Anti-CD16a single-domain antibody and use thereof

Abstract

Provided are an anti-CD16a single-domain antibody and a use thereof, relating to the field of immunology. The single-domain antibody is composed of heavy chains, and the heavy chains include a heavy chain CDR1 shown in any one of SEQ ID NO: 11 to SEQ ID NO: 13, a heavy chain CDR2 shown in any one of SEQ ID NO: 14 to SEQ ID NO: 17, and a heavy chain CDR3 shown in any one of SEQ ID NO: 18 to SEQ ID NO: 21. The present invention has the beneficial effects that biological genetic engineering technology is used to screen out a single-domain antibody specific to CD16a, and the antibody has good affinity.

Description

An anti-CD16a single-domain antibody and its use Technical Field

[0001] The present invention relates to a single-domain antibody capable of specifically binding to CD16a (hereinafter abbreviated as "CD16a single-domain antibody"), a pharmaceutical composition containing the single-domain antibody as an active ingredient, and pharmaceutical therapeutic uses thereof. Background Art

[0002] CD16a is a low-affinity, dominant activating transmembrane receptor expressed on NK cells, macrophages, and mast cells. It is a member of the immunoglobulin superfamily of transmembrane receptors. On NK cells, signal transduction occurs through the binding of the α chain of FcγRIIIA to immunoreceptor tyrosine-based activation motifs (ITAMs) containing the FcεRIγ chain and / or the T cell receptor (TCR) / CD3ζ chain.

[0003] Single-domain antibodies (sdAbs) are currently the smallest antibody molecules, with a molecular weight one-tenth that of intact antibodies. In addition to possessing the antigenic reactivity of intact antibodies, sdAbs also possess unique functional properties, such as small molecular weight, strong stability, good solubility, easy expression, weak immunogenicity, strong penetrability, strong targeting, and low production costs. These antibodies virtually overcome the drawbacks of traditional antibodies, such as long development cycles, low stability, and demanding storage conditions.

[0004] Therefore, it is particularly necessary to research and develop a CD16a antibody with a small molecular weight, good tumor penetration, and the ability to be freely assembled into bispecific or multispecific antibodies, while also possessing a high-affinity single-domain (hereinafter referred to as "single-domain").

[0005] Summary of the Invention

[0006] The invention purpose of this patent is to provide a single-domain antibody that can specifically bind to CD16a and its use.

[0007] The first aspect of the present invention provides an anti-CD16a single-domain antibody, which is composed of a heavy chain, and the heavy chain includes a heavy chain CDR1 shown in any one of SEQ ID NO: 11 to SEQ ID NO: 13, a heavy chain CDR2 shown in any one of SEQ ID NO: 14 to SEQ ID NO: 17, and a heavy chain CDR3 shown in any one of SEQ ID NO: 18 to SEQ ID NO: 21.

[0008] Preferably, the amino acid sequence of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 is one of the following (1)-(5):

[0009] (1) CDR1 shown in SEQ ID NO: 12, CDR2 shown in SEQ ID NO: 17, and CDR3 shown in SEQ ID NO: 21;

[0010] (2) CDR1 shown in SEQ ID NO: 12, CDR2 shown in SEQ ID NO: 14, and CDR3 shown in SEQ ID NO: 21;

[0011] (3) CDR1 shown in SEQ ID NO: 11, CDR2 shown in SEQ ID NO: 16, and CDR3 shown in SEQ ID NO: 20;

[0012] (4) CDR1 shown in SEQ ID NO: 11, CDR2 shown in SEQ ID NO: 16, and CDR3 shown in SEQ ID NO: 19;

[0013] (5) CDR1 shown in SEQ ID NO: 13, CDR2 shown in SEQ ID NO: 15, and CDR3 shown in SEQ ID NO: 18.

[0014] The above CDR combinations (1)-(5) correspond to the single-domain antibodies 23D1, 14A3, 4B5, 15E2, and 13G6, respectively.

[0015] All of the above sequences can be replaced by sequences having "at least 80% homology" to the sequence or sequences with only one or a few amino acid substitutions; preferably "at least 85% homology", more preferably "at least 90% homology", more preferably "at least 95% homology", and most preferably "at least 98% homology".

[0016] In one embodiment, one to five amino acid residues in any one or more CDRs of the heavy chain CDR1, CDR2, and CDR3 can be substituted with their conservative amino acids. Specifically, one to five amino acid residues in the heavy chain CDR1 can be substituted with their conservative amino acids; one to five amino acid residues in the heavy chain CDR2 can be substituted with their conservative amino acids; and one to five amino acid residues in the heavy chain CDR3 can be substituted with their conservative amino acids.

[0017] As used herein, the term "sequence homology" refers to the extent to which two (nucleotide or amino acid) sequences have identical residues at identical positions in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Thus, two copies of identical sequences have 100% homology.

[0018] In some embodiments, sequences that replace only one or a few amino acids compared to the aforementioned sequences, for example, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions, can also achieve the purpose of the invention. These variations include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and addition of one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. In fact, when determining the degree of sequence homology between two amino acid sequences or when determining the CDR1, CDR2 and CDR3 combination in a single domain antibody, technicians can consider so-called "conservative" amino acid substitutions. In the case of substitutions, the substitution will preferably be a conservative amino acid substitution. The conservative amino acid can generally be described as an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and the substitution has little or substantially no effect on the function, activity or other biological properties of the polypeptide. The conservative amino acid substitutions are common in the art. For example, conservative amino acid substitutions are substitutions of one or a few amino acids in the following groups (a)-(d) by another or a few amino acids in the same group: (a) polar negatively charged residues and their uncharged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. Particularly preferred conservative amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg or Gln; Phe is replaced by Met, Leu, or Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe or Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr, or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser. In addition, those skilled in the art will appreciate that the creativity of single-domain antibodies is embodied in the CDR1-3 regions, while the framework region sequences FR1-4 are not immutable. The sequences of FR1-4 can be conservative sequence variants of the sequences disclosed in the present invention.

[0019] The meaning of "anti-CD16a single-domain antibody" in the present invention includes not only complete single-domain antibodies, but also fragments, derivatives and analogs of the anti-CD16a single-domain antibody. As used herein, the terms "fragment", "derivative" and "analog" have the same meaning and refer to polypeptides that substantially maintain the same biological function or activity as the antibody of the present invention. The polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or protein sequence used to purify the polypeptide, or a fusion protein formed with an Fc tag). According to the teachings of this article, these fragments, derivatives and analogs are within the scope well known to those skilled in the art.

[0020] In a preferred embodiment, the antibody sequence further comprises a framework region FR; the framework region FR comprises the amino acid sequences of FR1, FR2, FR3 and FR4; the amino acid sequences of the framework region FR are:

[0021] FR1 or a variant of FR1 as shown in any one of SEQ ID NOs: 22-26, wherein the variant of FR1 comprises up to 5 amino acid substitutions in FR1;

[0022] FR2 or a variant of FR2 as set forth in any one of SEQ ID NOs: 27-29, wherein the variant of FR2 comprises up to 5 amino acid substitutions in FR2;

[0023] FR3 or a variant of FR3 as shown in any one of SEQ ID NOs: 30-34, wherein the variant of FR3 comprises up to 5 amino acid substitutions in FR3;

[0024] FR4 or a variant of FR4 shown in SEQ ID NO: 35, wherein the variant of FR4 comprises a substitution of up to 5 amino acids in the FR4.

[0025] The second aspect of the present invention is to provide an amino acid sequence of a single-domain antibody capable of binding to CD16a, wherein the amino acid sequence of the single-domain antibody is shown in SEQ ID NOs: 1-5, respectively, or the single-domain antibody has at least 80% sequence homology with the amino acid sequence of SEQ ID NOs: 1-5 and is capable of specifically binding to the CD16a protein, or the amino acid sequence of the single-domain antibody is compared with any one of SEQ ID NOs: 1-5, and at least one amino acid residue in the FR1, FR2, FR3 or FR4 sequence is substituted by a conservative amino acid.

[0026] In one embodiment, the anti-CD16a single domain antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to an amino acid sequence selected from SEQ ID NOs: 1-5, and is capable of specifically binding to CD16a protein.

[0027] The third aspect of the present invention is to provide an Fc fusion antibody or a humanized antibody of any of the aforementioned anti-CD16a single-domain antibodies.

[0028] The fourth aspect of the present invention is to provide a recombinant protein comprising any of the aforementioned anti-CD16a single-domain antibodies.

[0029] The fifth aspect of the present invention is to provide a bispecific antibody or a multispecific antibody comprising the single domain antibody according to any one of the foregoing items, wherein the single domain antibody serves as a first antigen binding portion that specifically binds to CD16a.

