Anti-CD16A antibody and its use

Abstract

The invention provides an anti-CD16A antibody and its use, wherein the antibody comprises heavy chain variable regions CDR1, CDR2, and CDR3 sequences shown in amino acid sequences having at least 95% identity with SEQ ID NO: 1, 2, and 3, respectively, and / or light chain variable regions CDR1, CDR2, and CDR3 sequences shown in amino acid sequences having at least 95% identity with SEQ ID NO: 4, 5, and 6, respectively.

Description

[Technical Field]

[0001] This invention relates to the field of biopharmaceuticals, and more specifically to anti-CD16A antibodies and their use. [Background technology]

[0002] Natural killer cells (NK cells) are a crucial component of the intrinsic immune system. Unlike T cells, NK cells do not express antigen-specific receptors. NK cells themselves possess broad tumor-killing capabilities and play a vital role in enhancing antibody and T cell responses. Currently, there are diverse forms of NK cell-based tumor immunotherapy, and a variety of techniques are employed. A decrease in the number or dysfunction of NK cells is associated with the progression of various cancers. "Cold tumors" are immunosensitive and express very few or no MHC I system molecules on their surface, making them largely unrecognizable by T cells, but they can be recognized and killed by NK cells. The introduction of this concept has elevated the status of NK cells in anti-tumor immunotherapy.

[0003] The CD16 molecule is an important label on the surface of NK cells and can activate the IgE NK cell receptor (FcεRIγ) and the CD3ζ immunoreceptor tyrosine activation motif (ITAM) to induce ADCC activity. For this reason, CD16 targets are preferentially selected in bispecific antibodies based on NK cell retargeting.

[0004] CD16 targets have been widely selected for bispecific antibodies based on NK cell retargeting (McCall et al., 1999, Gleason et al., 2014). AFM13 is a bispecific antibody against CD16A and CD30 molecules, promoting the killing of CD30+ non-Hodgkin lymphoma cells by NK cells (Reusch et al., 2014, Pahl et al., 2018). For other NK cell-related bispecific antibodies, researchers have constructed triplicate antibodies (NKCEs) using CD16, NKp46, and tumor targets. NKCEs have stronger in vitro killing capacity than clinically therapeutic monoclonal antibodies, while also exhibiting superior in vivo stability and tumor control capabilities (Gauthier et al., 2019).

[0005] Human CD16 can be divided into CD16A and CD16B. CD16A is mainly expressed in NK cells. CD16B is mainly produced by neutrophils, and a soluble form of the receptor is also present in human serum. CD16A has numerous alleles among individuals; for example, there is polymorphism at position 158. 158V CD16A 158F It has a higher affinity for the antibody Fc domain than CD16B. CD16B exhibits polymorphisms among people, i.e., CD16B NA1 CD16B NA2 and CD16B SH Therefore, previous CD16 antibodies (e.g., 3G8) basically recognize CD16 but do not specifically recognize CD16A. Thus, in the design of bispecific or polyspecific antibodies, it is necessary to develop an antibody that recognizes CD16A in order to specifically target NK cells. [Overview of the project] [Problems that the invention aims to solve]

[0006] This application is submitted based on the inventors' findings regarding the following problems and facts.

[0007] NK cells themselves possess broad tumor-killing capabilities and play an important role in enhancing antibody and T-cell responses. However, the CD16A protein is primarily expressed in NK cells, and CD16 targets are preferentially selected by bispecific antibodies based on NK cell retargeting.

[0008] The inventors of this application have successfully screened mouse-derived anti-CD16A monoclonal antibodies that have high binding activity to human or monkey CD16A proteins. Furthermore, the inventors have obtained a fully humanized anti-CD16A antibody by humanizing the constant region of the monoclonal antibody, leaving the CDR of the mouse-derived anti-CD16A monoclonal antibody, and further humanizing the frame region in the light chain variable region or heavy chain variable region of the chimeric antibody. This humanized antibody not only specifically targets and binds to human and monkey CD16A proteins, but also has the advantage of low immunogenicity, and can effectively treat and / or prevent CD16A-mediated autoimmune diseases and other related diseases.

[0009] Furthermore, the bispecific or polyspecific conjugating molecules prepared using the anti-CD16A antibody similarly specifically target and bind to human CD16A protein and monkey CD16 protein. Typically, based on the specificity of the bispecific or polyspecific conjugating molecule, these molecules can target NK cells to other antigens. The NK cell-mediated cytotoxicity then eliminates cells that produce such antigens, thereby treating various diseases such as tumors. [Means for solving the problem]

[0010] Therefore, in a first aspect of the present invention, the present invention provides an antibody or antigen-binding fragment. According to the examples of the present invention, the heavy chain variable regions CDR1, CDR2, CDR3 sequences and / or SEQ ID NO: 1, 2, and 3 are represented by amino acid sequences that have at least 95% identity with SEQ ID NO: 1, 2, and 3, respectively. It comprises light chain variable region CDR1, CDR2, and CDR3 sequences shown by amino acid sequences having at least 95% identity with NO: 4, 5, and 6 or 4, 5, and 6. According to an embodiment of the present invention, the antibody or antigen-binding fragment can bind to human or monkey CD16A protein and effectively treat or prevent CD16A-mediated related diseases.

[0011] According to an embodiment of the present invention, the antibody or antigen-binding fragment may further comprise at least one of the following additional technical features.

[0012] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises at least one of the heavy chain FR region and the light chain FR region.

[0013] According to an embodiment of the present invention, at least a part of at least one of the heavy chain FR region and the light chain FR region is derived from at least one of human-derived antibodies, primate-derived antibodies, mouse-derived antibodies, or their variants.

[0014] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises at least one of the heavy chain frame regions HFR1, HFR2, HFR3, and HFR4 sequences shown by SEQ ID NO: 7-10 respectively, or at least one of the heavy chain frame regions HFR1, HFR2, HFR3, and HFR4 sequences shown by SEQ ID NO: 15-18 respectively.

[0015] According to an embodiment of the present invention, the antibody or antigen-binding fragment comprises at least one of the light chain frame regions LFR1, LFR2, LFR3, and LFR4 sequences shown by SEQ ID NO: 11-14 respectively, or at least one of the light chain frame regions LFR1, LFR2, LFR3, and LFR4 sequences shown by SEQ ID NO: 19-22 respectively.

[0016] According to an embodiment of the present invention, the antibody or antigen-binding fragment includes at least one of the heavy chain framework region HFR1, HFR2, HFR3, and HFR4 sequences shown in SEQ ID NOs: 7 to 10, at least one of the light chain framework region LFR1, LFR2, LFR3, and LFR4 sequences shown in SEQ ID NOs: 11 to 14, or at least one of the heavy chain framework region HFR1, HFR2, HFR3, and HFR4 sequences shown in SEQ ID NOs: 15 to 18, and at least one of the light chain framework region LFR1, LFR2, LFR3, and LFR4 sequences shown in SEQ ID NOs: 19 to 22.

[0017] According to an embodiment of the present invention, the antibody or antigen-binding fragment includes a heavy chain variable region shown in SEQ ID NO: 23 or SEQ ID NO: 25, and / or a light chain variable region shown in SEQ ID NO: 24 or SEQ ID NO: 26.

[0018] According to an embodiment of the present invention, the antibody or antigen-binding fragment is 1) a heavy chain variable region shown in SEQ ID NO: 23 and a light chain variable region shown in SEQ ID NO: 24, or 2) a heavy chain variable region shown in SEQ ID NO: 25 and a light chain variable region shown in SEQ ID NO: 26. ID NO: 26.

[0019] According to an embodiment of the present invention, the antibody or antigen-binding fragment contains at least one of a heavy chain constant region and a light chain constant region, and at least a part of at least one of the heavy chain constant region and the light chain constant region is derived from at least one of a human-derived antibody, a primate-derived antibody, a mouse-derived antibody, or variants thereof.

[0020] According to an embodiment of the present invention, both the light chain constant region and the heavy chain constant region are derived from a mouse-derived IgG antibody or variants thereof or a human-derived IgG antibody or variants thereof.

[0021] According to embodiments of the present invention, both the light chain constant region and the heavy chain constant region are derived from mouse-derived IgG1 antibodies or their variants, or from human-derived IgG1 antibodies or their variants.

[0022] According to an embodiment of the present invention, the antibody has a heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 27 or 29 and / or a light chain constant region of the amino acid sequence shown in SEQ ID NO: 28 or 30.

[0023] According to an embodiment of the present invention, the antibody or antigen-binding fragment has a heavy chain of amino acid sequences shown in any of SEQ ID NO: 31, 33, and 35 and a light chain of amino acid sequences shown in any of SEQ ID NO: 32, 34, and 36.

[0024] According to embodiments of the present invention, the antibody or antigen-binding fragment has a heavy chain of the amino acid sequence shown in SEQ ID NO:31 and a light chain of the amino acid sequence shown in SEQ ID NO:32, the antibody or antigen-binding fragment has a heavy chain of the amino acid sequence shown in SEQ ID NO:33 and a light chain of the amino acid sequence shown in SEQ ID NO:34, or the antibody or antigen-binding fragment has a heavy chain of the amino acid sequence shown in SEQ ID NO:35 and a light chain of the amino acid sequence shown in SEQ ID NO:36.

[0025] According to embodiments of the present invention, the antibody or antigen-binding fragment includes a monoclonal antibody or a polyclonal antibody.

[0026] According to embodiments of the present invention, the monoclonal antibody comprises at least one of Fv, a single-chain antibody, Fab, a single-domain antibody, and a minimal recognition unit.

[0027] According to embodiments of the present invention, the antibody or its antigen-binding fragment can bind to the amino acid sequence shown in SEQ ID NO: 37 and / or 38.

[0028] In a second aspect of the present invention, the present invention proposes a bispecific binding molecule. According to an embodiment of the present invention, the molecule comprises a first binding region containing an antibody or antigen-binding fragment as described in the first aspect, and a second binding region having BCMA or B7H6 binding activity. The bispecific binding molecule according to the embodiment of the present invention can bind to human or monkey CD16A protein and BCMA protein, or to human or monkey CD16A protein and B7H6 protein, and can be used in scientific research, or can effectively treat or prevent CD16A and BCMA, or CD16A and B7H6-mediated related diseases.

[0029] As those skilled in the art will understand, the binding activity of the second binding region is not particularly limited and may have other binding activities, and the bispecific antibody has the antibody or antigen-binding fragment described in the first embodiment, and it is sufficient that both the antibody or antigen-binding fragment and the second binding region effectively exert their functions. Furthermore, it is possible to prepare more specific antibodies, such as triplicate, quadruple-specific, or quintuple-specific antibodies, using the antibody or antigen-binding fragment described in this application, and based on the multispecificity of the antibody, the antibody or antigen-binding fragment of the present invention can target NK cells to other antigens, and cells that produce such antigens are eliminated by the cytotoxic effect mediated by NK cells.

[0030] According to embodiments of the present invention, the bispecific binding molecule may further include at least one of the following additional technical features.

[0031] According to embodiments of the present invention, the bispecificity binding molecule includes a symmetric bispecificity binding molecule or an asymmetric bispecificity binding molecule.

[0032] According to embodiments of the present invention, the bispecificity binding molecule is an asymmetric bispecificity binding molecule.

[0033] According to an embodiment of the present invention, the antibody or antigen-binding fragment is an anti-CD16A single-chain antibody.

[0034] According to embodiments of the present invention, the second binding region is at least one of a full-length antibody having BCMA or B7H6 binding activity, Fv, a single-chain antibody, Fab, a single-domain antibody, and a minimal recognition unit.

[0035] According to embodiments of the present invention, the second binding region contains an anti-BCMA single-chain antibody or an anti-B7H6 single-chain antibody.

[0036] According to an embodiment of the present invention, the anti-CD16A single-chain antibody comprises an anti-CD16A antibody light chain variable region and an anti-CD16A antibody heavy chain variable region, wherein the anti-CD16A antibody heavy chain variable region has an amino acid sequence shown in SEQ ID NO: 23 or 25, and the anti-CD16A antibody light chain variable region has an amino acid sequence shown in SEQ ID NO: 24 or 26.

[0037] According to an embodiment of the present invention, the anti-CD16A single-chain antibody further comprises a linked peptide 1, the N-end of the linked peptide 1 is connected to the C-end of the variable region of the anti-CD16A antibody heavy chain, The C-end of linked peptide 1 is connected to the N-end of the anti-CD16A antibody light chain variable region, or the N-end of linked peptide 1 is connected to the C-end of the anti-CD16A antibody light chain variable region and the C-end of linked peptide 1 is connected to the N-end of the anti-CD16A antibody heavy chain variable region.

[0038] According to an embodiment of the present invention, the anti-BCMA single-chain antibody comprises an anti-BCMA antibody light chain variable region and an anti-BCMA antibody heavy chain variable region, wherein the anti-BCMA antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 59, and the anti-BCMA antibody light chain variable region has the amino acid sequence shown in SEQ ID NO: 60.

