0182] 1. Design and molecular construction of bispecific antibodies

[0183] 1.1. Structural Design

[0184] All constructs were based on the anti-CD16A antibody 12H7 (heavy chain / light chain) backbone and fused with the anti-5T4 VHH fragment. The VHH fragment was linked by a flexible linker (3 G4S sequences). Three antibody forms were designed, denoted by the names in Table 8 below. The specific details of the three antibodies are as follows.

[0185] Table 8

[0186] Antibody name VHH fusion location Heavy chain sequence Light chain sequence 12H7-5T4-VHH(VL-N) N-terminus of the light chain variable region QVQLQQSGAELVRPGTSVKVSCKASGYAFALFLIEWIKQGPGQGLEWIGVINPGSGVTNYNEKFKAKATLTADTSSSTAYMQLSSLTSDDSAVYFCARSGSYRYGFLYWGQGT LVTVSAASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:23) EVQLVESGGGEVQPGGSLRLSCAASERPFGTYAMGWFRQAPGKERDLVAAVSRNAGASYYAESVKGRFTISRDNAKNTMYLQMSSLRAEDTAVYYCAARSAAYSRSSEVYTGKDEYYYWGQGTLVTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASLGEEITLTCSASSTLSYMHWYQQKSGTSPKLLIYSTSNLASGVPSRFSGSGSGTFYSLTISSVEAEDAADYYCHQWSSYPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:24) 12H7-5T4-VHH(VL-C) C-terminus of the light chain constant region QVQLQQSGAELVRPGTSVKVSCKASGYAFALFLIEWIKQGPGQGLEWIGVINPGSGVTNYNEKFKAKATLTADTSSSTAYMQLSSLTSDDSAVYFCARSGSYRYGFLYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:23) QIVLTQSPAIMSASLGEEITLTCSASSTLSYMHWYQQKSGTSPKLLIYSTSNLASGVPSRFSGSGSGTFYSLTISSVEAEDAADYYCHQWSSYPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGGSGGGGSGGGGSEVQLVESGGGEVQPGGSLRLSCAASERPFGTYAMGWFRQAPGKERDLVAAVSRNAGASYYAESVKGRFTISRDNAKNTMYLQMSSLRAEDTAVYYCAARSAAYSRSSEVYTGKDEYYYWGQGTLVTVSS (SEQ ID NO:25) 12H7-5T4-VHH(Fc-C) C-terminal of the heavy chain Fc region QVQLQQSGAELVRPGTSVKVSCKASGYAFALFLIEWIKQGPGQGLEWIGVINPGSGVTNYNEKFKAKATLTADTSSSTAYMQLSSLTSDDSAVYFCARSGSYRYGFLYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSEVQLVESGGGEVQPGGSLRLSCAASERPFGTYAMGWFRQAPGKERDLVAAVSRNAGASYYAESVKGRFTISRDNAKNTMYLQMSSLRAEDTAVYYCAARSAAYSRSSEVYTGKDEYYYWGQGTLVTVSS (SEQ ID NO:26) QIVLTQSPAIMSASLGEEITLTCQIVLTQSPAIMSASLGEEITLTCSASSTLSYMHWYQQKSGTSPKLLIYSTSNLASGVPSRFSGSGSGTFYSLTISSVEAEDAADYYCHQWSSYPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:27)

[0187] 2. Construction and expression carrier

[0188] The heavy chain gene, light chain gene, and corresponding VHH fusion sequence encoding 12H7 were cloned into the eukaryotic expression vector pCDNA3.1. The antibody FC segment was mutated using LALAPG to remove ADCC effects. All sequences (including signal peptide, adapter, VHH, heavy / light chains) were verified to be correct by sequencing.

[0189] 3. Expression and detection of bispecific antibodies

[0190] HEK293 cells were co-transfected with heavy and light chain (or light / heavy chain containing VHH fusion structures) expression plasmids to achieve transient or stable expression of bispecific antibodies. The culture supernatant was collected and purified by Protein A / G affinity chromatography, following the same method as in Example 5. The purified samples were then analyzed by reduced and non-reduced SDS-PAGE to confirm that the molecular weight, assembly state, and subunit composition of each construct were consistent with the theoretical design, ensuring that the obtained bispecific antibodies possessed good purity and structural integrity for subsequent functional experiments.