[0030] In one embodiment, the bispecific antibody or multispecific antibody further comprises a binding portion that is specific for another tumor antigen besides CD16a;

[0031] Preferably, other tumor antigens besides CD16a include FOLR1, Her2, DEC205, CLEC9A, CEACAM5, CTLA4, CD3, CD7, CD11c, CD19, CD20, CD22, CD40, CD44, CD206, EGFR, EGFRvIII, fibroblast activation protein (FAP), CA9, MMP-2, PD-L1, SIRPa, Trop2, GPC1, GPC3, cMET, BCMA, VEGFR, Cladin18.2, Nkp46, CD30, NKG2D, IL-2Rβ, BCMA, CD123, TGF-β, CD38, IL-7, IL-8, FRα, NCR3, IL-15, Muc1, IL-16 or any other tumor antigen.

[0032] In one embodiment, the bispecific antibodies include but are not limited to FOLR1 / CD16a, Her2 / CD16a, DEC205 / CD16a, CLEC9A / CD16a, CEACAM5 / CD16a, CTLA4 / CD16a, CD3 / CD16a, CD7 / CD16a, CD11c / CD16a, CD19 / CD16a, CD20 / CD16a, CD22 / CD16a, CD40 / CD16a, CD44 / CD16a, CD206 / CD16a, EGFR / CD16a, EGFRvIII / CD16a, Fibroblast Activation Protein (FAP) / CD16a, CA9 / CD16a, MMP-2 / CD16a, PD-L1 / Bispecific antibodies such as CD16a, SIRPa / CD16a, Trop2 / CD16a, GPC1 / CD16a, GPC3 / CD16a, cMET / CD16a, BCMA / CD16a, VEGFR / CD16a, Cladin18.2 / CD16a, CD30 / CD16a, NKG2D / CD16a, IL-2Rβ / CD16a, BCMA / CD16a, CD123 / CD16a, TGF-β / CD16a, CD38 / CD16a, IL-7 / CD16a, IL-8 / CD16a, FRα / CD16a, NCR3 / CD16a, IL-15 / CD16a, Muc1 / CD16a or IL-16 / CD16a.

[0033] In one embodiment, the aforementioned multispecific antibody is a trispecific antibody, comprising a first antigen-binding portion that specifically binds CD16a, a second antigen-binding portion that specifically binds Nkp46, and a third antigen-binding portion that specifically binds other tumor antigens other than CD16a and Nkp46.

[0034] In one embodiment, the trispecific antibodies include but are not limited to FOLR1 / CD16a / Nkp46, Her2 / CD16a / Nkp46, DEC205 / CD16a / Nkp46, CLEC9A / CD16a / Nkp46, CEACAM5 / CD16a / Nkp46, CTLA4 / CD16a / Nkp46, CD3 / CD16a / Nkp46, CD7 / CD16a / Nkp46, CD11c / CD16a / Nkp46, CD19 / CD16a / Nkp46, CD20 / CD16a / Nkp46, CD22 / CD16a / Nkp46, CD40 / CD1 6a / Nkp46, CD44 / CD16a / Nkp46, CD206 / CD16a / Nkp46, EGFR / CD16a / Nkp46, EGFRvIII / CD16a / Nkp46, Fibroblast Activation Protein (FAP) / CD16a / Nkp46, CA9 / CD16a / Nkp46, MMP-2 / CD16a / Nkp46, PD-L1 / CD16a / Nkp46, SIRPa / CD16a / Nkp46, Trop2 / C D16a / Nkp46, GPC1 / CD16a / Nkp46, GPC3 / CD16a / Nkp46, cMET / CD16a / Nkp46, BCMA / CD16a / Nkp46, VEGFR / CD16a / N kp46, Cladin18.2 / CD16a / Nkp46, CD30 / CD16a / Nkp46, NKG2D / CD16a / Nkp46, IL-2Rβ / CD16a / Nkp46, BCMA / CD16a / Trispecific antibodies such as Nkp46, CD123 / CD16a / Nkp46, TGF-β / CD16a / Nkp46, CD38 / CD16a / Nkp46, IL-7 / CD16a / Nkp46, IL-8 / CD16a / Nkp46, FRα / CD16a / Nkp46, NCR3 / CD16a / Nkp46, IL-15 / CD16a / Nkp46, Muc1 / CD16a / Nkp46 or IL-16 / CD16a / Nkp46.

[0035] In a preferred embodiment, the multispecific antibody is a trispecific antibody comprising a first antigen-binding portion that specifically binds CD16a, a second antigen-binding portion that specifically binds Nkp46, and a third antigen-binding portion that specifically binds FOLR1;

[0036] the second antigen-binding portion has CDR1 set forth in SEQ ID NO: 63, CDR2 set forth in SEQ ID NO: 65, and CDR3 set forth in SEQ ID NO: 70;

[0037] the third antigen-binding portion has CDR1 set forth in SEQ ID NO: 58, CDR2 set forth in SEQ ID NO: 59, and CDR3 set forth in SEQ ID NO: 60;

[0038] Preferably, the second antigen binding moiety that specifically binds Nkp46 and the third antigen binding moiety that specifically binds FOLR1 are VHHs;

[0039] Preferably, the trispecific antibody is not fused to an Fc.

[0040] In one embodiment, the amino acid sequences of the aforementioned trispecific antibodies are shown in SEQ ID NOs: 47-51, respectively.

[0041] The sixth aspect of the present invention is to provide a nucleotide molecule encoding the aforementioned anti-CD16a single domain antibody or the aforementioned Fc fusion antibody or the aforementioned humanized antibody, whose nucleotide sequences are shown in SEQ ID NOs: 6-10, respectively, or the amino acid sequence encoded by the nucleotide sequence is the same as the amino acid sequence encoded by any one of SEQ ID NOs: 6-10, or has at least 95% sequence homology with any one of SEQ ID NOs: 6-10.

[0042] In one embodiment, the nucleic acid molecule encoding the anti-CD16a single domain antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to a nucleotide sequence selected from SEQ ID NOs: 6-10, and the anti-CD16a single domain antibody encoded thereby can specifically bind to the CD16a protein.

[0043] The seventh aspect of the present invention is to provide a nucleotide molecule encoding the aforementioned bispecific antibody or multispecific antibody, whose nucleotide sequence is shown in SEQ ID NO: 52-56, respectively, or the amino acid sequence encoded by the nucleotide sequence is the same as the amino acid sequence encoded by any one of SEQ ID NO: 52-56, or has at least 95% sequence homology with any one of SEQ ID NO: 52-56.

[0044] In one embodiment, the nucleotide molecule encoding the aforementioned bispecific antibody or multispecific antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a nucleotide sequence selected from SEQ ID NOs: 52-56, and the bispecific antibody or multispecific antibody encoded thereby can specifically bind to CD16a, FOLR1, or Nkp46 protein.

[0045] The eighth aspect of the present invention is to provide an expression vector comprising the aforementioned nucleotide molecule encoding an anti-CD16a single-domain antibody, Fc fusion antibody, or humanized antibody, or comprising the aforementioned nucleotide molecule encoding a bispecific antibody or multispecific antibody.

[0046] In a preferred embodiment, the expression vector used can be RJK-V4-hFC (a nucleotide molecule encoding an anti-CD16a single-domain antibody or its Fc fusion antibody or humanized antibody is integrated into RJK-V4-hFC by genetic engineering). Other general expression vectors can also be selected as needed.

[0047] A ninth aspect of the present invention provides a host cell capable of expressing the aforementioned anti-CD16a single-domain antibody, Fc fusion antibody, humanized antibody, bispecific antibody, or multispecific antibody, or an expression vector comprising the aforementioned host cell. Preferably, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0048] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell, including bacteria and fungi.

[0049] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or a combination thereof.

[0050] In another preferred embodiment, the prokaryotic cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or a combination thereof.

[0051] In another preferred embodiment, the eukaryotic cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, or a combination thereof.

[0052] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of insect cells such as fall armyworm, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or a combination thereof.

[0053] In another preferred embodiment, the host cell is a suspension ExpiCHO-S cell.

[0054] In another preferred embodiment, the host cell is a suspension 293F cell.

[0055] The tenth aspect of the present invention is to provide a recombinant protein comprising the aforementioned anti-CD16a single-domain antibody. The recombinant protein may be a single-domain antibody as shown in SEQ ID NOs: 1-5, or a single-domain antibody having at least 80% homology with SEQ ID NOs: 1-5, or a multi-epitope antibody, a bispecific antibody, a multispecific antibody, or a multivalent antibody; for example, the multi-epitope antibody may be composed of more than one sequence in SEQ ID NOs: 1-5; the multivalent antibody may be composed of one of the sequences in SEQ ID NOs: 1-5 repeated several times; the multispecific antibody includes but is not limited to a trispecific antibody and a tetraspecific antibody; in addition, the recombinant protein may be a fragment, derivative, or analog of the aforementioned antibody.

[0056] The eleventh aspect of the present invention is to provide a pharmaceutical composition comprising the aforementioned single-domain antibody that binds to CD16a, the aforementioned bispecific antibody, or the multispecific antibody, and a pharmaceutically acceptable carrier. Typically, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically determined based on the isoelectric point of the antibody (the pH of the aqueous carrier medium must deviate from the isoelectric point of the antibody and differ by approximately 2 from the isoelectric point of the antibody). The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous, transdermal (direct application to the affected area or application of a plaster).

