Anti-CD16a single-domain antibody and application thereof

Abstract

The invention belongs to the field of immunology, and relates to an anti-CD16a single-domain antibody and application thereof. The single-domain antibody is composed of heavy chains, and the heavy chains comprise a heavy chain CDR1 as shown in any one of SEQ ID NO: 11 to SEQ ID NO: 13, a heavy chain CDR2 as shown in any one of SEQ ID NO: 14 to SEQ ID NO: 17, and a heavy chain CDR3 as shown in any one of SEQ ID NO: 18 to SEQ ID NO: 21. Compared with the prior art, the invention has the beneficial effects that the specific single-domain antibody aiming at CD16a is screened by using a biological genetic engineering technology, and the affinity of the antibody is better.

Description

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 a pharmaceutical therapeutic use thereof. Background Art

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

[0003] Single-domain antibodies or single-domain antibodies (sdAb) are the smallest antibody molecules currently, with a molecular weight of 1 / 10 of that of complete antibodies. In addition to the antigenic reactivity of complete antibodies, single-domain antibodies also have some unique functional characteristics, such as small molecular weight, strong stability, good solubility, easy expression, weak immunogenicity, strong penetration, strong targeting, and low preparation cost, which almost perfectly overcome the defects of traditional antibodies such as long development cycle, low stability, and harsh storage conditions.

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

[0005] The invention objective of this patent is to provide a single domain antibody that can specifically bind to CD16a and its use.

[0006] 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-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.

[0007] Preferably, the amino acid sequence of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 is one of the following (1)-(5):

[0008] (1) CDR1 shown in SEQ ID NO: 12, CDR2 shown in SEQ ID NO: 17, and CDR3 shown in SEQ ID NO: 21;

[0009] (2) CDR1 shown in SEQ ID NO: 12, CDR2 shown in SEQ ID NO: 14, and CDR3 shown in SEQ ID NO: 21;

[0010] (3) CDR1 shown in SEQ ID NO: 11, CDR2 shown in SEQ ID NO: 16, and CDR3 shown in SEQ ID NO: 20;

[0011] (4) CDR1 shown in SEQ ID NO: 11, CDR2 shown in SEQ ID NO: 16, and CDR3 shown in SEQ ID NO: 19;

[0012] (5) CDR1 shown in SEQ ID NO:13, CDR2 shown in SEQ ID NO:15, and CDR3 shown in SEQ ID NO:18.

[0013] The above CDR combinations (1)-(5) correspond to the single domain antibodies 23D1, 14A3, 4B5, 15E2, and 13G6, respectively.

[0014] All of the above sequences can be replaced by sequences having "at least 80% homology" with the sequence or sequences with only one or a few amino acids replaced; preferably "at least 85% homology", more preferably "at least 90% homology", more preferably "at least 95% homology", and most preferably "at least 98% homology".

[0015] In one embodiment, one to five arbitrary amino acid residues in any one or more CDRs of the heavy chain CDR1, CDR2 and CDR3 can be replaced by their conservative amino acids, respectively. Specifically, in the heavy chain CDR1, 1 to 5 amino acid residues can be replaced by their conservative amino acids; in the heavy chain CDR2, 1 to 5 amino acid residues can be replaced by their conservative amino acids; in the heavy chain CDR3, 1 to 5 amino acid residues can be replaced by their conservative amino acids.

[0016] As used herein, the term "sequence homology" refers to the extent to which two (nucleotide or amino acid) sequences have identical residues at the same position in an alignment, and is usually expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Thus, two copies having exactly the same sequence have 100% homology.

[0017] In some embodiments, a sequence that replaces only one or a few amino acids compared to the aforementioned sequence, 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 variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and optimally 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or several (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 combination of CDR1, CDR2 and CDR3 in a single domain antibody, a technician may consider so-called "conservative" amino acid substitutions, and in the case of substitutions, the substitutions will preferably be conservative amino acid substitutions. The conservative amino acids, which can generally be described as amino acid substitutions 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 in which one or a few amino acids in the following groups (a)-(d) are replaced 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, Gln; Phe is replaced by Met, Leu, Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe, 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 know that the creativity of single-domain antibodies is embodied in the CDR1-3 region, while the framework region sequence FR1-4 is not unchangeable, and the sequence of FR1-4 can adopt the conservative sequence variant of the sequence disclosed in the present invention.