[0191] 4. Validation of antigen binding activity (ELISA and flow cytometry)

[0192] 4.1. ELISA method for verifying affinity

[0193] Recombinant human CD16A and recombinant human 5T4 proteins were coated onto 96-well plates, respectively. Serially diluted versions of three bispecific antibodies, along with controls (12H7 monoclonal antibody and anti-5T4 VHH), were added. Enzyme-labeled secondary antibody assays were performed. Binding curves were plotted, and the half-maximal binding concentration (EC50) for each antibody against CD16A and 5T4 was calculated. Results are shown in Tables 9-10 and... Figures 13-14 As shown.

[0194] Table 9. ECGs of the binding ability of different forms of antibodies to 5T4 antigen detected by ELISA method 50

[0195]

[0196] Table 10 ECGs of different antibody binding affinity to CD16 antigen detected by ELISA method 50

[0197]

[0198] from Figure 13It can be seen that when the anti-5T4 VHH fragment is fused to the N-terminus of the 12H7 antibody light chain (12H7-5T4-VHH(VL-N)), its binding ability to 5T4 is basically the same as that of free VHH, and the resulting EC 50 The similar values ​​indicate that this fusion method effectively maintains the original affinity of VHH for the 5T4 antigen. In contrast, when the VHH fragment was fused to the C-terminus of the light chain (VL-C) or the C-terminus of the Fc region (Fc-C), the binding affinity of the construct to 5T4 decreased to varying degrees, manifested as significantly weakened antigen-binding activity. This suggests that fusion at these locations may lead to spatial conformational constraints or insufficient flexibility in the linker region, thereby affecting the effective epitope recognition ability of VHH.

[0199] from Figure 14 It can be seen that the VHH fusion affects CD16A binding activity. Regardless of the VHH fusion position, all three constructs (VL-N, VL-C, and Fc-C) retain the specific binding ability of the 12H7 antibody framework to CD16A. No significant negative impact of VHH fusion on CD16A affinity was observed, indicating that the CD16A binding site was not disrupted. That is, VHH fusion did not disrupt the Fc region-mediated CD16A binding site, nor did it adversely affect its structural stability.

[0200] 4.2 Flow cytometry verification of dual binding

[0201] The A549 tumor cell line, which highly expresses the 5T4 molecule, was selected as the target cell line to evaluate the target binding ability of the prepared anti-5T4 antibody and its derived constructs. The test antibody was added to A549 cells in logarithmic growth phase and incubated to allow for sufficient binding of the antibody to the naturally occurring 5T4 antigen on the cell surface. After incubation, unbound antibody was removed by washing, and the cells were stained with fluorescently labeled secondary antibody. The antibody binding was then quantitatively analyzed by flow cytometry. Results are as follows: Figure 15 As shown.

[0202] from Figure 15 Further flow cytometry (FACS) analysis showed that the results were consistent with the overall trend of ELISA: the VL-N construct had the best binding ability to 5T4 on the cell surface, while the binding signal of the VL-C and Fc-C constructs was significantly weakened. These results together confirm that the fusion position of VHH has a significant impact on the binding ability to 5T4, but has no significant impact on the binding of CD16A.

[0203] 5. Killing activity detection

[0204] 5.1 Experimental Materials and Conditions

[0205] In this embodiment, the human non-small cell lung cancer cell line A549, which highly expresses 5T4, was selected as the target cell to evaluate the targeting binding and cytotoxic effects of bispecific antibodies. NK92-CD16A cells stably expressing CD16A were used as effector cells to mimic antibody-dependent cell-mediated cytotoxicity (ADCC) responses. After optimization through preliminary experiments, the effector-to-target ratio (E:T) was set to 2:1 to obtain the most discriminative cytotoxicity detection window.

[0206] 5.2 Experimental Procedures and Detection Methods

[0207] (1) A549 target cells in logarithmic growth phase were mixed with NK92-CD16A effector cells at a ratio of E:T = 2:1, and bispecific antibody constructs of different gradient concentrations were added. The system was incubated at 37°C and 5% CO2 for 24 hours to allow the bispecific antibody to fully bind to the 5T4 target site and mediate the activation of CD16A.