[0039] According to embodiments of the present invention, the anti-BCMA single-chain antibody further comprises a linked peptide 2, wherein the N end of the linked peptide 2 is connected to the C end of the heavy chain variable region of the anti-BCMA antibody, the C end of the linked peptide 2 is connected to the N end of the light chain variable region of the anti-BCMA antibody, or the N end of the linked peptide 2 is connected to the C end of the light chain variable region of the anti-BCMA antibody, and the C end of the linked peptide 2 is connected to the N end of the heavy chain variable region of the anti-BCMA antibody.

[0040] According to an embodiment of the present invention, the anti-B7H6 single-chain antibody comprises an anti-B7H6 antibody light chain variable region and an anti-B7H6 antibody heavy chain variable region, wherein the anti-B7H6 antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 61, and the anti-B7H6 antibody light chain variable region has the amino acid sequence shown in SEQ ID NO: 62.

[0041] According to embodiments of the present invention, the anti-B7H6 single-chain antibody further comprises a linked peptide 3, wherein the N end of the linked peptide 3 is connected to the C end of the heavy chain variable region of the anti-B7H6 antibody, the C end of the linked peptide 3 is connected to the N end of the light chain variable region of the anti-B7H6 antibody, or the N end of the linked peptide 3 is connected to the C end of the light chain variable region of the anti-B7H6 antibody, and the C end of the linked peptide 3 is connected to the N end of the heavy chain variable region of the anti-B7H6 antibody.

[0042] According to embodiments of the present invention, at least one of the linked peptide 1, linked peptide 2, and linked peptide 3 has an amino acid sequence (GGGGS)n, where n is an integer of 1 or more, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. As those skilled in the art will understand, ordinary flexible amino acid fragments or rigid amino acid fragments can be used as the usual linked peptides in the art.

[0043] According to an embodiment of the present invention, at least one of the linked peptide 1, linked peptide 2, and linked peptide 3 has the amino acid sequence shown in SEQ ID NO:44.

[0044] According to an embodiment of the present invention, the anti-CD16A single-chain antibody has the amino acid sequence shown in SEQ ID NO:41.

[0045] According to an embodiment of the present invention, the anti-BCMA single-chain antibody has the amino acid sequence shown in SEQ ID NO:42.

[0046] According to an embodiment of the present invention, the anti-B7H6 single-chain antibody has the amino acid sequence shown in SEQ ID NO:43.

[0047] According to embodiments of the present invention, the first antigen-binding region further comprises a first Fc peptide segment, the N-end of the first Fc peptide segment being connected to the C-end of the antibody or antigen-binding fragment.

[0048] According to embodiments of the present invention, the second antigen-binding region further comprises a second Fc peptide segment, and the N-terminus of the second Fc peptide segment is the anti-BCMA single-chain antibody or the anti It is attached to the C-end of the B7H6 single-chain antibody.

[0049] According to an embodiment of the present invention, the first Fc peptide segment has the amino acid sequence shown in SEQ ID NO:45.

[0050] According to an embodiment of the present invention, the second Fc peptide segment has the amino acid sequence shown in SEQ ID NO:46.

[0051] According to embodiments of the present invention, the first Fc peptide segment and the second Fc peptide segment are connected by a knob-into-hole structure. As those skilled in the art will understand, the amino acid sequences of the first Fc peptide segment and the second Fc peptide segment may be the same or different. For example, if the amino acid sequences of the first Fc peptide segment and the second Fc peptide segment are the same, they may be connected by a disulfide bond.

[0052] According to an embodiment of the present invention, the first antigen-binding region has the amino acid sequence shown in SEQ ID NO: 47, and the second antigen-binding region has the amino acid sequence shown in SEQ ID NO: 48 or 49.

[0053] The antibody or its antigen-binding fragment or the nucleic acid encoding the bispecific binding molecule of the present invention falls within the scope of the present invention, and based on its amino acid sequence, those skilled in the art can easily obtain the corresponding nucleic acid sequence.

[0054] Therefore, in a third aspect of the present invention, the present invention provides a nucleic acid molecule that encodes an antibody or antigen-binding fragment or the bispecific binding molecule described in the first aspect. The antibody or antigen-binding fragment encoded by the nucleic acid molecule in some specific embodiments of the present invention can bind to human or monkey CD16A protein to effectively treat or prevent CD16A-mediated related diseases, and the bispecific binding molecule encoded by the nucleic acid molecule can bind to human or monkey CD16A protein and BCMA protein, or to human or monkey CD16A protein and B7H6 protein to effectively treat or prevent CD16A-BCMA, or CD16A-B7H6-mediated related diseases.

[0055] According to embodiments of the present invention, the nucleic acid molecule may further include at least one of the following additional technical features.

[0056] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0057] Furthermore, the nucleic acids referred to in the specification and claims of this invention include, in practice, either or both of the complementary double strands, as can be understood by those skilled in the art. For convenience, although in many cases only one strand is shown in this specification and claims, the complementary other strand is also disclosed. In addition, nucleic acid sequences in this application include either DNA or RNA form, and disclosing one means disclosing the other.

[0058] In a fourth aspect of the present invention, the present invention provides an expression vector supporting the nucleic acid molecule described above. The expression vector may include a selectable control sequence that is operably connected to the nucleic acid molecule. The control sequence is one or more control sequences that can guide the expression of the nucleic acid molecule in a host. The expression vectors provided in the examples of the present invention can efficiently express the antibody or antigen-binding fragment in large quantities in suitable host cells.

[0059] In this specification, "operably connected" means that by connecting an exogenous gene to a vector, regulatory elements within the vector, such as transcriptional and translational regulatory sequences, can exert their function of regulating the transcription and translation of the expected exogenous gene. When connecting the nucleic acid molecule to the vector, the nucleic acid molecule and the regulatory elements on the vector can be connected directly or indirectly, and these regulatory elements should control the translation and expression of the nucleic acid molecule. Of course, these regulatory elements may be directly derived from the vector itself or may be exogenous, i.e., not derived from the vector itself. As those skilled in the art will understand, nucleic acid molecules for encoding antibodies or antigen-binding fragments can be inserted into different vectors independently, but it is common to see them inserted into the same vector. Commonly used vectors may be plasmids, bacteriophages, etc. For example, Plasmid-X plasmid.

[0060] In a fifth aspect of the present invention, the present invention provides a method for preparing the antibody or antigen-binding fragment or bispecific binding molecule described above, comprising the steps of introducing the expression vector described above into cells and culturing the cells under conditions suitable for protein expression and secretion to obtain the antibody or antigen-binding fragment or bispecific binding molecule. The methods provided in some specific embodiments of the present invention can effectively obtain the antibody or antigen-binding fragment or bispecific binding molecule in large quantities in vitro.

[0061] According to some specific embodiments of the present invention, the method for preparing the antibody or antigen-binding fragment or bispecificity-binding molecule described above may further include at least one of the following additional technical features.

[0062] According to some specific embodiments of the present invention, the cells are not particularly limited and prokaryotic or eukaryotic cells can be used.

[0063] According to some specific embodiments of the present invention, the cells are eukaryotic cells.

[0064] According to some specific embodiments of the present invention, the eukaryotic cells are mammalian cells. According to some specific embodiments of the present invention, when the cells are eukaryotic cells, for example mammalian cells, the expression efficiency of the recombinant antibody is high.

[0065] In a sixth aspect of the present invention, the present invention provides recombinant cells that can express the nucleic acids or expression vectors described above, or the antibodies or antigen-binding fragments or the bispecificity binding molecules described above. The recombinant cells are transfected or transformed with the expression vector. According to some specific embodiments of the present invention, the recombinant cells can efficiently express the antibodies or antigen-binding fragments or the bispecificity binding molecules in large quantities under appropriate conditions.

[0066] It should be noted that the recombinant cells described in the present invention are not particularly limited and may be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells may include Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis. The eukaryotic cells include fungi such as Pasteurella pastris, budding yeast, fission yeast, and Trichoderma, insect cells such as Spidoptera fulgiperda, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells. In some examples, the recombinant cells described in the present invention are preferably mammalian cells and include BHK cells, CHO cells, NSO cells, or COS cells, but do not include animal germ cells, fertilized eggs, or embryonic stem cells.

[0067] Furthermore, the "appropriate conditions" described in the specification of this application refer to conditions suitable for the expression of the antibody or antigen-binding fragment described in this application, or the bispecific binding molecule. To facilitate this, suitable conditions for the expression of an antibody or antigen-binding fragment or the bispecific binding molecule include a suitable transformation or transfection scheme, suitable transformation or transfection conditions, a healthy host cell state, a suitable host cell density, a suitable cell culture environment, and a suitable cell culture time. “Suitable conditions” are not particularly limited, and those skilled in the art will optimize the optimal conditions for the expression of the antibody or antigen-binding fragment according to the specific environment of their laboratory.

[0068] In a seventh aspect of the present invention, the present invention provides an immunoconjugate comprising the antibody or antigen-binding fragment or the bispecificity binding molecule described above, and a therapeutic agent. As described above, the antibody or antigen-binding fragment of the embodiment of the present invention can effectively bind to the CD16A protein, and the bispecific binding molecule can bind to human or monkey CD16A protein and BCMA protein, or to human or monkey CD16A protein and B7H6 protein, and thus can effectively treat or prevent related diseases mediated by CD16A and BCMA, or CD16A and B7H6. Similarly, an immune conjugate containing the antibody or antigen-binding fragment can bind to human or monkey CD16A protein, and an immune conjugate containing the bispecific binding molecule can similarly bind to human or monkey CD16A protein and BCMA protein, or to human or monkey CD16A protein and B7H6 protein, and such an immune conjugate has a good effect in preventing and / or treating CD16A-mediated diseases, or related diseases mediated by CD16A and BCMA, or CD16A and B7H6.

[0069] In an eighth aspect of the present invention, the present invention provides a composition comprising the antibody or antigen-binding fragment, bispecific binding molecule, nucleic acid molecule, expression vector, or recombinant cell described above. As described above, the antibody or antigen-binding fragment according to some specific embodiments of the present invention can effectively bind to human or monkey CD16A protein, the bispecific binding molecule can bind to human or monkey CD16A protein and BCMA protein, or to human or monkey CD16A protein and B7H6 protein, and to effectively suppress the proliferation of tumor cells, the composition comprising the above substance can similarly effectively bind to human or monkey CD16A protein, or to human or monkey CD16A protein and BCMA protein, or to human or monkey CD16A protein and B7H6 protein, and has a good effect in preventing and / or treating CD16A-mediated diseases, or related diseases mediated by CD16A and BCMA, or CD16A and B7H6, and the type of composition is not particularly limited and may be a food composition or a drug composition.

[0070] The compositions of the present invention can be administered in combination with each other or in combination with one or more other therapeutic compounds, for example, in combination with a chemotherapeutic agent. For this purpose, the compositions further contain a chemotherapeutic agent. The antibody or antigen-binding fragment or immunoconjugate of the present invention may be used in combination with a second therapeutic agent, and the reagents of the second therapeutic agent may include, but are not limited to, other reagents that inhibit CD16A activity (including other antibodies or antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and / or reagents that inhibit CD16A upstream or downstream signaling.

[0071] It should be noted that the composition may include a combination separated in time and / or space, and may work cooperatively to achieve the objectives of the present invention. For example, the components of the composition may be administered to a subject as a whole or separately. If the components of the composition are administered to a subject separately, each component may be administered to the subject simultaneously or sequentially.

[0072] In a ninth aspect of the present invention, the present invention relates to the antibody or antigen-binding fragment described above, and We present drugs comprising bispecific binding molecules, nucleic acid molecules, expression vectors, recombinant cells, or compositions. As described above, the antibody or antigen-binding fragment according to some specific embodiments of the present invention can effectively bind to human or monkey CD16A protein, and the bispecific binding molecule can bind to human or monkey CD16A protein and BCMA protein, or to human or monkey CD16A protein and B7H6 protein. Therefore, a drug comprising an active ingredient or a series of substances containing an effective amount of the antibody or antigen-binding fragment or the bispecific binding molecule can similarly effectively bind to human or monkey CD16A protein, or human or monkey CD16A protein and BCMA protein, or human or monkey CD16A protein and B7H6 protein, and has a good effect in preventing and / or treating CD16A-mediated diseases, or related diseases mediated by CD16A and BCMA, or CD16A and B7H6.

[0073] According to embodiments of the present invention, the drug may further include at least one of the following additional technical features.

[0074] According to embodiments of the present invention, the drug may further contain a pharmaceutically acceptable carrier.

[0075] As used herein, the term “effective dose” or “effective dosage” means an amount that can produce a function or activity in humans and / or animals and is acceptable to humans and / or animals.

[0076] The effective dose of the antibody or antigen-binding fragment or the bispecific binding molecule described in the present invention varies depending on the administration pattern and the severity of the disease being treated. The selection of a preferred effective dose can be determined by a person skilled in the art based on various factors (e.g., passing clinical trials). These factors include, but are not limited to, pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, and half-life, the severity of the disease the patient is trying to treat, the patient's weight, the patient's immune status, and the route of administration. For example, depending on the urgency of the treatment situation, the dose may be divided into several doses per day or the dose may be proportionally reduced.

[0077] As used herein, a “pharmaceutically acceptable” component is a substance that can be administered to humans and / or mammals without excessive side effects (such as toxicity, irritation, and allergies), i.e., has a reasonable benefit / risk ratio. The term “pharmaceutically acceptable carrier” refers to a vector for administering therapeutic agents, including various excipients and diluents.