[0018] 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 all 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 substitution 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 prolongs 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 proprotein 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 belong to the scope known to those skilled in the art.

[0019] 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 respectively:

[0020] 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;

[0021] 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;

[0022] 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;

[0023] 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.

[0024] 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 sequences of the single-domain antibodies are shown in SEQ ID NOs: 1-5, respectively, or the single-domain antibodies have at least 80% sequence homology with the amino acid sequences of SEQ ID NOs: 1-5 and are capable of specifically binding to 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 replaced by a conservative amino acid.

[0025] In one embodiment, the anti-CD16a single domain antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology with an amino acid sequence selected from SEQ ID NOs: 1-5, and is capable of specifically binding to CD16a protein.

[0026] 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.

[0027] The fourth aspect of the present invention is to provide a recombinant protein comprising any of the aforementioned anti-CD16a single-domain antibodies.

[0028] 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 of the foregoing items, wherein the single domain antibody serves as a first antigen binding portion that specifically binds to CD16a.

[0029] In one embodiment, the bispecific antibody or multispecific antibody further comprises a binding portion that is specific for other tumor antigens other than CD16a;

[0030] 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.

[0031] 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.

[0032] In one embodiment, the aforementioned 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.

[0033] 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 / 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 / Nkp 46. ​​SIRPa / CD16a / Nkp46, Trop2 / CD16a / Nkp46, GPC1 / CD16a / Nkp46, GPC3 / CD16a / Nkp46, cMET / CD16a / Nkp46, BCMA / CD16 a / Nkp46, VEGFR / CD16a / Nkp46, Cladin18.2 / CD16a / Nkp46, CD30 / CD16a / Nkp46, NKG2D / CD16a / Nkp46, IL-2Rβ / CD16a / Nkp 46, trispecific antibodies such as 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.

[0034] 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;

[0035] The second antigen-binding portion has CDR1 shown in SEQ ID NO: 63, CDR2 shown in SEQ ID NO: 65, and CDR3 shown in SEQ ID NO: 70;

[0036] The third antigen-binding portion has CDR1 shown in SEQ ID NO: 58, CDR2 shown in SEQ ID NO: 59, and CDR3 shown in SEQ ID NO: 60;

[0037] Preferably, the second antigen-binding portion that specifically binds Nkp46 and the third antigen-binding portion that specifically binds FOLR1 are VHHs;

[0038] Preferably, the trispecific antibody is not fused to Fc.

[0039] In one embodiment, the amino acid sequences of the foregoing trispecific antibodies are respectively as shown in SEQ ID NO: 47 - 51.

[0040] The sixth aspect of the present invention is to provide nucleotide molecules encoding the foregoing anti-CD16a single-domain antibody, or the foregoing Fc fusion antibody, or the foregoing humanized antibody, the nucleotide sequences of which are respectively as shown in SEQ ID NO: 6 - 10, or the amino acid sequences encoded by the said nucleotide sequences are the same as the amino acid sequences encoded by any one of SEQ ID NO: 6 - 10, or have at least 95% sequence homology with any one of SEQ ID NO: 6 - 10.

[0041] 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%, 100% sequence homology with the nucleotide sequence selected from SEQ ID NO: 6 - 10, and the anti-CD16a single-domain antibody encoded by it can specifically bind to the CD16a protein.

[0042] The seventh aspect of the present invention is to provide nucleotide molecules encoding the foregoing bispecific antibody or multispecific antibody, the nucleotide sequences of which are respectively as shown in SEQ ID NO: 52 - 56, or the amino acid sequences encoded by the said nucleotide sequences are the same as the amino acid sequences encoded by any one of SEQ ID NO: 52 - 56, or have at least 95% sequence homology with any one of SEQ ID NO: 52 - 56.