[0208] (2) After incubation, the activity of the remaining target cells was detected by the CCK-8 method. The specific killing rate of the antibody was obtained by calculating the change in cell survival rate, and then the cytotoxic efficacy of the bispecific antibody was evaluated.

[0209] 5.3 Validation of CD16A-dependent cell killing function

[0210] Experimental results showed that, at all tested antibody concentrations, the three different conformations of the 12H7-5T4-VHH bispecific antibody all exhibited significant, dose-dependent cytotoxic activity. This result demonstrates that:

[0211] (1) 12H7, as the parent antibody against CD16A, retains its CD16A binding and activation functions completely after being fused with anti-5T4VHH;

[0212] (2) The tandem design of the anti-CD16A and anti-5T4 functional modules in the same molecule effectively realizes the spatial bridging between target cells and effector cells, thereby inducing NK92-CD16A cells to produce a strong targeted cytotoxic response.

[0213] (3) This bispecific antibody successfully overcomes the structural defect that a single antibody cannot simultaneously achieve tumor targeting and effector cell recruitment, thus improving the killing efficiency of tumor cells.

[0214] 5.4 Effect of VHH fusion position on the killing activity of bispecific antibodies

[0215] By comparing the dose-response curves of bispecific antibodies obtained using three different fusion strategies, the results are as follows: Figure 16As shown, the fusion position of VHH has a significant impact on the overall cytotoxic efficacy of the antibody.

[0216] (1) Activity ranking

[0217] Under the same conditions, the cytotoxicity of the three constructs was compared, and the activities from highest to lowest were as follows:

[0218] 12H7-5T4-VHH(VL-N) > 12H7-5T4-VHH(VL-C) > 12H7-5T4-VHH(Fc-C)

[0219] Among them, the construct in which the anti-5T4 VHH is fused to the N-terminus (VL-N) of the 12H7 antibody light chain exhibits the best killing activity.

[0220] (2) EC 50 analyze

[0221] EC 50 The analysis results are shown in Table 11.

[0222] Table 11

[0223]

[0224] The 12H7-5T4-VHH (VL-N) construct exhibited the strongest cytotoxic effect, with its EC50... 50 For: EC 50 =0.003062 μg / mL; in comparison, the weakest Fc-C construct (12H7-5T4-VHH(Fc-C)) had an EC50 of 0.003062 μg / mL. 50 Gundam:

[0225] EC 50 = 0.03977 μg / mL; the difference between the two is about 13 times, indicating that the VL-N end fusion strategy is significantly better than the other two structural forms.

[0226] (3) Maximum lethality Emax

[0227] Despite the existence of EC 50 The difference lies in the fact that the 12H7-5T4-VHH (Fc-C) construct achieved the highest maximum killing rate (Emax = 92%) under high-dose conditions, indicating that this construct still possesses high effector cell mobilization capacity under fully activated conditions, but its overall sensitivity (ECG) is lower. 50 It is inferior to the VL-N construct.

[0228] Based on the above results, we can conclude that:

[0229] The fusion position of the anti-5T4 VHH fragment in the 12H7 antibody framework is an important structural factor that determines the final cytotoxic activity of this bispecific antibody.

[0230] The fusion mode of the light chain N-terminus (VL-N) provides the most favorable spatial conformation and molecular flexibility, enabling the VHH fragment to more effectively recognize tumor epitopes, thereby achieving optimal CD16A activation and cell killing ability.

[0231] In contrast, when VHH is fused to the C-terminus or Fc-terminus of the light chain, conformational limitations may lead to a decrease in antigen binding efficiency, thereby affecting overall cytotoxic activity.

[0232] Therefore, the 12H7-5T4-VHH (VL-N) bispecific antibody construction mode proposed in this invention shows significant advantages in terms of structural rationality, functional synergy, and cell killing efficiency, providing a more promising antibody engineering design strategy for clinical applications.