[0057] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the aforementioned single-domain antibody and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.

[0058] The twelfth aspect of the present invention is to provide a medicament for treating a disease, which comprises the aforementioned single-domain antibody for binding to CD16a protein, the aforementioned bispecific antibody or multispecific antibody as an active ingredient.

[0059] The thirteenth aspect of the present invention is to provide a kit for detecting CD16a levels, comprising the aforementioned anti-CD16a single domain antibody. In a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, and the like.

[0060] In a preferred embodiment, the kit includes an antibody that recognizes CD16a protein, a lysis medium for dissolving the sample, and general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0061] In a preferred embodiment, the kit further comprises a secondary antibody and an enzyme or fluorescent or radioactive label for detection, and a buffer.

[0062] In a preferred embodiment, the second antibody of the kit can be the aforementioned anti-CD16a single domain antibody (as an anti-antibody), and can be a single domain antibody, a monoclonal antibody, a polyclonal antibody or any other form of antibody.

[0063] The thirteenth aspect of the present invention provides a method for producing an anti-CD16a single domain antibody, comprising the steps of:

[0064] (a) culturing the host cell according to the ninth aspect of the present invention under conditions suitable for producing the single domain antibody, thereby obtaining a culture containing the anti-CD16a single domain antibody; and

[0065] (b) isolating or recovering the anti-CD16a single domain antibody from the culture; and

[0066] (c) optionally, purifying and / or modifying the single domain antibody against CD16a obtained in step (b).

[0067] The fourteenth aspect of the present invention is to provide use of the aforementioned anti-CD16a single-domain antibody, the aforementioned bispecific antibody or multispecific antibody or the aforementioned pharmaceutical composition in the preparation of a drug for treating a disease.

[0068] In a preferred embodiment, the disease is various disorders mediated by NK cells and associated with CD16a.

[0069] In a preferred embodiment, various NK cell-mediated CD16a-associated disorders include, but are not limited to, rheumatoid arthritis (RA), bone erosions, intraperitoneal abscesses, inflammatory bowel disease, allogeneic transplant rejection, psoriasis, angiogenesis, atherosclerosis, asthma, multiple sclerosis, systemic lupus erythematosus (SLE), ocular surface disorders (e.g., dry eye), ankylosing spondylitis, psoriatic arthritis, cancers (e.g., multiple myeloma and breast cancer).

[0070] In a preferred embodiment, the disease includes but is not limited to tumors, autoimmune diseases, metabolism-related diseases, and infectious diseases.

[0071] In a preferred embodiment, the infectious disease includes acute and chronic infectious diseases (eg, bacterial infection or viral infection).

[0072] In a preferred embodiment, the tumor includes solid tumors and hematological tumors.

[0073] In a preferred embodiment, tumors include, but are not limited to, tumors of epithelial origin (adenomas and various types of carcinomas, including adenocarcinomas, squamous cell carcinomas, transitional cell carcinomas, and others); bladder and urinary tract cancers; breast cancers; gastrointestinal cancers (including esophageal cancer, stomach cancer, small intestine cancer, colon cancer, rectal cancer, and anal cancer); liver (hepatocellular carcinoma); cancers of the gallbladder and biliary system, exocrine pancreas, and kidney; lung cancers (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchioloalveolar carcinoma, and mesothelioma); head and neck cancers (e.g., tongue cancer, oral cancer, laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, tonsil cancer, salivary gland cancer, nasal cavity cancer, and paranasal sinus cancer); cancers of the ovary, fallopian tube, peritoneum, vagina, vulva, penis, cervix, myometrium, and endometrium; thyroid cancer (e.g., follicular thyroid carcinoma); adrenal, prostate, skin, and adnexal cancers (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthomas, dysplastic nevi); hematologic malignancies (i.e., leukemias, lymphomas) and precancerous and borderline malignant diseases, including hematologic malignancies and disorders of the lymphoid lineage (e.g., acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell lymphomas such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, T-cell lymphomas and leukemias, natural killer (NK) cell lymphomas, Hodgkin lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma); , multiple myeloma, and post-transplant lymphoproliferative disorders) and hematologic malignancies and myeloid-related disorders (e.g., acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), hypereosinophilic syndrome, myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome, and promyelocytic leukemia); tumors of mesenchymal origin, such as sarcomas of soft tissue, bone, or cartilage, such as osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant central or peripheral nervous system tumors (e.g., astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pinealomas, and schwannomas); endocrine tumors (e.g., pituitary tumors, adrenal tumors, pancreatic islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary thyroid carcinoma); ocular and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, seminoma, dysgerminoma, hydatidiform mole, and choriocarcinoma); pediatric and embryonal tumors (e.g., medulloblastoma, neuroblastoma, Wilms tumor, and primitive neuroectodermal tumor); or congenital or other syndromes that predispose patients to malignancy (e.g., xeroderma pigmentosum).

[0074] In a preferred embodiment, the disease includes but is not limited to peripheral T-cell lymphoma, Hodgkin's lymphoma, mycosis fungoides, anaplastic large cell lymphoma, pancreatic cancer, gastroesophageal junction cancer, gastric cancer, hepatocellular carcinoma, renal clear cell carcinoma, biliary tract cancer, colorectal cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, diffuse large B-cell lymphoma, mantle cell lymphoma, marginal zone lymphoma, follicular lymphoma, multiple myeloma, acute myeloid leukemia, myelodysplastic syndrome, non-Hodgkin's lymphoma, B-cell blood cancer, viral infection. Beneficial effects

[0075] Compared with the prior art, the present invention has the following beneficial effects:

[0076] (1) The single-domain antibody of the present invention is specific for the CD16a protein with a correct spatial structure.

[0077] (2) The single-domain antibody obtained by the present invention has a flexible expression system selection and can be expressed in either a prokaryotic system or a eukaryotic system of yeast cells or mammalian cells. In addition, the expression cost in the prokaryotic expression system is low, which can reduce the subsequent production cost.

[0078] (3) The single-domain antibodies obtained by the present invention are simple to modify in multiple combinations. Multivalent and multispecific antibodies can be obtained by simple concatenation through genetic engineering. In addition, their immune heterogeneity is very low and they will not produce a strong immune response without humanization.

[0079] (4) The single-domain antibodies obtained by the present invention have a wider affinity range. Before affinity maturation, their affinity range can range from nM level to pM level, providing multiple options for antibodies with different uses in the future;

[0080] (5) The single-domain antibody against CD16a obtained by the present invention has strong antigen binding ability and specificity, and has excellent redirected killing ability, and can effectively mediate the ADCC effect. This single-domain antibody is combined with other antigen-binding moieties to form a bispecific or multispecific antibody, and can also be used as part of a chimeric antigen receptor (CAR), or assembled into any other form of an antibody.

[0081] The trispecific antibody FOLR1 / CD16a / Nkp46 (FOLR1 / CD16a / Nkp46 is only used as an embodiment, and CD16a can also be combined with other tumor surface antigen targets) prepared based on the CD16a single-domain antibody has an unexpectedly excellent ADCC effect without the addition of immunoglobulin Fc. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] In order to more clearly illustrate the technical solution of the present application, the following is a brief introduction to the drawings required for use in the embodiments. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0083] Figure 1 shows the enrichment of the library for screening antibodies targeting CD16a in Example 3, wherein the left side shows the enrichment of the library for screening antibodies targeting CD16a-V176, and the right side shows the enrichment of the library for screening antibodies targeting CD16a-F176;

[0084] FIG2 is a dose-effect curve (14A3) of the binding of the CD16a-hFc antibody to human CD16a (V176) in Example 12;

[0085] FIG3 is a binding dose-effect curve (23D1) of the CD16a-hFc antibody and human CD16a (V176) in Example 12;

[0086] FIG4 is a binding dose-effect curve of the CD16a-hFc antibody and human CD16a (V176) in Example 12 (15E2, 13G6);

[0087] FIG5 is a binding dose-effect curve (4B5) of the CD16a-hFc antibody to human CD16a (V176) in Example 12;

[0088] FIG6 is a binding dose-effect curve (4B5) of the CD16a-hFc antibody of Example 12 to human CD16a (F176);

[0089] FIG7 is a binding dose-effect curve of the CD16a-hFc antibody in Example 12 and human CD16a (F176) (15E2, 13G6);

[0090] FIG8 is a binding dose-effect curve of the CD16a-hFc antibody in Example 12 to human CD16a (F176) (14A3);

[0091] FIG9 is a binding dose-effect curve (23D1) of the CD16a-hFc antibody in Example 12 to human CD16a (F176);

[0092] Figure 10 shows the RKA test results of CD16a-hFc antibody (4B5);

[0093] FIG11 shows the RKA test results of CD16a-hFc antibody (15E2);

[0094] Figure 12 shows the RKA test results of CD16a-hFc antibodies (13G6, 14A3);

[0095] FIG13 shows the RKA test results of CD16a-hFc antibody (23D1);

[0096] FIG14 shows the ADCC test results of CD16a-hFc (4B5);

[0097] FIG15 shows the ADCC test results of CD16a-hFc (15E2);

[0098] FIG16 shows the ADCC test results of CD16a-hFc (13G6, 14A3);

[0099] FIG17 shows the ADCC test results of CD16a-hFc (23D1);

[0100] FIG18 shows the ADCC test results of the FOLR1 / Nkp46 / CD16a trispecific antibody;

[0101] Figures 19-21 show the ADCC test results of the FOLR1 / Nkp46 / CD16a trispecific antibody;

[0102] FIG22 shows the ADCC test results of the FOLR1 / Nkp46 bispecific antibody. DETAILED DESCRIPTION

[0103] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0104] As used herein, "single-domain antibodies" (sdAbs, also referred to as nanobodies or VHHs by Ablynx) are well known to those skilled in the art. Single-domain antibodies are antibodies whose complementary determining regions are part of a single domain polypeptide. Thus, single-domain antibodies comprise a single complementary determining region (a single CDR1, a single CDR2, and a single CDR3). Examples of single-domain antibodies are antibodies that have only heavy chains (which naturally do not contain light chains), single-domain antibodies derived from conventional antibodies, and engineered antibodies.