[0078] The drug of the present invention comprises a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to) saline, buffer, glucose, water, glycerin, ethanol, and combinations thereof. Typically, the drug formulation needs to be suitable for the method of administration, which may be oral, nasal, intradermal, subcutaneous, intramuscular, intravenous, or intraperitoneal. The dosage forms of the drug of the present invention are injectable, oral formulations (tablets, capsules, oral solutions), transdermal, and sustained-release formulations. For example, it can be prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. It is preferable that the drug be prepared under sterile conditions. The antibody or antigen-binding fragment can be administered by intravenous injection or injection or intramuscular or subcutaneous injection.

[0079] Of course, the anti-CD16A monoclonal antibody or bispecificity conjugate molecule described herein may also be prepared as part of a kit or other diagnostic reagent, if necessary.

[0080] In a tenth aspect of the present invention, the present invention provides a kit comprising the antibody or its antigen-binding fragment, a bispecificity binding molecule, a nucleic acid molecule, an expression vector, or recombinant cells as described above. As described above, the antibody or antigen-binding fragment according to some specific embodiments of the present invention can effectively bind to human or monkey CD16A protein, and the bispecific binding molecule can bind to human or monkey CD16A protein and BCMA protein, or to human or monkey CD16A protein and B7H6 protein. Therefore, a kit containing the antibody or antigen-binding fragment can effectively perform qualitative or quantitative detection of human or monkey CD16A protein, and a kit containing the bispecific binding molecule can effectively perform qualitative or quantitative detection of human or monkey CD16A protein and BCMA protein, or to human or monkey CD16A protein and B7H6 protein. The kit according to the present invention can be used, for example, in kits that utilize the specific binding performance of human or monkey CD16A to antibodies, such as immunoblotting or immunoprecipitation. These kits include any one or more of the following: an antagonist, an anti-CD16A antibody or drug reference material, a protein purification column, an immunoglobulin affinity purification buffer, a cell measurement diluent, a specification or literature. Anti-CD16A antibodies can be used in different types of diagnostic tests, for example, to detect the presence of various diseases or drugs, toxins, or other proteins in vitro or intracellularly. They can be used to test for related diseases, for example, by detecting the serum or blood of a subject, and may also be used in scientific research. Using the kit, human or monkey CD16A protein, or human or monkey CD16A protein and BCMA protein, or human or monkey CD16A protein and B7H6 protein can be detected in a test sample. Such related diseases may include CD16A-related diseases, such as autoimmune diseases or cancer. Of course, the antibodies or antigen-binding fragments provided herein can be used for radioimmunodetection and radioimmunotherapy of the above diseases. The bispecific binding molecules are also applicable to the above-mentioned usage scenarios and will not be repeated here.

[0081] The kit may typically include reagents such as a coating solution for detecting CD16A, or CD16A and BCMA, or CD16A and B7H6.

[0082] In an eleventh aspect of the present invention, the present invention proposes the use of the antibody or antigen-binding fragment nucleic acid molecule, expression vector, recombinant cell, or composition described above in the preparation of a drug for the prevention and / or treatment of CD16A-mediated related diseases. As described above, since the antibody or antigen-binding fragment according to some specific embodiments of the present invention can effectively bind to human or monkey CD16A protein, a drug containing an effective amount of the antibody or antigen-binding fragment or a series of substances can similarly effectively bind to human or monkey CD16A protein and have a good effect in preventing and / or treating CD16A-mediated diseases or related diseases mediated by CD16A and BCMA, or CD16A and B7H6.

[0083] According to embodiments of the present invention, the use for preparing the above drug may further include at least one of the following additional technical features. According to embodiments of the present invention, the related diseases mediated by CD16A include autoimmune diseases.

[0084] The aforementioned autoimmune diseases include at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cholangitis, multiple cerebrospinal sclerosis, and acute idiopathic polyneuritis.

[0085] In a twelfth aspect of the present invention, the present invention relates to the bispecific binding molecule, nucleic acid molecule, and The present invention proposes the use of current vectors, recombinant cells, or compositions in the preparation of drugs for the prevention and / or treatment of CD16A and BCMA, or CD16A and B7H6-mediated related diseases. As described above, the bispecific binding molecules according to some specific embodiments of the present invention bind to human or monkey CD16A protein and BCMA protein, or to human or monkey CD16A protein and B7H6 protein. Therefore, a drug containing an effective amount of the active component of the bispecific binding molecule or a set of substances thereof can similarly effectively bind to human or monkey CD16A protein and BCMA protein, or to human or monkey CD16A protein and B7H6 protein, and has a good effect in preventing and / or treating CD16A and BCMA, or CD16A and B7H6-mediated related diseases.

[0086] According to embodiments of the present invention, the use for preparing the above drug may further include at least one of the following additional technical features.

[0087] According to embodiments of the present invention, the related diseases mediated by CD16A and BCMA, or CD16A and B7H6, include cancer.

[0088] According to embodiments of the present invention, the cancer includes at least one of the following: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, transverse myasthenia, astrocytoma, neuroblastoma, and glioma.

[0089] In a thirteenth aspect of the present invention, the present invention provides the use of the antibodies or antigen-binding fragments, nucleic acid molecules, expression vectors, or recombinant cells described above in the preparation of a kit for detecting CD16A. As described above, the antibodies or antigen-binding fragments according to some specific embodiments of the present invention can effectively bind to human or monkey CD16A proteins and block the binding of the CD16A proteins to their receptors, so that the antibodies or antigen-binding fragments can be used to prepare a kit for detecting CD16A proteins, and the kit can effectively perform qualitative or quantitative detection of human or monkey CD16A proteins.

[0090] In a fourteenth aspect of the present invention, the present invention proposes the use of the bispecificity conjugate molecules, nucleic acid molecules, expression vectors, or recombinant cells described above in the preparation of kits for detecting CD16A and / or BCMA, or CD16A and / or B7H6. As described above, the bispecificity conjugate molecules according to some specific embodiments of the present invention can effectively conjugate human or monkey CD16A protein and BCMA protein, or human or monkey CD16A protein and B7H6 protein, so that the bispecificity conjugate molecules can be used to prepare kits for detecting CD16A and / or BCMA, or CD16A and / or B7H6, and that the kits can effectively perform qualitative or quantitative detection of human or monkey CD16A and / or BCMA, or CD16A and / or B7H6.

[0091] In a 15th aspect of the present invention, the present invention provides a method for treating or preventing a disease mediated by CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6. According to an embodiment of the present invention, the method comprises administering to a subject at least one of 1) an antibody or antigen-binding fragment described above, 2) a bispecific binding molecule described above, 3) a nucleic acid molecule described above, 4) an expression vector described above, 5) a recombinant cell described above, 6) a composition described above, and 7) a drug described above. As described above, the bispecific binding molecule effectively binds to human or monkey CD16A protein and BCMA protein, or to human or monkey CD16A protein and B7 Since the antibody or antigen-binding fragment can effectively bind to the H6 protein, and can bind to the human or monkey CD16A protein, it can effectively treat or prevent CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6-mediated related diseases, preferably autoimmune diseases or cancer, the method according to the embodiments of the present invention can effectively treat or prevent CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6-mediated related diseases, such as autoimmune diseases or cancer.

[0092] According to embodiments of the present invention, a method for treating or preventing the above-mentioned disease may further include at least one of the following additional technical features.

[0093] According to embodiments of the present invention, the related diseases mediated by CD16A include autoimmune diseases.

[0094] The aforementioned autoimmune diseases include at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cholangitis, multiple cerebrospinal sclerosis, and acute idiopathic polyneuritis.

[0095] According to embodiments of the present invention, the related diseases mediated by CD16A and BCMA, or CD16A and B7H6, include cancer.

[0096] According to embodiments of the present invention, the cancer includes at least one of the following: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, transverse myasthenia, astrocytoma, neuroblastoma, and glioma.

[0097] In a sixteenth aspect of the present invention, the present invention provides a method for diagnosing related diseases mediated by CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6. According to an example of the present invention, the method includes the steps of detecting CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in a test sample using at least one of the following: 1) an antibody or antigen-binding fragment described above, 2) a bispecificity binding molecule described above, 3) a nucleic acid molecule described above, 4) an expression vector described above, and 5) recombinant cells described above, and determining the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in the test sample based on the detection result of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6. The antibodies or antigen-binding fragments according to this application, or antibodies or antigen-binding fragments expressed in nucleic acid molecules, expression vectors, or recombinant cells, can all effectively bind to human or monkey CD16A protein, and the bispecific binding molecules, or bispecific binding molecules expressed in nucleic acid molecules, expression vectors, or recombinant cells, can all effectively bind to CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6. Therefore, by employing the method of this application, the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in a test sample derived from a test individual can be effectively detected, and related diseases caused by CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6F can be effectively diagnosed.

[0098] According to embodiments of the present invention, the above-mentioned diagnostic disease method is one of the following additional technical features. It is not necessary to include one more, but it may be included in addition.

[0099] According to an embodiment of the present invention, the fact that the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in the test sample is above the minimum standard for the disease is an indicator from patients suffering from related diseases caused by CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6. The minimum standard value was determined by performing a comparative analysis and verification of the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in test samples from a large number of individuals suffering from related diseases caused by CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6, and a large number of healthy individuals.

[0100] According to the embodiments of the present invention, the test sample includes at least one of blood, saliva, sweat, tissue, cells, serum, plasma, feces, and urine.

[0101] According to embodiments of the present invention, the related diseases mediated by CD16A include autoimmune diseases.

[0102] The aforementioned autoimmune diseases include at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cholangitis, multiple cerebrospinal sclerosis, and acute idiopathic polyneuritis.

[0103] According to embodiments of the present invention, the related diseases mediated by CD16A and BCMA, or CD16A and B7H6, include cancer.

[0104] According to embodiments of the present invention, the cancer includes at least one of the following: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, transverse myasthenia, astrocytoma, neuroblastoma, and glioma.

[0105] In a sixteenth aspect of the present invention, the present invention provides a method for staging related diseases mediated by CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6F. According to an example of the present invention, CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 is detected in a test sample using at least one of the following: 1) the antibody or antigen-binding fragment described above, 2) the bispecificity binding molecule described above, 3) the nucleic acid molecule described above, 4) the expression vector described above, and 5) the recombinant cells described above. Based on the detection results of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6, the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in the test sample is determined. The bispecific binding molecules submitted in this application, or bispecific binding molecules expressed by nucleic acid molecules, expression vectors, or recombinant cells, can all effectively bind to human or monkey CD16A and / or BCMA, or CD16A and / or B7H6. The antibodies or antigen-binding fragments, or antibodies or antigen-binding fragments expressed by nucleic acid molecules, expression vectors, or recombinant cells, can all effectively bind to human or monkey CD16A proteins. Therefore, by employing the method described in this application, the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in a test sample derived from a test individual can be effectively detected. Furthermore, the timing of related diseases caused by CD16A, CD16A and / or BCMA, or CD16A and / or B7H6 is evaluated based on the content of CD16A, CD16A and / or BCMA, or CD16A and / or B7H6.

[0106] According to embodiments of the present invention, the method for classifying the above-mentioned disease into stages may further include at least one of the following additional technical features.

[0107] According to embodiments of the present invention, the fact that the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in the test sample is above the standard level for stage IV tumor disease is an indicator from patients with stage IV tumor disease, and the fact that the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in the test sample is between the standard levels for stage IV and stage III tumor disease is an indicator from patients with stage III tumor disease. The content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in the test sample being between the standard levels for stage III and stage II tumors is an indicator from patients with stage II tumors, and the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in the test sample being between the standard levels for stage I and stage II tumors is an indicator from patients with stage I tumors. As those skilled in the art will understand, the levels of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6, vary depending on the type of tumor during stages I, II, III, and IV of the tumor. The stage of the tumor can be determined by comparing the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in the test sample with the standard levels of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 at the corresponding tumor stage, or by comparing the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in the test sample with the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in samples from individuals or populations at known disease stages. The standard levels for tumor stages I, II, III, and IV were determined by differential comparative analysis and verification of the CD16A, CD16A and / or BCMA, or CD16A and / or B7H6 content in test samples from a large number of individuals suffering from related diseases caused by the formation of the aforementioned blood vessels and a large number of healthy individuals.

[0108] According to the embodiments of the present invention, the test sample includes at least one of blood, saliva, sweat, tissue, cells, serum, plasma, feces, and urine.

[0109] According to embodiments of the present invention, the related diseases mediated by CD16A include autoimmune diseases.

[0110] The aforementioned autoimmune diseases include at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cholangitis, multiple cerebrospinal sclerosis, and acute idiopathic polyneuritis.

[0111] According to embodiments of the present invention, the related diseases mediated by CD16A and BCMA, or CD16A and B7H6, include cancer.

[0112] According to embodiments of the present invention, the cancers include hemangiomas, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, It includes at least one of the following: nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, transverse myasthenia, astrocytoma, neuroblastoma, and glioma.