[0233] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0234] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. An antibody that specifically binds to CD16A or an antigenic binding fragment thereof, comprising a first heavy chain variable region and a first light chain variable region, wherein the first heavy chain variable region contains HCDR1, HCDR2, and HCDR3, the sequences of HCDR1, HCDR2, and HCDR3 respectively comprising the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; and the first light chain variable region contains LCDR1, LCDR2, and LCDR3, the sequences of LCDR1, LCDR2, and LCDR3 respectively comprising the amino acid sequences shown in SEQ ID NO:4, STS, and SEQ ID NO:

5.

2. The antibody that specifically binds to CD16A or its antigenic binding fragment according to claim 1, characterized in that, The sequences of HCDR1, HCDR2, and HCDR3 are as shown in positions 26-33, 51-58, and 97-108 of SEQ ID NO:6, respectively, or have 90% or more homology with the sequences shown in positions 26-33, 51-58, and 97-108 of SEQ ID NO:6, respectively; the sequences of LCDR1, LCDR2, and LCDR3 contain positions 27-31, 49-51, and 88-97 of SEQ ID NO:7, respectively, or have 90% or more homology with the sequences shown in positions 27-31, 49-51, and 88-97 of SEQ ID NO:7, respectively. Preferably, the antibody that specifically binds to CD16A or its antigenic binding fragment comprises a first heavy chain and a first light chain, wherein the sequence of the first heavy chain is as shown in SEQ ID NO:6 or has 90% or more homology with it, and the sequence of the first light chain is as shown in SEQ ID NO:7 or has 90% or more homology with it.

3. An antibody that strongly binds to CD16B but weakly binds to CD16A, or an antigenic binding fragment thereof, comprising a second heavy chain variable region and a second light chain variable region, wherein the second heavy chain variable region contains HCDR4, HCDR5, and HCDR6, the sequences of said HCDR4, HCDR5, and HCDR6 respectively comprising the amino acid sequences shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; and the second light chain variable region contains LCDR4, LCDR5, and LCDR6, the sequences of said LCDR4, LCDR5, and LCDR6 respectively comprising the amino acid sequences shown in SEQ ID NO:11, WAS, and SEQ ID NO:

12.

4. The antibody or its antigenic binding fragment that strongly binds to CD16B but weakly binds to CD16A according to claim 3, characterized in that, The sequences of HCDR4, HCDR5, and HCDR6 are as shown in positions 26-33, 51-58, and 97-106 of SEQ ID NO:13, respectively, or have 90% or more homology with the sequences shown in positions 26-33, 51-58, and 97-106 of SEQ ID NO:13, respectively; the sequences of LCDR4, LCDR5, and LCDR6 are as shown in positions 27-32, 50-52, and 89-97 of SEQ ID NO:14, respectively, or have 90% or more homology with the sequences shown in positions 27-32, 50-52, and 89-97 of SEQ ID NO:14, respectively. Preferably, the antibody that strongly binds to CD16B but weakly binds to CD16A, or its antigenic binding fragment, comprises a second heavy chain and a second light chain, wherein the sequence of the second heavy chain is as shown in SEQ ID NO:13 or has 90% or more homology with it, and the sequence of the second light chain is as shown in SEQ ID NO:14 or has 90% or more homology with it.

5. A nucleic acid molecule encoding an antibody that specifically binds to CD16A as described in claim 1 or 2, or an antibody that strongly binds to CD16B but weakly binds to CD16A as described in claim 3 or 4, or an nucleic acid molecule that antigenically binds to CD16A.

6. A construct comprising the nucleic acid molecule of claim 5, preferably, the construct being a chimeric antigen construct or a vector.

7. A cell expressing an antibody that specifically binds to CD16A as described in claim 1 or 2, or an antigenic binding fragment thereof, an antibody that strongly binds to CD16B but weakly binds to CD16A as described in claim 3 or 4, or a chimeric antigen construct as described in claim 6.