[0105] Single domain antibodies can be derived from any species, including mice, humans, camels, llamas, goats, rabbits and cattle. For example, naturally occurring VHH molecules can be derived from antibodies provided by species of the Camelidae family (e.g., camels, dromedaries, llamas and guanacos). Like complete antibodies, single domain antibodies are capable of selectively binding to specific antigens. Single domain antibodies can contain only the variable domain of an immunoglobulin chain, which has CDR1, CDR2 and CDR3 and a framework region.

[0106] As used herein, the term "sequence homology" refers to the extent to which two (nucleotide or amino acid) sequences have identical residues at identical positions in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Thus, two copies of identical sequences have 100% homology.

[0107] As used herein, the term "Fc fusion antibody" refers to a novel protein produced by fusing the Fc segment of an antibody of interest with a functional protein molecule having biological activity using genetic engineering technology.

[0108] The term "humanized antibody" refers to an antibody obtained by fusing the heavy chain variable region of a target antibody (such as an animal antibody) with the constant region of a human antibody, or by transplanting the complementarity determining regions (CDR1-3 sequences) of a target antibody into the variable region of a human antibody, or by subjecting the target antibody to amino acid mutations based on the characteristics of the human antibody framework region (FR1-4). Humanized antibodies can be produced synthetically or by site-directed mutagenesis.

[0109] In the present invention, sequences with high sequence homology to the CDR1-3 sequences disclosed herein can also be used to generate single-domain antibodies against CD16a. In some embodiments, sequences having "at least 80% homology," or "at least 85% homology," "at least 90% homology," "at least 95% homology," or "at least 98% homology" to the sequences in SEQ ID NOs. 1-5 can also achieve the objectives of the invention.

[0110] In some embodiments, sequences that replace only one or a few amino acids compared to the sequences in SEQ ID NO. 1-5, for example, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions, can also achieve the purpose of the invention. In fact, when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2 and CDR3 in a single domain antibody, a technician may consider so-called "conservative" amino acid substitutions. In the case of substitutions, the substitution will preferably be a conservative amino acid substitution, which can generally be described as an amino acid substitution in which an amino acid residue is replaced by another amino acid residue with a similar chemical structure, and the substitution has little or substantially no effect on the function, activity or other biological properties of the polypeptide. The conservative amino acid substitutions are common in the art. For example, conservative amino acid substitutions are substitutions of one or a few amino acids in the following groups (a)-(d) by another or a few amino acids in the same group: (a) polar negatively charged residues and their uncharged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. Particularly preferred conservative amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg or Gln; Phe is replaced by Met, Leu, or Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe or Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr, or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser. In addition, those skilled in the art will appreciate that the creativity of single-domain antibodies is embodied in the CDR1-3 regions, while the framework region sequences FR1-4 are not immutable. The sequences of FR1-4 can be conservative sequence variants of the sequences disclosed in the present invention.

[0111] Preferred host cells of the present invention are bacterial cells, fungal cells or mammalian cells.

[0112] This patent uses genetic engineering technology to prepare target proteins and truncated forms of target proteins, and then immunizes the obtained antigen proteins into Bactrian camels in Alxa League, Inner Mongolia. After multiple immunizations, peripheral blood lymphocytes or spleen cells of the camels are obtained. Through genetic engineering, the camel-derived antibody variable region coding sequence is recombined into a phage display vector. Specific antibodies against the antigen protein are screened through phage display technology, and their ability to bind to the antigen and their application in the treatment of autoimmune diseases are further tested.

[0113] The above technical solution is now broken down into details and described in the form of specific embodiments:

[0114] Example 1: Preparation of human CD16a recombinant extracellular domain protein:

[0115] The human recombinant extracellular domain protein used in this patent was obtained through the company's own expression and purification. The expression vector design scheme for the human recombinant CD16a protein is as follows:

[0116] (1) The coding sequence of CD16a was retrieved from NCBI and its accession number is NM_000569.7. The amino acid sequence generated by this sequence is accession number NP_000560.6.

[0117] (2) The nucleotide sequences encoding amino acids 17 to 208 of CD16a (CD16a-V176, CD16a-F176) were cloned into the pcDNA3.4 vector by gene synthesis. The constructed vector was Sanger sequenced and compared with the original sequence. After confirmation, the recombinant plasmid was extensively extracted to remove endotoxins and transfected into suspension 293F cells for target protein expression and purification. The purity reached over 90%, meeting animal immunization requirements.

[0118] There are two antigens: one is CD16a-F176, which is amino acids 17 to 208, with amino acid 176 being F; the other is CD16a-V176, which is amino acids 17 to 208, with amino acid 176 being V. These two antigens are prepared and purified separately.

[0119] Example 2: Construction of a single domain antibody library against CD16a protein:

[0120] 1 mg of the purified human recombinant CD16a protein (CD16a-F176, CD16a-V176) obtained in Example 1 was mixed with an equal volume of Freund's complete adjuvant and used to immunize Bactrian camels in Alxa, Inner Mongolia, once a week for a total of seven consecutive immunizations. Except for the first immunization, the remaining six immunizations were performed using 1 mg of CD16a protein mixed with an equal volume of Freund's incomplete adjuvant. This immunization process is intended to centrally stimulate the camels to produce antibodies against the CD16a protein. CD16a-V176 and CD16a-F176 were not mixed, and the animals were immunized separately to obtain antibodies.

[0121] After the animals were immunized, 150 mL of peripheral blood lymphocytes were collected and RNA was extracted from the cells. The extracted total RNA was used to synthesize cDNA, and the VHH (antibody heavy chain variable region) was amplified using the cDNA as a template through a nested PCR reaction.

[0122] The pMECS vector and VHH fragment were then digested with restriction endonucleases, and the digested fragments and vector were linked. The linked fragments were electroporated into competent cells TG1 to construct a phage display library of CD16a protein and measure the library capacity. The library capacity was approximately 1×10 9 At the same time, the correct insertion rate of the target fragment in the library was detected by colony PCR identification.

[0123] The results showed that after PCR amplification of 30 colonies randomly selected from the library, 29 clones could amplify bands of the predicted size, and 1 clone amplified an incorrect band, so the correct insertion rate was 29÷30×100%≈96.7%.

[0124] Example 3: Screening of single domain antibodies against CD16a protein:

[0125] 200 μL of the recombinant TG1 cells in Example 2 were cultured in 2×TY medium, during which 40 μL of helper phage VCSM13 was added to infect the TG1 cells and cultured overnight to amplify the phage. The next day, the phage was precipitated with PEG / NaCl and the amplified phage was collected by centrifugation.

[0126] 500 μg of CD16a protein diluted in 100 mM NaHCO3 at pH 8.3 was coupled to an ELISA plate and placed at 4°C overnight. A negative control well (culture medium control) was also set up. The next day, 200 μL of 3% skim milk was added and the plate was blocked at room temperature for 2 h. After blocking, 100 μL of amplified phage library (approximately 2 × 10 11 phage particles) at room temperature for 1 hour; after 1 hour, the cells were washed 15 times with PBS + 0.05% Tween-20 to remove unbound phage.

[0127] Phages specifically bound to the CD16a protein were dissociated using trypsin at a final concentration of 25 mg / mL and infected with Escherichia coli TG1 cells in the logarithmic growth phase. The cells were cultured at 37°C for 1 hour to produce and collect phages for the next round of screening. The same screening process was repeated for one round to gradually enrich the phages.

[0128] When the enrichment multiple reaches more than 10 times, the enrichment effect is shown in Figure 1.

[0129] In Figure 1, P / N = the number of monoclonal bacteria grown after phage eluted from the positive wells in bio-panning infected with TG1 bacteria / the number of monoclonal bacteria grown after phage eluted from the positive wells infected with TG1 bacteria. This parameter will gradually increase after enrichment occurs; I / E = the total amount of phage added to the positive wells in each round of bio-panning / the total amount of phage eluted from the positive wells in each round of bio-panning. This parameter will gradually approach 1 after enrichment occurs.