[0113] In a 17th aspect of the present invention, the present invention provides a method for evaluating the prognosis of related diseases mediated by CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6. According to an example of the present invention, the method includes the steps of detecting CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in a test sample using at least one of the following: 1) a bispecific binding molecule described above, 2) an antibody or antigen binding fragment described above, 3) a nucleic acid molecule described above, 4) an expression vector described above, and 5) recombinant cells described above, and determining the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in the test sample based on the detection result of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6.As described above, the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 has a significant impact on cancer, and the prognosis of such diseases can be effectively evaluated by monitoring the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in the tissues or excretions, such as peripheral blood or urine, after treating individuals suffering from related diseases. For example, by comparing the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in the body of a subject before and after treatment, or by comparing the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in the body of a subject after treatment with the CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 levels of a normal or affected individual, as described in the present application. Isomer-binding molecules, or bispecific binding molecules expressed by nucleic acid molecules, expression vectors, or recombinant cells, can all effectively bind to CD16A and / or BCMA, or CD16A and / or B7H6. Since the antibodies or antigen-binding fragments, or antibodies or antigen-binding fragments expressed by nucleic acid molecules, expression vectors, or recombinant cells, can all effectively bind to human or monkey CD16A, by employing the method described herein, the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in a test sample derived from a test individual can be effectively detected, and the prognosis of related diseases caused by CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 can be evaluated based on the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6.

[0114] According to embodiments of the present invention, a method for evaluating the prognosis of the above-mentioned disease may further include at least one of the following additional technical features.

[0115] According to embodiments of the present invention, the test sample is derived from a patient suffering from a related disease mediated by CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6, either before or after treatment.

[0116] According to the embodiments of the present invention, the test sample includes at least one of blood, saliva, sweat, tissue, cells, serum, plasma, feces, and urine.

[0117] According to embodiments of the present invention, based on the content of CD16A, CD16A and / or BCMA, or CD16A and / or B7H6 in test samples of patients suffering from CD16A, CD16A and / or BCMA, or CD16A and / or B7H6-mediated related diseases before or after treatment, the prediction of CD16A, CD16A and / or BCMA, or CD16A and / or B7H6-mediated related diseases is determined. Determine the aftereffects.

[0118] According to embodiments of the present invention, a decrease in the content of CD16A, CD16A and / or BCMA, or CD16A and / or B7H6 in test samples from patients suffering from related diseases mediated by CD16A, CD16A and / or BCMA, or CD16A and / or B7H6 after treatment is an indicator of a favorable prognosis for the patient.

[0119] According to embodiments of the present invention, the related diseases mediated by CD16A include autoimmune diseases.

[0120] The aforementioned autoimmune diseases include at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cholangitis, multiple cerebrospinal sclerosis, and acute idiopathic polyneuritis.

[0121] According to embodiments of the present invention, the related diseases mediated by CD16A and BCMA, or CD16A and B7H6, include cancer.

[0122] According to embodiments of the present invention, the cancer includes at least one of the following: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, transverse myasthenia, astrocytoma, neuroblastoma, and glioma.

[0123] In an eighteenth aspect of the present invention, the present invention proposes the use of the bispecific binding molecules, antibodies or antigen-binding fragments, nucleic acid molecules, expression vectors, recombinant cells, compositions or drugs described above in the treatment or prevention of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6-mediated related diseases. As described above, the bispecific binding molecules have high binding activity to CD16A and / or BCMA, or CD16A and / or B7H6, and the antibodies or antigen-binding fragments can effectively bind to human or monkey CD16A, and can effectively treat or prevent CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6-mediated related diseases.

[0124] According to embodiments of the present invention, the above use may further include at least one of the following additional technical features.

[0125] According to embodiments of the present invention, the related diseases mediated by CD16A include autoimmune diseases.

[0126] The aforementioned autoimmune diseases include at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cholangitis, multiple cerebrospinal sclerosis, and acute idiopathic polyneuritis.

[0127] According to embodiments of the present invention, the related diseases mediated by CD16A and BCMA, or CD16A and B7H6, include cancer.

[0128] According to embodiments of the present invention, the cancer includes at least one of the following: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, transverse myasthenia, astrocytoma, neuroblastoma, and glioma.

[0129] In a 19th aspect of the present invention, the present invention proposes the use of the bispecificity binding molecules, antibodies or antigen-binding fragments, nucleic acid molecules, expression vectors or recombinant cells described above in the diagnosis of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6-mediated related diseases, in the staging of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6-mediated related diseases, in the evaluation of CD16A, or in the evaluation of the prognosis of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6-mediated related diseases. As described above, the bispecific binding molecules submitted in this application, or bispecific binding molecules expressed by nucleic acid molecules, expression vectors, or recombinant cells, can effectively bind to CD16A and / or BCMA, or CD16A and / or B7H6. The antibodies or antigen-binding fragments, or antibodies or antigen-binding fragments expressed by nucleic acid molecules, expression vectors, or recombinant cells, can effectively bind to human or monkey CD16A. Therefore, by employing the method described in this application, the content of CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 in a test sample derived from a test individual can be effectively detected, and the diagnosis, disease staging, and disease prognosis assessment of related diseases mediated by CD16A, or CD16A and / or BCMA, or CD16A and / or B7H6 can be effectively performed.

[0130] According to embodiments of the present invention, the above use may further include at least one of the following additional technical features.

[0131] According to embodiments of the present invention, the related diseases mediated by CD16A include autoimmune diseases.

[0132] The aforementioned autoimmune diseases include at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cholangitis, multiple cerebrospinal sclerosis, and acute idiopathic polyneuritis.

[0133] According to embodiments of the present invention, the related diseases mediated by CD16A and BCMA, or CD16A and B7H6, include cancer.

[0134] According to embodiments of the present invention, the cancer includes at least one of the following: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, transverse myasthenia, astrocytoma, neuroblastoma, and glioma.

[0135] In the present invention, "subject" or "individual" generally refers to, for example, primates and / or rodents, particularly mammals such as humans, monkeys, or mice. [Effects of the Invention]

[0136] The beneficial effects of this invention are as follows: 1) The mouse-derived anti-CD16A monoclonal antibody obtained in the present invention has higher CD16A protein binding activity than conventional CD16A monoclonal antibodies, and the CD16A protein includes human CD16A protein and monkey CD16A protein. 2) The humanized antibody obtained after humanizing the mouse-derived anti-CD16A monoclonal antibody similarly has higher CD16A protein binding activity than conventional CD16A monoclonal antibodies, the CD16A protein includes human CD16A protein and monkey CD16A protein, and the humanized antibody has low immunogenicity, is safer, and has longer-lasting efficacy.

[0137] Additional aspects and advantages of the present invention are partially shown in the following description, partially become apparent from the following description, or can be understood through practice of the present application. The above and / or additional aspects and advantages of the present invention will become apparent and easier to understand from the description of the embodiments combined with the following drawings. [Brief explanation of the drawing]

[0138] [Figure 1A] This figure shows the ELISA detection results of different concentrations of mouse-derived CD16A antibody (m40F5) according to the examples of the present invention, as they bind to the human CD16A158F protein. [Figure 1B] This figure shows the ELISA detection results of different concentrations of mouse-derived CD16A antibodies according to the examples of the present invention, as they bind to the human CD16A158V protein. [Figure 1C] This figure shows the ELISA detection results of different concentrations of mouse-derived CD16A antibody according to the examples of the present invention, as they bind to the monkey CD16 protein. [Figure 1D] This figure shows the ELISA detection results of different concentrations of mouse-derived CD16A antibody according to the examples of the present invention, as they bind to the human CD16BNA1 protein. [Figure 1E] This figure shows the ELISA detection results of different concentrations of mouse-derived CD16A antibody according to the examples of the present invention, as they bind to the human CD16BNA2 protein. [Figure 1F] This figure shows the ELISA detection results of different concentrations of mouse-derived CD16A antibody according to the examples of the present invention, as they bind to the human CD16BSH protein. [Figure 2A] This figure shows the detection results of different concentrations of mouse-derived CD16A antibodies according to the embodiments of the present invention, binding to CHO cells that overexpress human CD16A protein. [Figure 2B] This figure shows the detection results of different concentrations of mouse-derived CD16A antibodies according to the examples of the present invention, binding to CHO cells that overexpress cynomolgus monkey CD16A protein. [Figure 3A]This figure shows the ELISA detection results of different concentrations of humanized CD16A antibody (h40F5) according to the examples of the present invention, binding to the human CD16A158F protein. [Figure 3B] This figure shows the ELISA detection results of different concentrations of humanized CD16A antibody (h40F5) according to the examples of the present invention, binding to the human CD16A158V protein. [Figure 3C] This figure shows the ELISA detection results of different concentrations of humanized CD16A antibody (h40F5) according to the examples of the present invention, as they bind to monkey CD16 protein. [Figure 3D] This figure shows the ELISA detection results of different concentrations of humanized CD16A antibody (h40F5) according to the examples of the present invention, binding to the human CD16BNA1 protein. [Figure 3E] This figure shows the ELISA detection results of different concentrations of humanized CD16A antibody (h40F5) according to the examples of the present invention, binding to the human CD16BNA2 protein. [Figure 3F] This figure shows the ELISA detection results of humanized CD16A antibody (h40F5) at different concentrations according to the examples of the present invention, as they bind to human CD16BSH protein. [Figure 4A] This figure shows the detection results of different concentrations of CD16A×BCMA bispecific antibodies according to the embodiments of the present invention, showing their binding to the human CD16A protein. [Figure 4B] This figure shows the detection results of different concentrations of CD16A × BCMA bispecific antibodies according to the embodiments of the present invention, showing their binding to human BCMA protein. [Figure 5] This figure shows the results of killing and detecting different concentrations of CD16A × BCMA bispecific antibodies against NCI-H929 cells according to the examples of the present invention. [Figure 6A] This figure shows the detection results of different concentrations of CD16A×B7H6 bispecific antibodies according to the embodiments of the present invention, showing their binding to the human CD16A protein. [Figure 6B] This figure shows the detection results of different concentrations of CD16A×B7H6 bispecific antibodies according to the embodiments of the present invention, showing their binding to the human B7H6 protein. [Figure 7]This figure shows the results of killing and detecting HCT-15 cells with different concentrations of CD16A × BCMA bispecific antibodies according to the examples of the present invention. [Modes for carrying out the invention]

[0139] The embodiments of the present invention will be described in detail below. The embodiments described below are illustrative and are used only to interpret the present invention and are not intended to limit it.

[0140] Furthermore, the terms “first” and “second” are used solely for descriptive purposes and should not be understood as indicating the number of technical features of relative importance, whether explicitly or implicitly. Therefore, features limited by “first” and “second” may explicitly or implicitly include at least one such feature. Moreover, in this description of the invention, “multiple” means two or more unless otherwise specified.

[0141] The endpoints and any values ​​of the ranges disclosed herein should be understood to include values ​​close to such exact ranges or values, and not to be limited to such exact ranges or values. In the case of numerical ranges, the intervals between the endpoint values ​​of each range, between the endpoint values ​​of each range and individual point values, and between individual point values ​​combine to obtain one or more new numerical ranges, and these numerical ranges shall be deemed to be specifically disclosed in the specification.

[0142] To facilitate understanding of the present invention, several technical and scientific terms are specifically defined below. Unless expressly defined herein, all other technical and scientific terms used herein have meanings generally understood by those skilled in the art. Amino acid residue abbreviations are standard three-letter and / or one-letter codes referring to one of the 20 common L-amino acids used in this art.

[0143] The antibodies or antigen-binding fragments described in the present invention are usually prepared by biosynthesis. Based on the nucleotide sequences described in the present invention, those skilled in the art can easily prepare the coding nucleic acids of the present invention using various known methods. These methods include, but are not limited to, PCR and artificial DNA synthesis, and specific methods can be found in J. Sunbrook's "A Guide to Molecular Cloning Experiments."

[0144] The antibody or antigen-binding fragment described in the present invention is usually prepared by biosynthesis. Based on the nucleotide sequence described in the present invention, those skilled in the art can easily prepare the coding nucleic acid of the present invention using various known methods. These methods include, but are not limited to, PCR and artificial DNA synthesis, and specific methods can be found in J. Sunbrook's "A Guide to Molecular Cloning Experiments." In one embodiment of the present invention, the coding nucleic acid sequence of the present invention can be constructed by a method of stepwise synthesizing the nucleotide sequence and extending it by repeated PCR. The antibody or antigen fragment is numbered and defined by the Kabat numbering system.

[0145] In this specification, the term "monoclonal antibody" refers to an antibody having a single antigen-binding site.

[0146] In this specification, the term "bispecific antibody" refers to an antibody having two different antigen-binding sites.

[0147] In this specification, the terms “mutant” or “variant” may refer to a molecule obtained by making a mutation involving one or more nucleotides or amino acids in a naturally occurring or artificially produced molecule.