[0043] 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%, 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, and Nkp46 proteins.

[0044] 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.

[0045] 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 means), and other general expression vectors can also be selected as needed.

[0046] The ninth aspect of the present invention is to provide 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.

[0047] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell, including bacteria and fungi.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] In another preferred embodiment, the eukaryotic cell is 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.

[0052] In another preferred embodiment, the host cell is a suspension ExpiCHO-S cell.

[0053] In another preferred embodiment, the host cell is a suspension 293F cell.

[0054] 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 the aforementioned single-domain antibody shown in SEQ ID NO: 1-5, or a single-domain antibody having at least 80% homology with SEQ ID NO: 1-5, or a multi-epitope antibody, a bispecific antibody, a multi-specific antibody, and a multivalent antibody; for example, the multi-epitope antibody may be composed of more than one sequence in SEQ ID NO: 1-5; the multivalent antibody may be composed of one of the sequences in SEQ ID NO: 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, and analog of the aforementioned antibody.

[0055] The eleventh aspect of the present invention is to provide a pharmaceutical composition comprising the aforementioned single domain antibody binding to CD16a, the aforementioned bispecific antibody or multispecific antibody, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally determined according to the isoelectric point of the antibody (the pH of the aqueous carrier medium needs to deviate from the isoelectric point of the antibody and differ from the isoelectric point of the antibody by about 2). The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous, transdermal (directly applied to the affected area or applied as a plaster).

[0056] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) 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 preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.

[0057] 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.

[0058] The thirteenth aspect of the present invention is to provide a kit for detecting CD16a levels, which contains the aforementioned anti-CD16a single domain antibody. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, and the like.

[0059] In a preferred embodiment, the kit includes an antibody that recognizes CD16a protein, a lysis medium for dissolving the sample, and common 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.

[0060] In a preferred embodiment, the kit further contains a secondary antibody and an enzyme or fluorescent or radioactive label for detection, and a buffer.

[0061] In a preferred embodiment, the second antibody of the kit can be the aforementioned single domain antibody against CD16a (as an anti-antibody), and can be a single domain antibody, a monoclonal antibody, a polyclonal antibody or any other form of an antibody.

[0062] The thirteenth aspect of the present invention provides a method for producing an anti-CD16a single domain antibody, comprising the steps of:

[0063] (a) culturing the host cell according to the ninth aspect of the present invention under conditions suitable for producing a single domain antibody, thereby

[0064] 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 the 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 erosion, intraperitoneal abscess, inflammatory bowel disease, allogeneic transplant rejection, psoriasis, angiogenesis, atherosclerosis, asthma, multiple sclerosis, systemic lupus erythematosus (SLE), ocular surface disorders (e.g., dry eyes), ankylosing spondylitis, psoriatic arthritis, cancer (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, infectious diseases include acute and chronic infectious diseases (eg, bacterial infections or viral infections).

[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, gastric 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 cancer, and paranasal sinus cancer); ovarian, fallopian tube, peritoneal, vaginal, vulvar, penis, cervix, myometrium, endometrial-related cancers thyroid cancer (e.g., follicular thyroid cancer); adrenal, prostate, skin and adnexal related cancers (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevus); hematological malignancies (i.e., leukemias, lymphomas) and precancerous hematological disorders and borderline malignant neoplastic diseases, including hematological malignancies and lymphoid lineage related disorders (e.g., acute lymphoblastic leukemia ALL, chronic lymphocytic leukemia CLL, B-cell lymphomas such as diffuse large B-cell lymphoma DLBCL, follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphomas and leukemias, natural killer NK-cell lymphoma, Hodgkin's lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma , multiple myeloma, and post-transplant lymphoproliferative disorder) as well as hematological malignancies and myeloid-related diseases (e.g., acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), hypereosinophilic syndrome, myeloproliferative diseases 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 sarcoma, Ewing 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, 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 tumors); or congenital or other syndromes that predispose the patient 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.