[0130] Example 4: Screening of specific positive clones for CD16a using phage enzyme-linked immunosorbent assay (ELISA):

[0131] Three rounds of screening were performed for single-domain antibodies against CD16a protein according to the screening method in Example 3 above. The phage enrichment factor of anti-CD16a protein reached 10 or greater. After the screening, 384 single colonies were selected from the positive clones obtained by screening and inoculated into 96-deep-well plates in 2×TY medium containing 100 μg / mL ampicillin. A blank control was set up. After culturing at 37°C to the logarithmic phase, IPTG was added to a final concentration of 1 mM and cultured overnight at 28°C.

[0132] Crude antibody was obtained using the osmotic swelling method. CD16a recombinant protein was released into 100 mM NaHCO₃ (pH 8.3), and 100 μg of protein was coated on an ELISA plate overnight at 4°C. 100 μL of the crude antibody extract was transferred to the antigen-added ELISA plate and incubated at room temperature for 1 hour. Unbound antibody was washed with PBST, and 100 μL of a 1:2000 diluted Mouse Anti-HA tag Antibody (HRP) (Thermo Fisher) was added and incubated at room temperature for 1 hour. Unbound antibody was washed with PBST, and horseradish peroxidase colorimetric solution was added. After incubation at 37°C for 15 minutes, stop solution was added, and absorbance was read at 450 nm on a microplate reader.

[0133] When the OD value of the sample well is more than 5 times greater than that of the control well, it is determined to be a positive clone well; the bacteria in the positive clone well are transferred and shaken into LB medium containing 100 μg / mL ampicillin to extract the plasmid and perform sequencing.

[0134] The gene sequences of each clone were analyzed using the sequence alignment software VectorNTI. Strains with identical CDR1, CDR2, and CDR3 sequences were considered the same clone, while strains with different sequences were considered different clones. Ultimately, a single-domain antibody specific for the CD16a protein was obtained.

[0135] The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, which constitutes the entire VHH. The obtained single-domain antibody recombinant plasmid can be expressed in a prokaryotic system to ultimately obtain a single-domain antibody protein.

[0136] Among them, the single-domain antibody clones obtained by screening with antigen CD16a-F176 include 15E2 and 4B5; the single-domain antibody clones obtained by screening with antigen CD16a-V176 include 14A3, 23D1, and 13G6.

[0137] Other single-domain antibody clones (sequences not shown) were also screened using antigens CD16a-F176 and CD16a-V176: 18E4, 10B6, 8G8, 15A7, and 24C6W.

[0138] The CDR and FR sequences of the five single-domain antibodies are shown in Tables 1-7, and the amino acid sequences and nucleotide sequences of the five single-domain antibodies are shown in Tables 8 and 9, respectively.

[0139] Table 1 CDR1 sequences of five single-domain antibodies

[0140] Table 2 CDR2 sequences of five single-domain antibodies

[0141] Table 3 CDR3 sequences of five single-domain antibodies

[0142] Table 4 FR1 sequences of five single-domain antibodies

[0143] Table 5 FR2 sequences of five single domain antibodies

[0144] Table 6 FR3 sequences of five single domain antibodies

[0145] Table 7 FR4 sequences of five single domain antibodies

[0146] Table 8 Amino acid sequences of five single domain antibodies

[0147] Table 9 Nucleic acid sequences of 5 single domain antibodies

[0148] Example 5: Construction of humanized FOLR1 / Nkp46 bispecific antibody domains

[0149] Referring to the methods of Examples 1-4, single domain antibodies of FOLR1 and Nkp46 were screened and obtained, namely FOLR1-4F4 single domain antibody and Nkp46-7F10 single domain antibody; the specific screening process was different from that of Examples 1-4, except that the antigen preparation process was different.

[0150] Preparation of recombinant human Nkp46 extracellular domain protein:

[0151] The human recombinant extracellular domain protein used in this patent was obtained through the company's own expression and purification. The expression vector design scheme for the human recombinant Nkp46 protein is as follows:

[0152] (1) The coding sequence of Nkp46 was retrieved from NCBI and its accession number is BC064806.1. The amino acid sequence generated by this sequence is accession number AAH64806.1.

[0153] (2) The nucleotide sequence encoding amino acids 22 to 254 of Nkp46 was cloned into the vector pcDNA3.4 by gene synthesis. The constructed vector was subjected to Sanger sequencing and compared with the original sequence. After confirmation, the recombinant plasmid was extensively extracted to remove endotoxins and transfected into suspension 293F cells for target protein expression and purification. The purity reached over 90%, meeting animal immunization requirements.

[0154] Preparation of recombinant human FOLR1 extracellular domain protein:

[0155] The human recombinant extracellular domain protein used in this patent was obtained through the company's own expression and purification. The expression vector design scheme for the human recombinant FOLR1 protein is as follows:

[0156] (1) The coding sequence of FOLR1 was retrieved from NCBI and its accession number is NM_000802.3. The amino acid sequence generated by this sequence is accession number NP_000793.1.

[0157] (2) The nucleotide sequence encoding amino acids 25 to 233 of FOLR1 was cloned into pcDNA3.4 by gene synthesis. The constructed vector was Sanger sequenced and compared with the original sequence. After confirmation, the recombinant plasmid was extracted in large quantities to remove endotoxins and transfected into suspension 293F cells for target protein expression and purification. The purity reached over 90%, meeting animal immunization requirements.

[0158] The sequences of the screened FOLR1-4F4 single-domain antibodies are shown in Tables 10 and 11. Humanized modifications were performed on this basis, and the sequence of the humanized 4F4 is shown in Table 12. The modified regions include FR1, CDR1, CDR2, CDR3, and FR4, as shown in the underlined sections in Table 12.

[0159] The sequences of the screened Nkp46-7F10 single-domain antibodies are shown in Tables 13 and 14. Humanized modifications were performed on this basis, and the sequence of the humanized 7F10 is shown in Table 15. The modified region includes CDR3, as shown in the underlined portion of Table 15.

[0160] Humanized 4F4 and humanized 7F10 were linked to form humanized FOLR1 / Nkp46, the sequences of which are shown in Table 16, wherein humanized 4F4 is represented by amino acids 1-125 of SEQ ID NO: 45, the linker between humanized 4F4 and humanized 7F10 is represented by amino acids 126-140 of SEQ ID NO: 45 (GGGGSGGGGSGGGGS), and humanized 7F10 is represented by amino acids 141-267 of SEQ ID NO: 45.

[0161] Table 10 CDR and FR sequences of FOLR1 (4F4)

[0162] Table 11 Amino acid sequence and nucleic acid sequence of FOLR1 (4F4)

[0163] Table 12 CDR and FR sequences of FOLR1 (humanized 4F4)

[0164] Table 13 CDR and FR sequences of Nkp46 (7F10)

[0165] Table 14 Amino acid sequence of Nkp46 (7F10)

[0166] Table 15 CDR and FR sequences of Nkp46 (humanized 7F10)

[0167] Table 16 Amino acid and nucleic acid sequences of humanized FOLR1 / Nkp46 dual antibody

[0168] Example 6: Purification and expression of CD16a protein-specific single-domain antibodies in host bacteria Escherichia coli

[0169] The plasmids (pMECS-VHH) of the different clones obtained by sequencing analysis in Example 4 were electroporated into Escherichia coli HB2151 and plated on LB+amp+glucose plates containing ampicillin and glucose, and cultured at 37°C overnight. Single colonies were selected and inoculated into 5 mL of LB culture medium containing ampicillin and cultured in a shaking incubator at 37°C overnight.

[0170] Inoculate 1 mL of overnight culture into 330 mL of TB medium and culture at 37°C with a shaker. When the OD600nm value reaches 0.6-0.9, add 1 M IPTG and culture overnight at 28°C with a shaker. Centrifuge to collect the E. coli and obtain a crude antibody extract using the osmotic swelling method.

[0171] The single domain antibody was purified by nickel column affinity chromatography.

[0172] Example 7: Construction of a eukaryotic expression vector for an anti-CD16a single-domain antibody Fc fusion antibody

[0173] (1) Subcloning the target sequence obtained in Example 4 into a eukaryotic expression vector: The antibodies screened in Example 4 were subjected to Sanger sequencing to obtain their nucleotide sequences;

[0174] (2) The above nucleotide sequence was synthesized into the vector RJK-V4-hFC designed and modified by our company by sequence synthesis to obtain a recombinant eukaryotic expression vector. The modification method of the vector is as described in Example 11;

[0175] (3) transforming the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culturing and performing plasmid extraction to remove endotoxins;

[0176] (4) Sequencing the extracted plasmid;

[0177] (5) The confirmed recombinant vector is prepared for subsequent eukaryotic cell transfection and expression, and the Fc protein of VHH is expressed by the method of Example 8 or 9, and the above-mentioned antibody is purified by the method of Example 10.