[0148] The term "complementary determination region," "CDR," or "CDR sequence" refers to the amino acid sequence responsible for antigen binding in an antibody, typically the amino acid residues around 23-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable region, and around 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable region (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of This includes and / or is derived from "highly variable rings" (e.g., amino acid residues around 26-32(LI), 50-52(L2) and 91-96(L3) in the light chain variable region, and around 26-32(H1), 53-55(H2) and 96-101(H3) in the heavy chain variable region (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0149] In this specification, the term "identity" refers to the determination of the proportion of identical amino acids or nucleotides between two amino acid or nucleic acid sequences using conventional methods when describing an amino acid or nucleic acid sequence relative to a reference sequence, for example, Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York), and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research). See Foundation, Washington, DC. For sequence comparison and measurement of sequence identity, see the homology comparison algorithm of Needleman et al. (1970) J.Mol.Biol.48:443, the local homology algorithm of Smith et al. (1981) Adv.Appl.Math.2:482, the similarity search method of Pearson et al. (1988) Proc.Natl.Acad.Sci.85:2444, and Smith-Waterman. This includes many algorithms such as the algorithms in Meth.Mol.Biol.70:173-187(1997), and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol.215:403-410). Computer programs utilizing these algorithms are also available, and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266:460-480(1996)), or GAP, BESTFIT, BLAST Altschul et al., FASTA, and TFASTA, which are available in the Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA, and in CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0150] A person skilled in the art can obtain a variant of the antibody or its functional fragment sequence by substituting, adding, and / or deleting one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids into the sequence of the present invention, provided that the antibody activity is not substantially impaired (at least 95% of the activity is retained). This variant is considered to fall within the protected scope of the present invention. For example, an amino acid having similar properties may be substituted in the variable region. The variant sequence described in the present invention has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. The sequence identity described in this invention can be measured using sequence analysis software. For example, the computer program BLAST, particularly BLASTP or TBLASTN, with default parameter settings can be used. All amino acid sequences referred to in this invention are shown in an N-terminal to C-terminal manner.

[0151] As described above, the monoclonal antibody of the present invention may be a full-length antibody, or it may consist only of a functional fragment (e.g., Fab, F(ab')2, or scFv fragment), or it may be modified to affect its function. The present invention includes anti-CD16A antibodies with modified glycan patterns. In some uses, modifications to remove undesirable glycan sites are useful, or the antibody may lack a fucose moiety in the oligosaccharide chain, for example, to enhance antibody-dependent cytotoxicity (ADCC) function. In other uses, galactosation modifications may be performed to alter complement-dependent cytotoxicity (CDC).

[0152] In this specification, the term "full-length antibody" refers to a tetrameric structure in which two identical light chains and two identical heavy chains are linked by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE).

[0153] As used in this text, the term "functional fragment" refers in particular to antibody fragments such as CDR-transplanted antibodies, Fab, Fab', F(ab')2, Fv or scFv, nanoantibodies, or any fragments whose half-life can be extended by chemical modification or liposome encapsulation, wherein the chemical modification refers to the addition of, for example, poly(alkylene) glycol, such as polyethylene glycol ("polyethylene glycolation, PEGylation") (polyethylene glycolated fragments called Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG or Fab'-PEG) ("PEG" is polyethylene glycol), and the fragment has CD16A binding activity. Preferably, the functional fragment consists of or includes a portion of the heavy chain variable region or light chain variable region of the derived antibody, wherein the portion of the sequence is sufficient to maintain binding specificity and sufficient affinity similar to the derived antibody, preferably at least equal to 1 / 100 of the affinity for CD16A, and more preferably at least equal to 1 / 10 of the affinity for the derived antibody. Such functional fragments contain at least three amino acids, preferably the 5th, 10th, 15th, 25th, 50th, and 100th consecutive amino acids of the antibody sequence from which they are derived.

[0154] In the present invention, unless otherwise stated, the term "antigen-binding fragment" as used usually means an antigen-binding antibody fragment, and may include a part of a complete antibody, generally an antigen-binding region or variable region, such as a CDR-transplanted antibody, Fab, Fab', F(ab')2, Fv or scFv, a nano-antibody, etc.

[0155] In this specification, the term "CDR-transplanted antibody" refers to an antibody in which the CDR of one type of monoclonal antibody is transplanted into the variable region of another type of antibody. For example, by transplanting the CDR of a mouse-derived monoclonal antibody into the variable region of a human-derived antibody, the human-derived antibody acquires the antigen-binding specificity of the mouse-derived monoclonal antibody in place of the human-derived antibody CDR, while simultaneously reducing its heterogeneity.

[0156] In this specification, the terms "Fab antibody" or "Fab" usually refer to an antibody containing only Fab molecules, which consist of a heavy chain of VH and CH1 and a complete light chain, with the light and heavy chains connected by a single disulfide bond.

[0157] In this specification, the term "nano-antibody" (single-domain antibody or VHH antibody) first refers to the antigen-binding immunoglobulin (variable) region (Ha) of a "heavy-chain antibody" (i.e., an "antibody lacking a light chain"). Mers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). These antibodies contain only the heavy chain variable region (VH) and the normal CH2 and CH3 regions, and bind specifically to the antigen via the heavy chain variable region.

[0158] In this specification, the term "Fv antibody" usually refers to an antibody in which only the light chain variable region (VL) and heavy chain variable region (VH) are non-covalently linked, and the antibody molecule is the minimal functional fragment that leaves a complete antigen-binding site.

[0159] In this specification, the term "single-chain antibody" or "scFv" refers to a fragment in which the antibody heavy chain variable region and light chain variable region are linked by a short-chain peptide.

[0160] In this specification, the "knob-in-hole structure" is achieved by forming a knob-hole mutation in the CH3 region of the constant region of the antibody heavy chain, facilitating heavy chain occlusion and forming heterodimers. For example, in this application, this is achieved by mutating amino acids in the CH3 domain of the constant region of the human IgG1 heavy chain (T366S, L368A, Y407V, Y349C mutations in one chain, i.e., a "hole," and T366W, S354C mutations in the other chain, i.e., a "knob"). The amino acid numbering here is performed according to the Kabat numbering system. For example, "T366S" means that the T amino acid numbered at position 366 according to the Kabat numbering system is replaced with an S amino acid.

[0161] Table 1 shows in detail the amino acid or nucleic acid sequence according to the present invention.

[0162] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15] [Table 1-16] [Table 1-17] [Examples]

[0163] The present invention will be described in detail below with reference to examples. Examples or test examples will not specify the details. If the experimental method is not specified, it should be carried out under normal conditions.

[0164] The means of the present invention will be interpreted below in combination with the examples. Those skilled in the art will understand that the following examples are used solely to illustrate the present invention and should not be considered to limit the scope of the invention. Where no specific techniques or conditions are shown in the examples, they should be carried out in accordance with the techniques or conditions described in the literature in the art or in accordance with the product specifications. Where the manufacturer of the reagents or equipment used is not indicated, they are conventional products available on the market.

[0165] Example 1: Construction of CHO-K1 cells overexpressing human and cynomolgus monkey CD16A.

[0166] HEK293T cells were seeded in a T150 culture flask and cultured in DMEM complete medium. After overnight culture, endotoxin-free psPAX2, pMD2.G, and pCDH-CMV- Add either the FCGR3A-IRES-puro vector (a coding sequence (SEQ ID NO: 57) with human CD16A protein (SEQ ID NO: 37) inserted between the polyclonal sites of the pCDH-CMV-MCS-IRES-puro vector) or the cynomolgus monkey protein (SEQ ID NO: 38) coding sequence (SEQ ID NO: 58) vector in a ratio of 7:3:10 to 1.5 mL of Opti-MEM (Gibco, product no. 31985070), then add 100 μL of P3000 transfection reagent. Add 100 μL of Lipofectamine 3000 (thermo, product no. L3000008) transfection reagent to 1.5 mL of Opti-MEM medium and mix thoroughly. The DNA diluent and liposome diluent were thoroughly mixed in a 1:1 volume ratio, incubated at room temperature for 5-10 minutes, and then added to 293T cells. The cells were cultured for 48 hours to obtain the viral supernatant. The viral supernatant was centrifuged at 2000 g at 4°C for 10 minutes, the supernatant was removed, filtered through a 0.45 μm filter, and then PEG8000 solution (Shanghai Biotechnology) was added. The mixture was thoroughly mixed and left overnight at 4°C. Next, the mixture was centrifuged at 2200 g for 90 minutes until a white precipitate formed at the bottom of the centrifuge tube. The virus was then resuspended in sterile PBS buffer.

[0167] Add polybrene (sigma, TR-1003) (final concentration 8 μg / mL) to DME / F12 medium, mix thoroughly, and then add an appropriate amount of virus solution. Place 2E5 CHO-K1 cells into a 24-well plate, add the medium containing the virus, incubate for 8 hours, and then replace with fresh medium. After 48 hours, detect the CD16A expression level on the surface of CHO-K1 cells by flow cytometry. After the appearance of positive cells, perform limiting dilution, i.e., digest the cells to a dilution density of 4 cells per 1 ml, inoculate into a 96-well flat-bottom plate, add 200 μl to each well, and culture for 2 weeks until the cells clearly form a single cell cluster. Then, detect the CD16A expression level on the surface of each clonal cell by flow cytometry. All positive cells are CHO-K1 cells that overexpress the desired human CD16A or cynomolgus monkey CD16.

[0168] Example 2: Preparation of anti-human CD16A hybridoma monoclonal antibody

[0169] In this invention, anti-human CD16A monoclonal antibodies are produced by mouse immunization. For the experiment, female, 6-week-old C57BL / 6 mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were used, and human CD16A extracellular domain protein (ACRO, CDA-H5220) was used as the immune antigen. For the initial immunization, the antigen was emulsified with Freund's complete adjuvant (Sigma, F5881), and intraperitoneal immunization was performed, administering 100 μg to each mouse by intraperitoneal injection. For subsequent immunization, the antigen was mixed with the Ribi adjuvant system (Sigma, S6322), and intraperitoneal immunization was performed once every two weeks after the initial immunization, for a total of three immunizations. Serum from the immunized mice was collected and antibody titer detection was performed using ELISA, following the standard procedure in this field.

[0170] Based on the antibody titer detection results, mice with high serum antibody titers were selected and spleen cell fusion was performed. The selected mice were sprint-immunized 72 hours before fusion, and intraperitoneal injection was administered using a mixture of the Ribi adjuvant system and the above antigens. Splenocyte lymphocytes and myeloma cells Sp2 / 0 (ATCC, CRL-8287) were fused using an optimized PEG-mediated fusion step to obtain hybridoma cells. The fused hybridoma cells were resuspended in HAT complete medium (RPMI-1640 medium with 20% FBS, 1×HAT and 1×OPI), dispensed into 96-well cell culture plates, and incubated at 37°C in 5% CO2. On day 5 after fusion, 50 μL / well of HAT complete medium was added. Seven to eight days after fusion, the culture medium was completely replaced based on the cell proliferation density. The medium was HT complete medium (containing 20% ​​FBS, 1×HT and 1×OPI in RPMI-1640 medium), added at 200 μL / well.

[0171] On days 10-11 after fusion, flow cytometry-based binding detection was performed based on the cell proliferation density. The medium in the positive wells was changed, and the cells were scaled up to 24-well plates in a timely manner according to the cell density. After re-examination of the cell lines transferred to 24-well plates, preservation and initial subcloning were performed. Cells that were positive in the initial subcloning were preserved, and a second subcloning was performed. Cells that were positive in the second subcloning were preserved and subjected to protein expression. Both subcloning and preservation procedures are standard techniques in this field. Antibodies were further prepared using serum-free cell culture methods, and mouse-derived antibodies were purified by protein G affinity chromatography for subsequent detection of functional activity.