8. A bispecific antibody, comprising the antibody specifically binding to CD16A as described in claim 1 or 2, or the antibody strongly binding to CD16B but weakly binding to CD16A as described in claim 3 or 4, and the antibody antigenically binding fragment thereof. Antibodies or antigenic fragments that bind to tumor antigens, or antibodies or antigenic fragments that bind to inflammatory factors, wherein... The tumor antigen is preferably HER2, PD-L1, CD33, PSMA, FAP, ROR1, HER3, CD19, CD20, BCMA, CD22, CD38, CD123, 5T4, or EGFR; Preferably, the bispecific antibody comprises the antibody that specifically binds to CD16A as described in claim 1 or 2, or its antigenic binding fragment, and the antibody or antigenic fragment that binds to tumor antigen 5T4. More preferably, the bispecific antibody is 12H7-5T4-VHH(VL-N), 12H7-5T4-VHH(VL-C), or 12H7-5T4-VHH(Fc-C). The heavy chain sequences of 12H7-5T4-VHH(VL-N) and 12H7-5T4-VHH(VL-C) are shown in SEQ ID NO:

23. The heavy chain sequence of 12H7-5T4-VHH(VL-N) is shown in SEQ ID NO:

24. The light chain sequence of 12H7-5T4-VHH(VL-C) is shown in SEQ ID NO:

25. The heavy chain sequence of 12H7-5T4-VHH(Fc-C) is shown in SEQ ID NO:

26. The light chain sequence of 12H7-5T4-VHH(Fc-C) is shown in SEQ ID NO:

27.

9. A trispecific antibody comprising an antibody or antigenic binding fragment thereof that specifically binds to CD16A as described in claim 1 or 2, an antibody or antigenic binding fragment thereof that targets an immunomodulatory factor, or an antibody or antigenic fragment thereof that binds to a tumor antigen, preferably wherein the immunomodulatory factor comprises CD3, IL-15, IL21, IL12, IL10 or PD-1.

10. A combination target polyclonal antibody comprising the antibody that specifically binds to CD16A as described in claim 1 or 2, or the antigenic binding fragment thereof; the antibody that strongly binds to CD16B but weakly binds to CD16A as described in claim 3 or 4, or the antigenic binding fragment thereof; and an antibody or antigenic binding fragment that binds to an immunomodulatory factor.

11. A pharmaceutical composition comprising an antibody that specifically binds to CD16A as described in claim 1 or 2, an antibody that strongly binds to CD16B but weakly binds to CD16A as described in claim 3 or 4, or a cell-expressed antibody as described in claim 7.

12. The pharmaceutical composition according to claim 11, characterized in that, The pharmaceutical composition further includes nanoparticles, small molecule compounds, ADC drugs, or immune cells. When the pharmaceutical composition includes nanoparticles, the pharmaceutical composition is a drug delivery carrier. Preferably, the immune cells are CAR-NK or CAR-T cells.

13. The use of the antibody that specifically binds to CD16A or the antigenic binding fragment thereof according to claim 1 or 2, the antibody that strongly binds to CD16B but weakly binds to CD16A or the antigenic binding fragment thereof according to claim 3 or 4, the nucleic acid molecule according to claim 5, the construct according to claim 6, the cell according to claim 7, the bispecific antibody according to claim 8, the trispecific antibody according to claim 9, the combined target polyclonal antibody according to claim 10, or the pharmaceutical composition according to claim 11 or 12 in the preparation of anticancer drugs, anti-infective disease drugs, inflammation-related disease drugs, autoimmune-related disease drugs, immune complex-related disease drugs, or hematological disease drugs; Preferably, the infectious disease is HIV, HBV, or bacterial sepsis; or Preferably, the inflammation-related diseases include inflammatory diseases and inflammatory diseases caused by systemic immune disorders, such as allergic purpura, systemic lupus erythematosus, rheumatoid arthritis, acute respiratory distress syndrome (ARDS), or inflammatory bowel disease (such as Crohn's disease); or Preferably, the autoimmune-related disease is rheumatoid arthritis or systemic lupus erythematosus; or Preferably, the blood disease is immune thrombocytopenic purpura (ITP) or neutrophil-related disease.

14. The use of the antibody that specifically binds to CD16A according to claim 1 or 2, or the antigenic binding fragment thereof that strongly binds to CD16B but weakly binds to CD16A according to claim 3 or 4, in detecting cells expressing CD16A or CD16B, or in preparing reagents for detecting cells expressing CD16A or CD16B.

15. The use of the antibody that specifically binds to CD16A according to claim 1 or 2, or its antigenic binding fragment, in activating ADCC of NK cells or in preparing reagents for activating ADCC of NK cells.

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