[0178] Example 8: Expression of single domain antibodies against CD16a protein in suspension ExpiCHO-S cells

[0179] (1) 3 days before transfection, 2.5×10 5 / mL cell passaging and expansion culture of ExpiCHO-S TM The calculated volume of cells was transferred to a fresh, pre-warmed 120 mL (final volume) of ExpiCHO TM The cells were cultured in a 500 mL shake flask containing expression medium; the cell concentration reached approximately 4 × 106 -6×10 6 Viable cells / mL;

[0180] (2) One day before transfection, TM The cells were diluted to a concentration of 3.5 × 10 6 viable cells / mL, and cells were cultured overnight;

[0181] (3) On the day of transfection, measure the cell density and percentage of viable cells. The cell density should reach about 7×10 6 -10×10 6 Viable cells / mL;

[0182] (4) Use fresh ExpiCHO preheated to 37℃ TM Dilute the cells to 6 × 10 6 The calculated required cell volume was transferred to a fresh, pre-warmed 100 mL (final volume) of ExpiCHO TM in a 500 mL shake flask containing expression medium;

[0183] (5) Gently invert to mix ExpiFectamine TM CHO reagent, use 3.7mL OptiPRO TM Dilute ExpiFectamine in culture medium TM CHO reagent, swirl or mix;

[0184] (6) Use 4 mL of refrigerated OptiPRO TM Dilute the plasmid DNA in the culture medium and vortex to mix;

[0185] (7) Incubate the ExpiFectamine CHO / plasmid DNA (the plasmid DNA is the Fc fusion antibody eukaryotic expression vector of the anti-CD16a single domain antibody prepared in Example 7) complex at room temperature for 1-5 minutes, then gently add it to the prepared cell suspension, gently shaking the flask during the addition process;

[0186] (8) The cells were cultured with shaking at 37°C, 8% CO2, and humidified air;

[0187] (9) On the first day after transfection (18-22 hours later), add 600ul of ExpiFectamine TM CHO Enhancer and 24mL ExpiCHO feed.

[0188] (10) Collect the supernatant approximately 8 days after transfection (cell viability is less than 70%).

[0189] Example 9: Expression of single domain antibodies against CD16a protein in suspension 293F cells

[0190] Recombinant single domain antibody expression experimental process (taking 500mL shake flask as an example):

[0191] (1) 3 days before transfection, 2.5×10 5 For passage and expansion of 293F cells, the calculated volume of cells was transferred to a 500 mL shake flask containing 120 mL (final volume) of fresh pre-warmed OPM-293CD05 Medium. The cell concentration reached approximately 2 × 10 6 -3×10 6 viable cells / mL.

[0192] (2) On the day of transfection, measure the cell density and percentage of viable cells. The cell density should reach about 2×10 6 -3×10 6 viable cells / mL.

[0193] (3) Dilute the cells to 1×10 6 The calculated volume of cells was transferred to a 500 mL shake flask containing 100 mL (final volume) of fresh pre-warmed culture medium.

[0194] (4) Dilute PEI (1 mg / mL) with 4 mL of Opti-MEM medium and mix thoroughly by vortexing or pipetting. Dilute plasmid DNA (the plasmid DNA is the eukaryotic expression vector for the Fc fusion antibody of the anti-CD16a single-domain antibody prepared in Example 7) with 4 mL of Opti-MEM medium, mix thoroughly by vortexing, and filter through a 0.22 μm filter. Incubate at room temperature for 5 min.

[0195] (5) Add the diluted PEI reagent to the diluted DNA and mix thoroughly by inverting. Incubate the PEI / plasmid DNA complex at room temperature for 15-20 minutes, then gently add it to the prepared cell suspension, gently shaking the flask during the addition process.

[0196] (6) The cells were cultured at 37°C, 5% CO2, and shaking at 120 rpm.

[0197] (7) Add 5 mL of OPM-CHO PFF05 feed at 24 h and 72 h after transfection.

[0198] (8) Collect the supernatant approximately 7 days after transfection (cell viability is less than 70%).

[0199] Example 10: Purification of single domain antibodies against CD16a protein

[0200] (1) The protein expression supernatant obtained in Example 8 or 9 was filtered through a 0.45 μm disposable filter to remove insoluble impurities;

[0201] (2) Purifying the filtrate by affinity chromatography using a protein purifier, utilizing the ability of human Fc to bind to Protein A, and using agarose filler coupled to Protein A for purification;

[0202] (3) The filtrate is passed through a Protein A prepacked column at a flow rate of 1 mL / min. During this step, the target protein in the filtrate will bind to the filler.

[0203] (4) washing the impurity proteins bound to the column with low-salt and high-salt buffers;

[0204] (5) Separate the target protein bound to the column with a low pH buffer;

[0205] (6) The eluate was quickly added to a pH 9.0 Tris-HCl solution for neutralization;

[0206] (7) The neutralized protein solution was dialyzed and analyzed by SDS-PAGE to confirm that the protein purity was above 95% and the concentration was above 0.5 mg / mL, and then stored at low temperature for future use.

[0207] Example 11: Construction of single-domain antibody eukaryotic expression vector RJK-V4-hFC

[0208] The target vector RJK-V4-hFC, commonly used for nanobodies, was modified by our company based on the Invitrogen commercial vector pCDNA3.4 (vector data link: https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / pcdna3_4_topo_ta_cloning_kit_man.pdf) by fusing the Fc region of the human IgG1 heavy chain coding sequence. This vector contains the hinge (CH2) and CH3 regions of the IgG1 heavy chain. The specific modification plan is as follows:

[0209] (1) Select the restriction enzyme sites XbaI and AgeI on pcDNA3.4;

[0210] (2) A multiple cloning site (MCS) and a 6×His tag were introduced at the 5′ and 3′ ends of the Fc fragment coding sequence, respectively, by overlapping PCR;

[0211] (3) amplifying the above fragment by PCR using a pair of primers with XbaI and AgeI restriction sites, respectively;

[0212] (4) using restriction endonucleases XbaI and AgeI to digest the recombinant DNA fragments in pcDNA3.4 and (3), respectively;

[0213] (5) The digested vector and the inserted fragment were ligated with T4 ligase, and the ligated product was transformed into Escherichia coli, amplified, and sequenced to obtain a recombinant plasmid.

[0214] Example 12: Antibody antigen binding dose-effect curve determination

[0215] This example was performed using a standard enzyme-linked immunosorbent assay (ELISA) procedure.

[0216] (1) Coat with 50 μL of 1 μg / mL human CD16a-V176 or human CD16a-F176 protein at 4°C overnight.

[0217] (2) Wash the plate; add 200 μL of 5% milk and block at 37°C for 2 h.

[0218] (3) Dilute the VHH-Fc to 2 μg / mL, and then dilute the antibody 5-fold in a gradient, for a total of 8 concentration gradients. The VHH-Fc herein refers to the Fc-fused single-domain antibody purified from Example 10.

[0219] (4) Wash the plate; add 50 μL of the single domain antibody diluted in step (3), duplicate wells, and incubate at 37°C for 1 h.

[0220] (5) Wash the plate; add 50 μL of HRP-Goat anti hIgG secondary antibody and incubate at 37°C for 30 min.

[0221] (6) Wash the plate several times; add 50 μL of TMB that has been restored to room temperature and react at room temperature for 15 minutes in the dark.

[0222] (7) Add 50 μL of stop solution (1N HCl) and read the result using a microplate reader.

[0223] (8) Curves were drawn and EC50 was calculated, as shown in Figures 2-9, where hIgG refers to an isotype control, an immunoglobulin molecule that does not bind to any target and is commercially available. Figures 2-5 are the dose-effect curves for the binding of each single-domain antibody to human CD16a (V176), and Figures 6-9 are the dose-effect curves for the binding of each single-domain antibody to human CD16a (F176).

[0224] As can be seen from Figures 2-9, the single-domain antibodies of the present invention all have good affinity and strong specificity for CD16a protein.

[0225] Example 13: Expression and purification of tool antibody (Tab)

[0226] The Tabs used in the embodiments of the present invention include: Tab1, 50NI, the sequence of which is from CN101583625B; Tab3, AFM13, the sequence of which is from CN110461357A; Tab4, AFM24, the sequence of which is from CN110461357A; and FOLR1-Tab1, farletuzumab.

[0227] The sequence was codon-optimized for mammalian cell expression by General Biosystems (Anhui) Co., Ltd. and cloned into the pcDNA3.1 vector. After resistance screening, plasmid-positive bacteria were selected for amplification and the plasmid was extracted using a plasmid extraction kit (Macherey Nagel, Cat# 740412.50). 100 μg of plasmid (40 μg heavy chain + 60 μg light chain) was added per 100 mL of cells and transiently expressed using PEI in 293F cells (culture medium: FreeStyle 293 Expression medium, Thermo, Cat#12338026 + F-68, Thermo, Cat#24040032). 6 to 24 hours after transfection, 5% volume of 10% Peptone (Sigma, Cat#P0521-100G) was added and cultured at 8% CO2, 130 rpm for approximately 7 to 8 days. When cell viability dropped to 50%, the expression supernatant was harvested and purified using a Protein A (GE, Cat#17-5438-02) gravity column. After dialysis against PBS, the concentration was determined using Nanodrop, the purity was determined using SEC, and the binding capacity was verified by indirect ELISA.