[0172] Example 3: ELISA binding experiment of mouse-derived CD16A antibody

[0173] The ELISA experiment is used to detect the binding properties of the mouse-derived CD16A antibody m40F5 obtained in Example 1. Human CD16A 158F (ACRO biosystems, CDA-H5220), Human CD16A 158V(ACRO biosystems, CD8-H52H4), cynomolgus CD16 (ACRO biosystems, FC6-C52H9), human CD16B NA1 (ACRO biosystems, CDB-H5227), human CD16B NA2 (ACRO biosystems, CDB-H5222) and human CD16B SH (Sino Biological, 11046-H08H2) was diluted to 2 μg / mL with coating buffer (35 mM NaHCO3, 15 mM Na2CO3, pH 9.6), and 100 μL of each was added to each well of an enzyme-linked plate and left overnight at 4°C. Then, it was washed three times with PBST (0.05% Tween 20-PBS, pH 7.2). 300 μL of blocking buffer (1% BSA, 0.05% Tween20-PBS, pH 7.2) was added to the plate and left standing at room temperature for 2 h. It was further washed three times with PBST. Mouse-derived antibody m40F5 or control antibody LS21 (WO2006 / 125668 EN) was added to each well and incubated at room temperature for 1 hour. Next, it was washed three times with PBST. 100 μL of HRP-goat anti-mouse IgG secondary antibody (boster, product number BA1051) diluted with blocking buffer was added to each well and incubated at room temperature for 1 hour. It was washed three times with PBST, TMB was added to each well, and after reacting for 2 - 5 minutes at room temperature under light-shielded conditions, the reaction was stopped with 2 M sulfuric acid in each well, and finally the OD450 value was read with a microplate reader. Figure 1A shows that the mouse-derived m40F5 antibody of the present invention can bind to human CD16A 158F and Figure 1B shows that the mouse-derived m40F5 antibody of the present invention can also bind to human CD16A 158V and Figure 1C shows that the mouse-derived m40F5 of the present invention can bind to cynomolgus CD16, and Figure 1D shows that the mouse-derived m40F5 of the present invention does not bind to human CD16B NA1 and Figure 1E shows that the mouse-derived 40F5 of the present invention does not bind to human CD16B NA2 and Figure 1F shows that the mouse-derived 40F5 of the present invention does not bind to human CD16B SH and Figure 1F shows that the mouse-derived 40F5 of the present invention does not bind to human CD16B

[0174] Example 4: Flow cytometry binding experiment of mouse-derived CD16A antibody

[0175] 2 × 10⁶ CHO-K1 cells overexpressing human CD16A or cynomolgus monkey CD16 obtained in Example 1 were mixed in PBS. 6 Diluted to 1 / mL, added 100 μL / tube to a 1.5 mL EP tube, then added 10 μL / tube of goat serum and blocked at 4°C for 30 min. Add a concentration gradient (5-fold dilution, with the highest final concentration being 10 μg / mL) of CD16A antibody and control antibody mIgG, and incubate at 4°C for 30 min. Add 1 mL of PBS to the EP tube, centrifuge at 3500 rpm for 5 min at 4°C, discard the supernatant, and wash once more with PBS. After centrifugation, discard the supernatant, resuspend the cells in PBS at 100 μL / tube, add 0.1 μL / tube of Alexa-647 labeled goat anti-mouse secondary antibody (Invitrogen), and incubate in the dark at 4°C for 30 min. Wash twice with PBS, centrifuge, and discard the supernatant. Resuspend the cells in PBS at 200 μL / tube and detect by flow cytometry. The results in Figures 2A and 2B demonstrate that the mouse-derived m40F5 antibody can bind to CHO-K1 cells overexpressing human CD16A and cynomolgus monkey CD16, and explain that the mouse-derived CD16A antibody can bind to the CD16A protein on the cell surface.

[0176] Example 5: Hybridoma cell sequencing

[0177] A total of 10 m40F5 antibody candidate hybridoma cells were screened in the above example. 6After culturing, cells are collected by centrifugation at 800 rpm for 10 minutes, total RNA is extracted using the Trizol kit (Invitrogen), and a cDNA library (Invitrogen) is synthesized by reverse transcription using the total RNA as a template. The nucleic acid sequence of the variable region of the m40F5 antibody corresponding to hybridoma cells is amplified by PCR using the cDNA as a template. The primer sequences used in the PCR amplification reaction are complementary to the first frame region or signal peptide region and constant region of the antibody variable region (see Larrick, J.W., et al., (1990) Scand.J. Immunol., 32, 121-128 and Coloma, J.J., et al., (1991) BioTechniques, 11, 152-156). PCR amplification was performed in a 50 μL reaction system, with 2 μL of cDNA, 5 μL of 10× PCR buffer, 2 μL (5 μM) of upstream and downstream primers, 2 μL of dNTPs, 1 μL of Taq enzyme (Takara, Ex Taq), and 38 μL of H2O added to each system. Initial denaturation was performed at 95°C for 5 mins, followed by temperature cycling and PCR amplification. The reaction conditions were denaturation at 94°C for 30 S, annealing at 58°C for 45 S, extension at 72°C for 50 S, for a total of 32 cycles, followed by extension at 72°C for 7 mins. After sequencing the amplified product, the heavy chain variable region (SEQ ID NO: 23) and light chain variable region (SEQ ID NO: 24) sequences of the mouse monoclonal antibody m40F5 were obtained.

[0178] Example 6 Humanization of anti-human CD16A monoclonal antibody

[0179] 6.1 Selection of variable region frames and recovery mutations in CD16A monoclonal antibodies

[0180] Based on Example 5, by comparing the IMGT human antibody heavy / light chain variable region gene database with MOE software, heavy or light chain variable region gene sequences with high homology to the mouse-derived monoclonal antibody m40F5 were selected as templates. The CDRs of the mouse-derived monoclonal antibody were then transplanted into the corresponding human-derived templates to form a variable region sequence in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Amino acid residues were determined and annotated by the Kabat numbering system.

[0181] To maintain the three-dimensional structure of the CDR region, residues on the VL-VH binding interface and close to the CDR are used. Furthermore, restorative mutations are introduced into residues embedded within the protein and residues that directly interact with the CDR, ensuring that the activity of the variable region is not affected.

[0182] 6.2 Expression of CD16A humanized antibodies

[0183] After selecting the variable region frame of the above CD16A monoclonal antibody and performing a restorative mutation, the heavy chain variable region sequence of the resulting humanized CD16A antibody h40F5 is shown in SEQ ID NO: 25, and the light chain variable region sequence is shown in SEQ ID NO: 26. The nucleotide sequence encoding the humanized antibody heavy chain (SEQ ID NO: 39) and the nucleotide sequence encoding the humanized antibody light chain (SEQ ID NO: 40) are recombined into the pTT5 plasmid using molecular biology techniques.

[0184] CD16A antibodies are prepared by rapidly transfecting ExpiCHO-S cells (Gibco, product number A29127) with the above-mentioned humanized anti-human CD16A antibody h40F5 light chain and heavy chain-carrying pTT5 vector. The day before transfection, ExpiCHO-S cells are raised to a cell density of (3-4) × 10⁶. 6 The solution was adjusted to 1 / mL and cultured overnight at 37°C with 8% CO2 and vibration at 120 rpm. On the day of transfection, the cells were 7 × 10⁶. 6 ~1 × 10 7When the cells have grown to 6 × 10¹ / mL and the viability exceeds 95%, prepare for transfection and use fresh, preheated ExpiCHO medium (Gibco, product number A2910002) to transfer the cells. 6 Dilute the above humanized anti-human CD16A antibody h40F5 light and heavy chains to 1 / mL, and transfect ExpiCHO-S cells with the pTT5 plasmid and ExpiFectamine CHO transfection reagent (Gibco, product number A29129) in a 2:1 molar ratio. Culture at 37°C, 8% CO2, and 120 rpm with vibration. After 18-22 hours of transfection, immediately add the transfected cells after thoroughly mixing ExpiFectamine CHO Enhancer and ExpiCHO Feed, mix thoroughly, and culture at 32°C, 5% CO2, and 120 pm with vibration. On day 5 after transfection, add 8 mL of ExpiCHO Feed back to the cells, mix thoroughly, and continue culturing. Observe changes in cell number and cell viability daily. When cell viability falls below 80% or after 10-14 days of culture, centrifuge to obtain the cells. The expression supernatant was filtered through a 0.22 μm filtration membrane, and antibodies containing the Fc domain were captured from the expression supernatant using a Mabselect prism A affinity chromatography column (GE Corporation, product number 17549854). After equilibrating the column with pH 7.2 phosphate buffer, the supernatant was passed through the affinity chromatography column, washed with washing buffer (100 mM citrate, pH 2.7), and finally concentrated and replaced with PBS buffer. The purified antibodies were identified as having an SDS-PAGE purity of 95% or higher, and ultimately humanized h40F5 antibody (heavy chain shown as SEQ ID NO: 35, light chain shown as SEQ ID NO: 36) was obtained.

[0185] 6.3 Affinity Verification of Humanized Antibody h40F5

[0186] The affinity between humanized h40F5 antibody and human CD16A antigen, and between human CD16B and monkey CD16 antigen were tested using BIACORE. The results are shown in Table 2, and the obtained humanized antibody and CD16A antigen showed good affinity.

[0187] [Table 2]

[0188] Example 7: ELISA binding experiment of humanized antibody h40F5

[0189] ELISA experiments were used to detect the binding properties of the humanized antibody h40F5, with wild-type human-derived IgG1 antibody as a control. Human CD16A 158F (ACRO biosystems, CDA-H5220), Human CD16A 158V (ACRO biosystems, CD8-H52H4), Cynomolgus monkey CD16 (ACRO biosystems, FC6-C52H9), Human CD16B NA1 (ACRO biosystems, CDB-H5227), Human CD16B NA2 (ACRO biosystems, CDB-H5222) and human CD16B SH Dilute (Yiqiao Shenzhou, 11046-H08H2) to 2 μg / mL with coating buffer (35 mM NaHCO3, 15 mM Na2CO3, pH 9.6), add 100 μL to each well of an enzyme-conjugated plate, and leave overnight at 4°C. Then wash three times with PBST (0.05% Tween 20-PBS, pH 7.2). Add 300 μL of blocking buffer (1% BSA, 0.05% Tween 20-PBS, pH 7.2) to the plate and let stand at room temperature for 2 hours. Wash three times with PBST. Add a concentration gradient of humanized anti-human CD16A antibody h40F5 or human-derived IgG1 antibody to each well and incubate at room temperature for 1 hour. Then wash three times with PBST. Add 100 μL of HRP-goat anti-human IgG secondary antibody (Jackson ImmunoResearch, 109-036-170) diluted in blocking buffer to each well and incubate at room temperature for 1 hour. Wash three times with PBST, add TMB to each well, and incubate in the dark at room temperature for 2-5 minutes. Stop the reaction in each well with 2M sulfuric acid, and finally read the OD450 value with a microplate reader. Figure 3A shows the humanized h40F5 antibody of the present invention reacting with human CD16A158F Figure 3B shows that the humanized h40F5 antibody of the present invention can bind to human CD16A. 158V Figure 3C shows that the humanized h40F5 of the present invention can bind to cynomolgus monkey CD16, and Figure 3D shows that the humanized h40F5 of the present invention can bind to human CD16B. NA1 Figure 3E shows that the humanized h40F5 of the present invention does not bind to human CD16B. NA2 Figure 3F shows that it does not bind to human CD16B. SH This indicates that it does not bind with [the specified object].

[0190] Example 8: Preparation of a bispecific antibody of CD16A × BCMA and detection of its binding activity and cell-killing ability.

[0191] Based on the experimental results of Examples 1-7, the inventors designed a bispecific antibody CD16A×BCMA and detected its binding activity and cell-killing ability.

[0192] 8.1 Design and preparation of bispecific antibodies of CD16A × BCMA

[0193] The antibody contains two monovalent units, one of which is in the form of anti-CD16A scFv-Fc, with its heavy chain variable region and light chain variable region amino acid sequences derived from the sequence of the humanized antibody h40F5 obtained in Example 6 of the present invention, and the other monovalent unit is in the form of anti-BCMA scFv-Fc (with a sequence derived from US009273141B2), and the dual specific The sex antibody is named CD16A×BCMA. This bispecific antibody contains two polypeptide chains, one containing a CD16A single-chain antibody scFv (SEQ ID NO: 41) and the other containing a BCMA single-chain antibody scFv (SEQ ID NO: 42). The constant regions of the two heavy chains are derived from human-derived antibody IgG1. Because this molecule has a unique asymmetric structure, different amino acid mutations were introduced into the constant regions of the two chains to reduce the formation of homogenous dimers. At the same time, a (L234A / L235A) mutation was also introduced into the constant region of the heavy chain to prevent crosslinking activation caused by the Fcγ receptor.

[0194] The method for preparing the bispecific antibody is as described in Example 6.2, and the bispecific antibody is prepared by combining plasmid mixing ratios (1:1 or other ratios) and undergoing a one-step affinity purification. The associated sequences of the bispecific antibody CD16A×BCMA are shown in Table 3.

[0195] [Table 3]

[0196] 8.2 Measurement of binding activity (ELISA) between CD16A x BCMA bispecific antibody and antigen.

[0197] Human BCMA (ACRO biosystems, BCA-H522y) or human CD16A antigen (ACRO biosystems, CDD-H52W1) was diluted to 2 μg / mL in coating buffer (35 mM NaHCO3, 15 mM Na2CO3, pH 9.6). 100 μL of this solution was added to each well of an enzyme-conjugated plate and left overnight at 4°C. The plate was then washed three times with PBST (0.05% Tween 20-PBS, pH 7.2). 300 μL of blocking buffer (1% BSA, 0.05% Tween 20-PBS, pH 7.2) was added to the plate and allowed to stand at room temperature for 2 hours. The plate was then washed three times with PBST. The corresponding bispecific antibody or control antibody hIgG1 was added to each well and incubated at room temperature for 1 hour. The precipitate was then washed three times with PBST. 100 μL of HRP-goat anti-human IgG secondary antibody (Jackson ImmunoResearch, 109-036-170), diluted in blocking buffer, was added to each well and incubated at room temperature for 1 hour. The wells were washed three times with PBST, TMB was added to each well, and the wells were incubated in the dark for 2–5 minutes at room temperature. The reaction was then stopped in each well with 2M sulfuric acid, and the OD450 values ​​were finally read using a microplate reader. Figures 4A and 4B show that the CD16A×BCMA of the present invention can bind to both CD16A and BCMA antigens.