[0075] Beneficial Effects

[0076] Compared with the prior art, the beneficial effects of the present invention are:

[0077] (1) The single domain antibody of the present invention is specific to the CD16a protein with a correct spatial structure.

[0078] (2) The single-domain antibody obtained in the present invention has a flexible expression system selection, and can be expressed in 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 later production cost.

[0079] (3) The single-domain antibodies obtained by the present invention are easy 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.

[0080] (4) The single domain antibody obtained by the present invention has a wider affinity range. Before affinity maturation, its affinity range can range from nM level to pM level, providing multiple options for antibodies with different uses in the later stage;

[0081] (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 ADCC effect. The single-domain antibody is combined with other antigen binding parts 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.

[0082] 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 on the basis of CD16a single domain antibody, in the absence of adding immunoglobulin Fc, its mediated ADCC effect is still unexpectedly excellent. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] In order to more clearly illustrate the technical solution of the present application, the drawings required for use in the embodiments are briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0084] Figure 1 The enrichment of the library for screening the antibody targeting CD16a in Example 3, wherein the left side shows the enrichment of the library for screening the antibody targeting CD16a-V176, and the right side shows the enrichment of the library for screening the antibody targeting CD16a-F176;

[0085] Figure 2 is the binding dose-effect curve of the CD16a-hFc antibody and human CD16a (V176) in Example 12 (14A3);

[0086] Figure 3 is the binding dose-effect curve (23D1) of the CD16a-hFc antibody and human CD16a (V176) in Example 12;

[0087] Figure 4 The binding dose-effect curve of the CD16a-hFc antibody and human CD16a (V176) in Example 12 (15E2, 13G6);

[0088] Figure 5 is the binding dose-effect curve of the CD16a-hFc antibody and human CD16a (V176) in Example 12 (4B5);

[0089] Figure 6 is the binding dose-effect curve (4B5) of the CD16a-hFc antibody in Example 12 and human CD16a (F176);

[0090] Figure 7 The binding dose-effect curve of the CD16a-hFc antibody in Example 12 and human CD16a (F176) (15E2, 13G6);

[0091] Figure 8 is the binding dose-effect curve (14A3) of the CD16a-hFc antibody in Example 12 and human CD16a (F176);

[0092] Fig. 9 is the binding dose-effect curve (23D1) of the CD16a-hFc antibody in Example 12 and human CD16a (F176);

[0093] Fig.10 The RKA test results for CD16a-hFc antibody (4B5);

[0094] Fig.11 The results of the RKA test for CD16a-hFc antibody (15E2);

[0095] Fig.12 The results of the RKA test for CD16a-hFc antibodies (13G6, 14A3);

[0096] Fig.13 The results of the RKA test for CD16a-hFc antibody (23D1);

[0097] Fig.14 ADCC test results of CD16a-hFc (4B5);

[0098] Fig.15 ADCC test results of CD16a-hFc (15E2);

[0099] Fig.16 ADCC test results of CD16a-hFc (13G6, 14A3);

[0100] Fig.17 ADCC test results of CD16a-hFc (23D1);

[0101] Fig.18 The ADCC test results of the FOLR1 / Nkp46 / CD16a trispecific antibody;

[0102] Figure 19-21 The ADCC test results of the FOLR1 / Nkp46 / CD16a trispecific antibody;

[0103] Fig. 22 These are the ADCC test results of the FOLR1 / Nkp46 bispecific antibody. DETAILED DESCRIPTION

[0104] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0105] As used herein, "single domain antibodies" (sdAb, also referred to as nanobodies or VHHs by developer 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 with only heavy chains (which naturally do not comprise light chains), single domain antibodies derived from conventional antibodies, and engineered antibodies.

[0106] 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 camelid species (e.g., camels, dromedaries, llamas and guanacos). Like complete antibodies, single domain antibodies are able to selectively bind to specific antigens. Single domain antibodies can contain only the variable domains of immunoglobulin chains, which have CDR1, CDR2 and CDR3 and framework regions.