[0228] The concentration of Tab1, Tab3, Tab4 and FOLR1-Tab1 obtained by the method is not less than 2 mg / ml and the purity is greater than 95%.

[0229] Example 14: Detection of the RKA effect of CD16a-hFc antibody

[0230] The purpose of this example is to perform RKA testing on an Fc-fusion single-domain antibody specific for CD16a. The Fc-fusion single-domain antibody was purified from Example 10. The cells used were P815 cells. RKA stands for Redirected Killing Assay. The experimental steps are as follows:

[0231] (1) Collect P815 cells by centrifugation.

[0232] (2) Resuspend P815 cells in assay buffer (RPMI-1640 + 1% FBS) and adjust the cell density to 2×10 5 cells / ml.

[0233] (3) Place 50 μl of cell suspension into each well of a 96-well plate.

[0234] (4) Tab1 and the single domain antibody samples to be tested were diluted in a 10-fold gradient starting from 4×10 μg / ml.

[0235] (5) Add the gradient diluted antibody solution to the cell suspension and incubate for 0.5 hours.

[0236] (6) Collect PBMCs by centrifugation and adjust the cell density to 2.5×10 6 cells / ml, and 100 μl of PBMC cell suspension was added to each well.

[0237] (7) Incubate at 37°C, 5% CO2 for 15 hours.

[0238] (8) Centrifuge the cell plate at 2000 rpm for 3 minutes, and take 50 μl of the supernatant and add it to a new 96-well plate.

[0239] (9) Add 50 μl of LDH detection reagent to each well and use FlexStation3 to detect OD492 and OD650.

[0240] According to the formula, target cell killing rate % = (sample-E / T) / (MAX-MIN);

[0241] According to the target cell killing rate and concentration, four-parameter fitting was performed to calculate the EC50 concentration of ADCC mediated by each antibody.

[0242] The RKA effect detection results are shown in Figures 10 to 13. It can be seen that the CD16a single domain antibodies of the present invention have strong redirected killing ability.

[0243] Example 15: ADCC effect of CD16a-hFc antibody

[0244] The ADCC effect of the CD16a-hFc of the present invention was determined using a reporter gene method, and the steps are as follows:

[0245] (1) Add 25 μl of assay buffer to a 96-well plate;

[0246] (2) Tab, hIgG, and VHH-hFc samples were prepared into solutions with a maximum concentration of 10 μg / mL and subjected to 10-fold gradient dilution to obtain 7 concentrations. VHH-hFc was obtained by purifying the Fc fusion protein of the single-domain antibody against CD16a protein prepared in Example 9 (expressed in 293F cells) in Example 10.

[0247] (3) Add the gradient diluted antibody solution to the cell culture wells in equal volumes of the cell suspension;

[0248] (4) For the sample wells and E / T wells (antibody concentration is 0), Jurkat-NFAT-luc-FcγRIIIa cells were collected and added to the cell culture wells at a density of 20,000 cells per well;

[0249] (5) After 6 h of incubation, cell killing was detected using the One-Glo kit and luminescence was read;

[0250] (6) Calculate the fold of induction = (sample-BG) / (E / T-BG)

[0251] Based on the target cell killing rate and concentration, a four-parameter fitting was performed to calculate the EC50 concentration of ADCC mediated by each antibody, as shown in Figures 14-17. As can be seen from Figures 14-17, each CD16a-hFc antibody has an ADCC effect.

[0252] Example 16 Preparation of trispecific antibody FOLR1 / CD16a / Nkp46

[0253] The preparation process of trispecific antibodies includes the following steps:

[0254] (1) The nucleotide sequence in Table 18 was synthesized into pCDNA3.4 to obtain a recombinant eukaryotic expression vector;

[0255] (2) transforming the recombinant eukaryotic expression vector constructed in step (1) into DH5α Escherichia coli, culturing and performing plasmid extraction to remove endotoxins;

[0256] (3) Sequencing the extracted plasmid;

[0257] (4) The confirmed recombinant vector is prepared for subsequent eukaryotic cell transfection and expression.

[0258] A trispecific antibody was assembled from FOLR1 / Nkp46 and an anti-CD16a single domain antibody, with a structure shown in Figure 18 (FOLR1 VHH-linker-Nkp46 VHH-linker-CD16a VHH) and amino acid sequences shown in SEQ ID NOs: 47-51. The trispecific antibody was not additionally linked to an Fc.

[0259] In SEQ ID NOs:47-51, humanized 4F4 is represented by amino acids 1-125, the linker between humanized 4F4 and humanized 7F10 is represented by amino acids 126-140 (GGGGSGGGGSGGGGS), humanized 7F10 is represented by amino acids 141-267, and the linker between humanized 7F10 and CD16a single domain antibody is represented by amino acids 268-282 (GGGGSGGGGSGGGGS).

[0260] All linkers in this specification are not limited to specific sequences, and other linkers used in the prior art for constructing antibodies can be used.

[0261] In SEQ ID NO:47, amino acids 283-408 are CD16a single domain antibody 4B5, in SEQ ID NO:48, amino acids 283-409 are CD16a single domain antibody 15E2, in SEQ ID NO:49, amino acids 283-409 are CD16a single domain antibody 13G6, in SEQ ID NO:50, amino acids 283-408 are CD16a single domain antibody 14A3, and in SEQ ID NO:51, amino acids 283-408 are CD16a single domain antibody 23D1.

[0262] Table 17 Amino acid sequences of trispecific antibodies

[0263] Table 18 Nucleic acid sequences of trispecific antibodies

[0264] Example 17 ADCC Effect of FOLR1 / Nkp46 / CD16a Trispecific Antibody

[0265] The ADCC effect of the trispecific antibody of the present invention was determined using the LDH method, and the steps are as follows:

[0266] (1) SK-OV-3 cells at passage 3-4 after recovery were collected and plated into 96-well plates at 10,000 cells per well;

[0267] (2) Tab, hIgG, FOLR1 / Nkp46, and trispecific antibodies were prepared at a maximum concentration of 10 μg / mL and diluted 10-fold to obtain 7 concentrations;

[0268] (3) Add the gradient diluted antibody solution to the cell culture wells in equal volumes of the cell suspension;

[0269] (4) For sample wells and E / T wells (antibody concentration is 0), collect PBMC cells and add 250,000 cells per well to the cell culture wells at twice the volume of the target cell suspension; for MAX wells, add lysis buffer twice the volume of the target cell suspension to each well; for MIN wells, add assay buffer twice the volume of the target cell suspension to each well;

[0270] (5) After 6 h of incubation, cell killing was detected using an LDH kit and the absorbance was read;

[0271] (6) Target cell killing rate % = (sample - E / T) / (MAX - MIN);

[0272] (7) Based on the target cell killing rate and concentration, four-parameter fitting was performed to calculate the EC50 concentration of ADCC mediated by each antibody.

[0273] Based on the target cell killing rate and concentration, a four-parameter fitting was performed to calculate the EC50 concentration of ADCC mediated by each antibody, as shown in Figures 19 to 22. As can be seen from Figures 19 to 22, each trispecific antibody has a good ADCC effect.

[0274] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the scope of protection of the present invention.

Claims

1. An anti-CD16a single domain antibody, characterized in that: The single-domain antibody is composed of a heavy chain, which includes a heavy chain CDR1 shown in any one of SEQ ID NO:11-SEQ ID NO:13, a heavy chain CDR2 shown in any one of SEQ ID NO:14-SEQ ID NO:17, and a heavy chain CDR3 shown in any one of SEQ ID NO:18-SEQ ID NO:

21.

2. The single domain antibody of CD16a according to claim 1, characterized in that: The amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are one of the following (1)-(5): (1) CDR1 shown in SEQ ID NO: 12, CDR2 shown in SEQ ID NO: 17, and CDR3 shown in SEQ ID NO: 21; (2) CDR1 shown in SEQ ID NO: 12, CDR2 shown in SEQ ID NO: 14, and CDR3 shown in SEQ ID NO: 21; (3) CDR1 shown in SEQ ID NO: 11, CDR2 shown in SEQ ID NO: 16, and CDR3 shown in SEQ ID NO: 20; (4) CDR1 shown in SEQ ID NO: 11, CDR2 shown in SEQ ID NO: 16, and CDR3 shown in SEQ ID NO: 19; (5) CDR1 shown in SEQ ID NO:13, CDR2 shown in SEQ ID NO:15, and CDR3 shown in SEQ ID NO:

18.