[0198] 8.3 Cell Killing Experiment

[0199] Multiple myeloma NCI-H929 cells were marked as CellTrace Violet (thermo, C34557), and the cell density was set to 1 × 10⁻⁶. 5 Adjust to / mL. PBMCs to a cell density of 1 × 10⁻¹⁶. 6The solution was adjusted to / mL, mixed PBMCs and marked NCI-H929 cells in a 1:1 ratio, and then added to a 96-well round-bottom plate. The bispecific antibody was then added in a concentration gradient and mixed uniformly. After incubation at 37°C for 24 hours, the mixture was transferred to a flow tube via a 200-mesh nylon net, 7AAD was added, and detection was performed 10 minutes later to characterize the killing efficiency by the percentage of CTV+7AAD+ cells. The results in Figure 5 show that the killing efficiency of PBMCs against NCI-H929 multiple myeloma cells gradually increased with increasing concentration of the CD16A×BCMA bispecific antibody.

[0200] Example 9: Preparation of a bispecific antibody of CD16A × B7H6 and its binding activity and cell-killing ability. Detection

[0201] Based on the experimental results of Examples 1-6, the inventors designed a bispecific antibody CD16A×B7H6 and detected its binding activity and cell-killing ability.

[0202] 9.1 Design and preparation of bispecific antibodies of CD16A×B7H6

[0203] The antibody contains two monovalent units, one of which is in the form of anti-CD16A scFv-Fc, where the light chain variable region and heavy chain variable region of CD16A scFv are derived from the humanized sequence of Example 6 of the present invention, and the other monovalent unit is in the form of anti-B7H6 scFv-Fc (the B7H6 scFv sequence is derived from CN114395045A), and this bispecific antibody is named CD16A×B7H6. The bispecific antibody contains two polypeptide chains, one a heavy chain containing a CD16A single-chain antibody scFv (SEQ ID NO: 41) and the other a heavy chain containing a B7H6 single-chain antibody scFv (SEQ ID NO: 43). Because the molecule has a special asymmetric structure, different amino acid mutations were introduced into the constant regions of the two chains to reduce the formation of homogenous dimers. Simultaneously, to prevent cross-linking activation caused by the Fcγ receptor, a mutation (L234A / L235A) was introduced into the heavy chain constant region.

[0204] For the preparation of the CD16A×B7H6 bispecific antibody, refer to Example 6.2. By combining plasmid mixing ratios (1:1 or other ratios), the CD16A×B7H6 bispecific antibody was prepared through a one-step affinity purification. The associated sequences of the said bispecific antibody CD16A×B7H6 are shown in Table 4.

[0205] [Table 4]

[0206] 9.2 Measurement of bispecific antibody-antigen binding activity (ELISA)

[0207] Human B7H6 (ACRO biosystems, B76-H52H8) or human CD16A antigen (ACRO biosystems, product number CDA-H5220) was diluted to 2 μg / mL in coating buffer (35 mM NaHCO3, 15 mM Na2CO3, pH 9.6). 100 μL of this solution was added to each well of an enzyme-conjugated plate and left overnight at 4°C. The plate was then washed three times with PBST (0.05% Tween 20-PBS, pH 7.2). 300 μL of blocking buffer (1% BSA, 0.05% Tween 20-PBS, pH 7.2) was added to the plate and allowed to stand at room temperature for 2 hours. The plate was then washed three times with PBST. The corresponding bispecific antibody or control antibody hIgG1 was added to each well and incubated at room temperature for 1 hour. The precipitate was then washed three times with PBST. 100 μL of HRP-goat anti-human IgG secondary antibody (Jackson ImmunoResearch, 109-036-170), diluted in blocking buffer, was added to each well and incubated at room temperature for 1 hour. After incubation, the precipitate was washed three times with PBST, TMB was added to each well, and the mixture was allowed to react in the dark at room temperature for 2–5 minutes. The reaction was then stopped in each well with 2M sulfuric acid, and the OD450 values ​​were finally read using a microplate reader. Figures 6A and 6B show that the bispecific antibody CD16A×B7H6 of the present invention can effectively bind to both the CD16A and B7H6 antigens.

[0208] 9.3 Cell Killing Experiment

[0209] After digestion, HCT-15 cells from colorectal cancer were counted, and the cell density was determined to be 2 × 10⁻⁶. 5 Adjust to / mL. Open the RTCA instrument (Agilent), select instrument type DP, select experimental pattern, and fill in cell information and drug information. Enter the schedule setting experimental step, add 50 μL of fresh medium (89% RPMI 1640 medium + 10% fetal bovine serum + 1% penicillin streptomycin) to the plate, then place it in the instrument, close it, and click Step 1 to start. After completing Done, remove the plate, add 100 μL of cell suspension, let it stand at room temperature for 15-30 mins to prevent edge effect, place it in the instrument, and click Start. When the logarithmic growth phase is reached, pause and add 50 μL of PBMC (4 × 10 6 Add ( / mL), add the bispecific antibody CD16A×B7H6 or control antibody hIgG in a concentration gradient, click start, and analyze after a certain period of time. The results in Figure 7 show that the killing efficiency of PBMCs against colorectal cancer HCT-15 cells gradually increased with increasing concentration of the CD16A×B7H6 bispecific antibody, and that CD16A×B7H6 kills PBMCs against B7H6 + It demonstrates that it effectively promotes the specific killing of tumor cells.

[0210] In this specification, any reference to terms such as “one embodiment,” “several embodiments,” “example,” “specific example,” or “several examples” means that a particular feature, structure, material, or property described with reference to such embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the general expressions of the above terms do not necessarily apply to the same embodiment or example. In addition, any particular feature, structure, material, or property described may be incorporated in an appropriate manner in any one or more embodiments or examples. Furthermore, those skilled in the art can combine and combine the various embodiments or examples and the features relating to the various embodiments or examples described herein without contradiction.

[0211] Although embodiments of the present invention have been presented and described, these embodiments are illustrative and should not be understood as limiting the present invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations are possible in the above embodiments within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment, Each of the following is a heavy chain variable region CDR1, CDR2, CDR3 sequence, and / or an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, 2, and 3. An antibody or antigen-binding fragment characterized by containing light chain variable regions CDR1, CDR2, and CDR3 sequences, respectively, which are represented by amino acid sequences having at least 95% identity with SEQ ID NO: 4, 5, and 6.

2. The antibody or antigen-binding fragment according to claim 1, characterized by comprising the heavy chain variable region CDR1 sequence shown in SEQ ID NO: 1, the heavy chain variable region CDR2 shown in SEQ ID NO: 2, the heavy chain variable region CDR3 shown in SEQ ID NO: 3, the light chain variable region CDR1 shown in SEQ ID NO: 4, the light chain variable region CDR2 shown in SEQ ID NO: 5, and the light chain variable region CDR3 shown in SEQ ID NO:

6.

3. The antibody or antigen-binding fragment according to claim 2, characterized in that it comprises at least one of a heavy chain FR region and a light chain FR region.

4. The antibody or antigen-binding fragment according to claim 3, characterized in that at least one portion of the heavy chain FR region and the light chain FR region is derived from at least one of human-derived antibodies, primate-derived antibodies, and mouse-derived antibodies or variants thereof.

5. At least one of the heavy chain frame regions HFR1, HFR2, HFR3, and HFR4 sequences shown in SEQ ID NO: 7 to 10, or The antibody or antigen-binding fragment according to claim 4, characterized in that it comprises at least one of the heavy chain frame regions HFR1, HFR2, HFR3, and HFR4 sequences, respectively, as indicated by SEQ ID NO: 15 to 18.

6. At least one of the light chain frame regions LFR1, LFR2, LFR3, and LFR4 sequences shown in SEQ ID NO: 11 to 14, or The antibody or antigen-binding fragment according to claim 4, characterized in that it comprises at least one of the light chain frame regions LFR1, LFR2, LFR3, and LFR4 sequences shown in SEQ ID NO: 19 to 22, respectively.

7. At least one of the heavy chain frame regions HFR1, HFR2, HFR3, and HFR4 sequences shown in SEQ ID NO: 7 to 10, respectively, SEQ ID NO: At least one of the light chain frame regions LFR1, LFR2, LFR3, and LFR4 sequences shown in 11-14, or At least one of the heavy chain frame regions HFR1, HFR2, HFR3, and HFR4 sequences shown in SEQ ID NO: 15 to 18, respectively, SEQ ID NO: The antibody or antigen-binding fragment according to any one of claims 5 to 6, characterized in that it comprises at least one of the light chain frame regions LFR1, LFR2, LFR3, and LFR4 sequences shown in 19 to 22.

8. The heavy chain variable region indicated in SEQ ID NO: 23 or SEQ ID NO: 25, and / or The antibody or antigen-binding fragment according to claim 7, characterized by comprising a light chain variable region indicated by SEQ ID NO: 24 or SEQ ID NO:

26.

9. 1) Heavy chain variable region shown in SEQ ID NO: 23, and SEQ ID NO: The light chain variable region shown in 24, or 2) The antibody or antigen-binding fragment according to claim 8, characterized by comprising a heavy chain variable region indicated by SEQ ID NO: 25 and a light chain variable region indicated by SEQ ID NO:

26.

10. The antibody or antigen-binding fragment according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment comprises at least one heavy chain constant region and at least a portion of the heavy chain constant region and at least one of the light chain constant regions is derived from at least one of human-derived antibodies, primate-derived antibodies, mouse-derived antibodies, or variants thereof.

11. The antibody or antigen-binding fragment according to claim 10, characterized in that both the light chain constant region and the heavy chain constant region are derived from a mouse-derived IgG antibody or a variant thereof, or a human-derived IgG antibody or a variant thereof.

12. The antibody or antigen-binding fragment according to claim 11, characterized in that both the light chain constant region and the heavy chain constant region are derived from a mouse-derived IgG1 antibody or a variant thereof, or a human-derived IgG1 antibody or a variant thereof.

13. The antibody or antigen-binding fragment according to claim 12, characterized in that the antibody has a heavy chain constant region of the amino acid sequence shown in SEQ ID NO: 27 or 29 and / or a light chain constant region of the amino acid sequence shown in SEQ ID NO: 28 or 30.

14. The antibody or antigen-binding fragment according to claim 10, characterized in that the antibody or antigen-binding fragment has a heavy chain of an amino acid sequence shown in any of SEQ ID NO: 31, 33, and 35 and a light chain of an amino acid sequence shown in any of SEQ ID NO: 32, 34, and 36.

15. The antibody or antigen-binding fragment has a heavy chain of the amino acid sequence shown in SEQ ID NO: 31 and a light chain of the amino acid sequence shown in SEQ ID NO:

32. The antibody or antigen-binding fragment has a heavy chain of the amino acid sequence shown in SEQ ID NO: 33 and a light chain of the amino acid sequence shown in SEQ ID NO: 34, or The antibody or antigen-binding fragment according to claim 10, characterized in that the antibody or antigen-binding fragment has a heavy chain of the amino acid sequence shown in SEQ ID NO: 35 and a light chain of the amino acid sequence shown in SEQ ID NO:

36.

16. The antibody or antigen-binding fragment according to claim 1, characterized in that it comprises a monoclonal antibody or a polyclonal antibody.

17. The antibody or antigen-binding fragment according to claim 16, characterized in that the monoclonal antibody comprises at least one of a full-length antibody, Fv, single-chain antibody, Fab, single-domain antibody, and minimum recognition unit.

18. The antibody or antigen-binding fragment according to claim 16, characterized in that the antibody or antigen-binding fragment can bind to the amino acid sequence shown in SEQ ID NO: 37 and / or 38.

19. It is a bispecific binding molecule, A first binding region comprising an antibody or antigen-binding fragment according to any one of claims 1 to 18, A bispecific binding molecule characterized by comprising a second binding region having BCMA or B7H6 binding activity.

20. The double specificity binding molecule according to claim 19, characterized in that the double specificity binding molecule includes a symmetric double specificity binding molecule or an asymmetric double specificity binding molecule.

21. The double specificity binding molecule according to claim 19, characterized in that the double specificity binding molecule is an asymmetric double specificity binding molecule.

22. The bispecific binding molecule according to claim 19, characterized in that the antibody or antigen-binding fragment is an anti-CD16A single-chain antibody.

23. The bispecific binding molecule according to claim 22, characterized in that the second binding region comprises at least one of a full-length antibody having BCMA or B7H6 binding activity, Fv, a single-chain antibody, Fab, a single-domain antibody, and a minimal recognition unit.

24. The bispecific binding molecule according to claim 23, characterized in that the second binding region contains an anti-BCMA single-chain antibody or an anti-B7H6 single-chain antibody.

25. The anti-CD16A single-chain antibody comprises an anti-CD16A antibody light chain variable region and an anti-CD16A antibody heavy chain variable region, wherein the anti-CD16A antibody heavy chain variable region has an amino acid sequence shown in SEQ ID NO: 23 or 25, and the anti-CD16A antibody light chain variable region has an amino acid sequence shown in SEQ ID NO: 24 or 26, characterized in that the bispecificity binding molecule according to 24.

26. The bispecificity binding molecule according to claim 25, wherein the anti-CD16A single-chain antibody further comprises a linking peptide 1, the N end of the linking peptide 1 being connected to the C end of the heavy chain variable region of the anti-CD16A antibody, the C end of the linking peptide 1 being connected to the N end of the light chain variable region of the anti-CD16A antibody, or the N end of the linking peptide 1 being connected to the C end of the light chain variable region of the anti-CD16A antibody, and the C end of the linking peptide 1 being connected to the N end of the heavy chain variable region of the anti-CD16A antibody.