3. The anti-CD16a single domain antibody according to claim 1, characterized in that: The single domain antibody further comprises a framework region FR; the framework region FR comprises the amino acid sequences of FR1, FR2, FR3 and FR4; the amino acid sequences of the framework region FR are respectively: FR1 or a variant of FR1 as shown in any one of SEQ ID NOs: 22-26, wherein the variant of FR1 comprises a substitution of up to 5 amino acids in FR1; FR2 or a variant of FR2 as shown in any one of SEQ ID NOs: 27-29, wherein the variant of FR2 comprises a substitution of up to 5 amino acids in FR2; FR3 or a variant of FR3 as shown in any one of SEQ ID NOs: 30-34, wherein the variant of FR3 comprises a substitution of up to 5 amino acids in FR3; FR4 or a variant of FR4 as shown in SEQ ID NO:35, wherein the variant of FR4 comprises a substitution of up to 5 amino acids in the FR4.

4. An anti-CD16a single domain antibody, characterized in that: The amino acid sequence of the single-domain antibody is shown in any one of SEQ ID NOs: 1-5, or the amino acid sequence of the single-domain antibody is compared with any one of SEQ ID NOs: 1-5, and at least one amino acid residue in FR1, FR2, FR3 or FR4 sequence is replaced by a conservative amino acid. 5 . The Fc fusion antibody or humanized antibody of the anti-CD16a single domain antibody according to any one of claims 1 to 4 .

6. A recombinant protein, characterized in that The recombinant protein comprises the anti-CD16a single domain antibody according to any one of claims 1 to 4.

7. A bispecific antibody or a multispecific antibody, characterized in that: It comprises the single domain antibody according to any one of claims 1 to 4 as a first antigen binding part that specifically binds to CD16a.

8. The bispecific antibody or multispecific antibody according to claim 7, characterized in that: It also contains binding portions that are specific for tumor antigens other than CD16a; Preferably, other tumor antigens besides CD16a include FOLR1, Her2, DEC205, CLEC9A, CEACAM5, CTLA4, CD3, CD7, CD11c, CD19, CD20, CD22, CD40, CD44, CD206, EGFR, EGFRvIII, fibroblast activation protein (FAP), CA9, MMP-2, PD-L1, SIRPa, Trop2, GPC1, GPC3, cMET, BCMA, VEGFR, Cladin18.2, Nkp46, CD30, NKG2D, IL-2Rβ, BCMA, CD123, TGF-β, CD38, IL-7, IL-8, FRα, NCR3, IL-15, Muc1, IL-16 or any other tumor antigen.

9. The bispecific antibody or multispecific antibody according to claim 7, characterized in that: The bispecific antibodies include FOLR1 / CD16a, Her2 / CD16a, DEC205 / CD16a, CLEC9A / CD16a, CEACAM5 / CD16a, CTLA4 / CD16a, CD3 / CD16a, CD7 / CD16a, CD11c / CD16a, CD19 / CD16a, CD20 / CD16a, CD22 / CD16a, CD40 / CD16a, CD44 / CD16a, CD206 / CD16a, EGFR / CD16a, EGFRvIII / CD16a, fibroblast activation protein (FAP) / CD16a, CA9 / CD16a, MMP-2 / CD16a, and PD-L1 / CD16a. , SIRPa / CD16a, Trop2 / CD16a, GPC1 / CD16a, GPC3 / CD16a, cMET / CD16a, BCMA / CD16a, VEGFR / CD16a, Cladin18.2 / CD16a, CD30 / CD16a, NKG2D / CD16a, IL-2Rβ / CD16 a. BCMA / CD16a, CD123 / CD16a, TGF-β / CD16a, CD38 / CD16a, IL-7 / CD16a, IL-8 / CD16a, FRα / CD16a, NCR3 / CD16a, IL-15 / CD16a, Muc1 / CD16a or IL-16 / CD16a bispecific antibody.

10. The bispecific antibody or multispecific antibody according to claim 7, characterized in that: wherein the multispecific antibody is a trispecific antibody, comprising a first antigen-binding portion that specifically binds to CD16a, a second antigen-binding portion that specifically binds to Nkp46, and a third antigen-binding portion that specifically binds to other tumor antigens other than CD16a and Nkp46; The trispecific antibodies include FOLR1 / CD16a / Nkp46, Her2 / CD16a / Nkp46, DEC205 / CD16a / Nkp46, CLEC9A / CD16a / Nkp46, CEACAM5 / CD16a / Nkp46, CTLA4 / CD16a / Nkp46, CD3 / CD16a / Nkp46, CD7 / CD16a / Nkp46, CD11c / CD16a / Nkp46, CD19 / CD16a / Nkp46, CD20 / CD 16a / Nkp46, CD22 / CD16a / Nkp46, CD40 / CD16a / Nkp46, CD44 / CD16a / Nkp46, CD206 / CD16a / Nkp46, EGFR / CD16a / Nkp46, EGFRvIII / CD16a / Nkp46, fibroblast activation protein (FAP) / CD16a / Nkp46, CA9 / CD16a / Nkp46, MMP-2 / CD16a / Nkp46, PD-L1 / CD16a / Nkp46, S IRPa / CD16a / Nkp46, Trop2 / CD16a / Nkp46, GPC1 / CD16a / Nkp46, GPC3 / CD16a / Nkp46, cMET / CD16a / Nkp46, BCMA / CD16a / N kp46, VEGFR / CD16a / Nkp46, Cladin18.2 / CD16a / Nkp46, CD30 / CD16a / Nkp46, NKG2D / CD16a / Nkp46, IL-2Rβ / CD16a / Nkp46 , BCMA / CD16a / Nkp46, CD123 / CD16a / Nkp46, TGF-β / CD16a / Nkp46, CD38 / CD16a / Nkp46, IL-7 / CD16a / Nkp46, IL-8 / CD16a / Nkp46, FRα / CD16a / Nkp46, NCR3 / CD16a / Nkp46, IL-15 / CD16a / Nkp46, Muc1 / CD16a / Nkp46 or IL-16 / CD16a / Nkp46 trispecific antibodies.

11. The bispecific antibody or multispecific antibody according to claim 7, characterized in that: wherein the multispecific antibody is a trispecific antibody, comprising a first antigen binding portion that specifically binds CD16a, a second antigen binding portion that specifically binds Nkp46, and a third antigen binding portion that specifically binds FOLR1; The second antigen binding portion has CDR1 set forth in SEQ ID NO:63, CDR2 set forth in SEQ ID NO:65, and CDR3 set forth in SEQ ID NO:70; The third antigen binding portion has CDR1 set forth in SEQ ID NO:58, CDR2 set forth in SEQ ID NO:59, and CDR3 set forth in SEQ ID NO:60; Preferably, the second antigen binding moiety that specifically binds Nkp46 and the third antigen binding moiety that specifically binds FOLR1 are VHH; Preferably, the trispecific antibody is not fused to Fc.

12. The bispecific antibody or multispecific antibody according to claim 10, characterized in that: The amino acid sequences of the trispecific antibodies are shown in SEQ ID NOs: 47-51, respectively.

13. A nucleotide molecule encoding the single domain antibody against Nkp46 according to any one of claims 1 to 4, characterized in that: The nucleotide sequence is shown in any one of SEQ ID NOs: 6-10, or the amino acid sequence encoded by the nucleotide sequence is the same as the amino acid sequence encoded by any one of SEQ ID NOs: 6-10.

14. A nucleotide molecule encoding a bispecific antibody or a multispecific antibody according to any one of claims 7 to 12, characterized in that: The nucleotide sequence is shown in any one of SEQ ID NOs: 52-56, or the amino acid sequence encoded by the nucleotide sequence is the same as the amino acid sequence encoded by any one of SEQ ID NOs: 52-56.

15. An expression vector, characterized in that: It comprises a nucleotide molecule encoding the anti-CD16a single domain antibody according to any one of claims 1 to 4, or the Fc fusion antibody or humanized antibody according to claim 5, or a nucleotide molecule encoding the bispecific antibody or multispecific antibody according to any one of claims 7 to 12, or a nucleotide molecule according to claim 13 or claim 14.

16. A host cell, characterized in that: It can express the single domain antibody against CD16a according to any one of claims 1 to 4, the Fc fusion antibody or humanized antibody according to claim 5, or the bispecific antibody or multispecific antibody according to any one of claims 7 to 12, or it contains the expression vector according to claim 15.

17. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises an anti-CD16a single domain antibody selected from any one of claims 1 to 4 or a bispecific antibody or a multispecific antibody according to any one of claims 7 to 12, and a pharmaceutically acceptable carrier.

18. A medicament for treating a disease, characterized in that: It comprises the anti-CD16a single domain antibody according to any one of claims 1 to 4 or the bispecific antibody or multispecific antibody according to any one of claims 7 to 12 as an active ingredient.

19. Use of the anti-CD16a single domain antibody according to any one of claims 1 to 4, the bispecific antibody or multispecific antibody according to any one of claims 7 to 12, or the pharmaceutical composition according to claim 17 in the preparation of a medicament for treating a disease.

20. The use according to claim 19, characterized in that: The disease includes a tumor.

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