27. The anti-BCMA single-chain antibody comprises an anti-BCMA antibody light chain variable region and an anti-BCMA antibody heavy chain variable region, wherein the anti-BCMA antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 59, and the anti-BCMA antibody light chain variable region has the amino acid sequence shown in SEQ ID NO: 60, characterized in that the bispecific binding molecule according to 26.

28. The bispecificity binding molecule according to claim 27, wherein the anti-BCMA single-chain antibody further comprises a linking peptide 2, the N end of the linking peptide 2 being connected to the C end of the heavy chain variable region of the anti-BCMA antibody, the C end of the linking peptide 2 being connected to the N end of the light chain variable region of the anti-BCMA antibody, or the N end of the linking peptide 2 being connected to the C end of the light chain variable region of the anti-BCMA antibody, and the C end of the linking peptide 2 being connected to the N end of the heavy chain variable region of the anti-BCMA antibody.

29. The anti-B7H6 single-chain antibody comprises an anti-B7H6 antibody light chain variable region and an anti-B7H6 antibody heavy chain variable region, wherein the anti-B7H6 antibody heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 61, and the anti-B7H6 antibody light chain variable region has the amino acid sequence shown in SEQ ID NO: 62, characterized in that the bispecificity binding molecule according to 28.

30. The bispecificity binding molecule according to claim 29, wherein the anti-B7H6 single-chain antibody further comprises a linking peptide 3, the N end of the linking peptide 3 being connected to the C end of the heavy chain variable region of the anti-B7H6 antibody, the C end of the linking peptide 3 being connected to the N end of the light chain variable region of the anti-B7H6 antibody, or the N end of the linking peptide 3 being connected to the C end of the light chain variable region of the anti-B7H6 antibody, and the C end of the linking peptide 3 being connected to the N end of the heavy chain variable region of the anti-B7H6 antibody.

31. The bispecific binding molecule according to claim 30, characterized in that at least one of the linked peptide 1, linked peptide 2, and linked peptide 3 has an amino acid sequence (GGGGS)n, where n is an integer of 1 or more.

32. The bispecific binding molecule according to claim 31, characterized in that n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

33. The bispecific binding molecule according to claim 31, characterized in that at least one of the linked peptide 1, linked peptide 2, and linked peptide 3 has the amino acid sequence shown in SEQ ID NO:

44.

34. The bispecific binding molecule according to claim 22, characterized in that the anti-CD16A single-chain antibody has the amino acid sequence shown in SEQ ID NO:

41.

35. The bispecific binding molecule according to claim 24, characterized in that the anti-BCMA single-chain antibody has the amino acid sequence shown in SEQ ID NO:

42.

36. The bispecific binding molecule according to claim 24, characterized in that the anti-B7H6 single-chain antibody has the amino acid sequence shown in SEQ ID NO:

43.

37. The bispecific binding molecule according to claim 19, wherein the first antigen-binding region further comprises a first Fc peptide segment, and the N-end of the first Fc peptide segment is connected to the C-end of the antibody or antigen-binding fragment.

38. The bispecific binding molecule according to claim 37, wherein the second antigen-binding region further comprises a second Fc peptide segment, and the N-end of the second Fc peptide segment is connected to the C-end of the anti-BCMA single-chain antibody or the anti-B7H6 single-chain antibody.

39. The bispecific binding molecule according to claim 38, characterized in that the first Fc peptide segment has the amino acid sequence shown in SEQ ID NO:

45.

40. The bispecific binding molecule according to claim 39, characterized in that the second Fc peptide segment has the amino acid sequence shown in SEQ ID NO:

46.

41. The bispecific binding molecule according to claim 40, characterized in that the first Fc peptide segment and the second Fc peptide segment are connected by a knob-into-hole structure.

42. The bispecific binding molecule according to claim 19, characterized in that the first antigen-binding region has the amino acid sequence shown in SEQ ID NO: 47, and the second antigen-binding region has the amino acid sequence shown in SEQ ID NO: 48 or 49.

43. A nucleic acid molecule characterized by encoding an antibody or antigen-binding fragment according to any one of claims 1 to 18 or a bispecific binding molecule according to any one of claims 19 to 42.

44. An expression vector characterized by supporting the nucleic acid molecule described in claim 43.

45. Recombinant cells characterized by being able to carry a nucleic acid molecule according to claim 43, an expression vector according to claim 44, or an antibody or antigen-binding fragment according to any one of claims 1 to 18, or a bispecific binding molecule according to any one of claims 19 to 42.

46. The recombinant cells are obtained by introducing the expression vector described in claim 44 into host cells, as described in claim 45.

47. The recombinant cell according to claim 46, characterized in that the recombinant cell is a eukaryotic cell.

48. The recombinant cell according to claim 46, characterized in that the recombinant cell is a mammalian cell.

49. A composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 18, a bispecific binding molecule according to any one of claims 19 to 42, a nucleic acid molecule according to claim 43, an expression vector according to claim 44, or a recombinant cell according to any one of claims 45 to 48.

50. A drug comprising an antibody or antigen-binding fragment according to any one of claims 1 to 18, a bispecific binding molecule according to any one of claims 19 to 42, a nucleic acid molecule according to claim 43, an expression vector according to claim 44, a recombinant cell according to any one of claims 45 to 48, or a composition according to claim 49.

51. A kit characterized by containing an antibody or antigen-binding fragment according to any one of claims 1 to 18, a bispecific binding molecule according to any one of claims 19 to 42, a nucleic acid molecule according to claim 43, an expression vector according to claim 44, or recombinant cells according to any one of claims 45 to 48.

52. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 18, a nucleic acid molecule according to claim 43, an expression vector according to claim 44, a recombinant cell according to any one of claims 45 to 48, or a composition according to claim 49 in the preparation of a drug for the prevention and / or treatment of CD16A-mediated related diseases.

53. The use according to claim 52, characterized in that the related diseases mediated by the aforementioned CD16A include autoimmune diseases.

54. The use according to claim 52, characterized in that the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cholangitis, multiple cerebrospinal sclerosis, and acute idiopathic polyneuritis.

55. A bispecific binding molecule according to any one of claims 19 to 42, or a nucleus according to claim 43. Use of an acid molecule, the expression vector according to claim 44, the recombinant cell according to any one of claims 45 to 48, or the composition according to claim 49, in the preparation of a drug for the prevention and / or treatment of CD16A and BCMA, or CD16A and B7H6-mediated related diseases.

56. The use according to claim 55, characterized in that the related diseases mediated by CD16A and BCMA, or CD16A and B7H6, include cancer.

57. The use according to claim 55, characterized in that the cancer includes at least one of the following: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, transverse myasthenia, astrocytoma, neuroblastoma, and glioma.

58. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 18, a nucleic acid molecule according to claim 43, an expression vector according to claim 44, or recombinant cells according to any one of claims 45 to 48 in the preparation of a CD16A detection kit.

59. Use of a bispecific binding molecule according to any one of claims 19 to 42, a nucleic acid molecule according to claim 43, an expression vector according to claim 44, or recombinant cells according to any one of claims 45 to 48 in the preparation of a kit for detecting CD16A and / or BCMA, or CD16A and / or B7H6.

60. Use of an antibody or antigen-binding fragment according to any one of claims 1 to 18, a nucleic acid molecule according to claim 43, an expression vector according to claim 44, a recombinant cell according to any one of claims 45 to 48, a composition according to claim 49, or a drug according to claim 50 in the prevention and / or treatment of a CD16A-mediated related disease.

61. The use according to claim 60, characterized in that the related diseases mediated by the aforementioned CD16A include autoimmune diseases.

62. The use according to claim 61, characterized in that the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cholangitis, multiple cerebrospinal sclerosis, and acute idiopathic polyneuritis.

63. Use of a bispecific binding molecule according to any one of claims 19 to 42, a nucleic acid molecule according to claim 43, an expression vector according to claim 44, a recombinant cell according to any one of claims 45 to 48, a composition according to claim 49, or a drug according to claim 50, in the prevention and / or treatment of related diseases mediated by CD16A and BCMA, or CD16A and B7H6.

64. The use according to claim 63, characterized in that the related diseases mediated by CD16A and BCMA, or CD16A and B7H6, include cancer.

65. The use according to claim 64, characterized in that the cancer includes at least one of the following: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, transverse myasthenia, astrocytoma, neuroblastoma, and glioma.

66. A method for preventing and / or treating a CD16A-mediated related disease, comprising the step of administering to a patient an antibody or antigen-binding fragment according to any one of claims 1 to 18, a nucleic acid molecule according to claim 43, an expression vector according to claim 44, a recombinant cell according to any one of claims 45 to 48, a composition according to claim 49, or a drug according to claim 50, in a pharmaceutically acceptable dose.

67. A method for diagnosing a CD16A-mediated related disease, comprising the steps of detecting CD16A in a test sample using at least one of the antibodies or antigen-binding fragments described in any one of claims 1 to 18, the nucleic acid molecule described in claim 43, the expression vector described in claim 44, or the recombinant cells described in any one of claims 45 to 48, and determining the CD16A content in the test sample based on the CD16A detection result.

68. The method according to 67, characterized in that the CD16A content in the test sample is below the minimum standard for the disease, and the test sample is an indicator from patients suffering from related diseases caused by CD16A.

69. A method for evaluating the prognosis of a CD16A-mediated related disease, comprising the steps of detecting CD16A in a test sample using at least one of the antibodies or antigen-binding fragments described in any one of claims 1 to 18, the nucleic acid molecule described in claim 43, the expression vector described in claim 44, or the recombinant cells described in any one of claims 45 to 48; determining the CD16A content in the test sample; and evaluating the prognosis of a CD16A-related disease based on the CD16A content.

70. The method according to 67 or 69, characterized in that the test sample comprises at least one of blood, saliva, sweat, tissue, cells, blood, serum, plasma, feces, and urine.

71. The method according to any one of claims 66, 67, or 69, characterized in that the related disease mediated by the CD16A includes autoimmune diseases.

72. The method according to 71, characterized in that the autoimmune disease includes at least one of the following: systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, scleroderma, dermatomyositis, autoimmune hemolytic anemia, thyroid autoimmune disease, ulcerative colitis, chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent diabetes mellitus, myasthenia gravis, ulcerative colitis, chronic atrophic gastritis with pernicious anemia, pulmonary hemorrhagic nephritis syndrome, pemphigus vulgaris, bullous pemphigoid, primary biliary cholangitis, multiple cerebrospinal sclerosis, and acute idiopathic polyneuropathy.

73. A method for preventing and / or treating a CD16A-BCMA or CD16A-B7H6-mediated related disease, comprising the step of administering to a patient a pharmaceutically acceptable dose of a bispecific binding molecule according to any one of claims 19 to 42, a nucleic acid molecule according to claim 43, an expression vector according to claim 44, a recombinant cell according to any one of claims 45 to 48, a composition according to claim 49, or a drug according to claim 50.

74. A method for diagnosing CD16A-BCMA or CD16A-B7H6-mediated related diseases, comprising using at least one of the bispecific binding molecule described in any one of claims 19 to 42, the nucleic acid molecule described in claim 43, the expression vector described in claim 44, or the recombinant cell described in any one of claims 45 to 48 to diagnose CD16A-BCMA or CD16A-B7H6-mediated related diseases in a test sample. A method comprising the steps of detecting CD16A and BCMA, or CD16A and B7H6, and determining the content of CD16A and BCMA, or CD16A and B7H6 in the test sample based on the detection results of CD16A and BCMA, or CD16A and B7H6.

75. The method according to 74, characterized in that the content of CD16A and BCMA, or CD16A and B7H6 in the test sample is below the minimum standard for the disease, and this is an indicator from patients suffering from related diseases caused by CD16A and BCMA, or CD16A and B7H6.

76. A method for evaluating the prognosis of a related disease mediated by CD16A and BCMA, or CD16A and B7H6, comprising the steps of: detecting CD16A and BCMA, or CD16A and B7H6 in a test sample using at least one of the bispecific binding molecules described in any one of claims 19 to 42, the nucleic acid molecule described in claim 43, the expression vector described in claim 44, or the recombinant cells described in any one of claims 45 to 48; determining the content of CD16A and BCMA, or CD16A and B7H6 in the test sample; and evaluating the prognosis of a related disease caused by CD16A and BCMA, or CD16A and B7H6 based on the content of CD16A and BCMA, or CD16A and B7H6.

77. The method according to 74 or 76, characterized in that the test sample comprises at least one of blood, saliva, sweat, tissue, cells, blood, serum, plasma, feces, and urine.

78. The method according to any one of claims 73, 74, or 76, characterized in that the related disease mediated by CD16A and BCMA, or CD16A and B7H6, includes cancer.

79. The method according to 78, characterized in that the cancer includes at least one of the following: hemangioma, gastric cancer, liver cancer, lung cancer, breast cancer, colon cancer, nasopharyngeal cancer, bladder cancer, cervical cancer, prostate cancer, bone cancer, skin cancer, thyroid cancer, kidney cancer, esophageal cancer, melanoma, fibrosarcoma, transverse myasthenia, astrocytoma, neuroblastoma, and glioma.

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