[0107] As used herein, the term "sequence homology" refers to the extent to which two (nucleotide or amino acid) sequences have identical residues at the same position in an alignment, and is usually expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Thus, two copies having exactly the same sequence have 100% homology.

[0108] 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.

[0109] 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 an antibody obtained by transplanting the complementary determining region (CDR1-3 sequence) of a target antibody into the variable region of a human antibody, or an antibody obtained by mutating the amino acids of the target antibody according to the characteristics of the human antibody framework region (FR1-4). Humanized antibodies can be synthesized or by site-directed mutagenesis.

[0110] In the present invention, sequences with high sequence homology to CDR1-3 disclosed in the present invention can also obtain single domain antibodies against CD16a. In some embodiments, sequences with "at least 80% homology" or "at least 85% homology", "at least 90% homology", "at least 95% homology", "at least 98% homology" to the sequences in SEQ ID NO.1-5 can achieve the purpose of the invention.

[0111] 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 which case 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 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 in which one or a few amino acids in the following groups (a)-(d) are replaced 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, Gln; Phe is replaced by Met, Leu, Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe, 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 know that the creativity of single-domain antibodies is embodied in the CDR1-3 region, while the framework region sequence FR1-4 is not unchangeable, and the sequence of FR1-4 can adopt the conservative sequence variant of the sequence disclosed in the present invention.

[0112] Preferred host cells of the present invention are bacterial cells, fungal cells or mammalian cells.

[0113] This patent is to prepare the target protein and the truncated form of the target protein through genetic engineering technology, and then immunize the Alxa Bactrian camel in Inner Mongolia with the obtained antigen protein. After multiple immunizations, the peripheral blood lymphocytes or spleen cells of the camel are obtained. The camel-derived antibody variable region coding sequence is recombined into a phage display vector through genetic engineering. 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.

[0114] The above technical solution is now broken down and explained in detail in the form of specific embodiments:

[0115] Example 1: Preparation of human CD16a recombinant extracellular domain protein:

[0116] The human recombinant extracellular domain protein used in this patent is obtained by the company's own expression and purification. The expression vector design scheme of human recombinant CD16a protein is as follows:

[0117] (1) The coding sequence of CD16a was retrieved from NCBI and its accession number is NM_000569.7. The accession number of the amino acid sequence encoded by this sequence is NP_000560.6.

[0118] (2) The nucleotide sequence encoding the 17th to 208th amino acids of CD16a (CD16a-V176, CD16a-F176) 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 extracted in large quantities to remove endotoxins and transfected into suspended 293F cells for target protein expression and purification. The purity reached more than 90%, meeting the requirements for animal immunization.

[0119] There are two kinds of antigens, one is the 17th to 208th amino acids of CD16a, the 176th amino acid is F, referred to as CD16a-F176; the other is the 17th to 208th amino acids of CD16a, the 176th amino acid is V, referred to as CD16a-V176. The two antigens are prepared and purified separately.

[0120] Example 2: Construction of a single domain antibody library against CD16a protein:

[0121] 1 mg of the human recombinant CD16a protein (CD16a-F176, CD16a-V176) purified in Example 1 was mixed with an equal volume of Freund's complete adjuvant, and the Bactrian camels of Alxa, Inner Mongolia were immunized once a week for a total of 7 consecutive immunizations. Except for the first immunization, the remaining six immunizations were performed by mixing 1 mg of CD16a protein with an equal volume of Freund's incomplete adjuvant for animal immunization. The immunization process is to centrally stimulate the camels to produce antibodies against the CD16a protein. CD16a-V176 and CD16a-F176 were not mixed, and animals were immunized separately to obtain antibodies.

[0122] After the animal immunization, 150 mL of peripheral blood lymphocytes were extracted from the camel, and RNA was extracted from the cells. The extracted total RNA was used to synthesize cDNA, and VHH (antibody heavy chain variable region) was amplified using cDNA as a template through a nested PCR reaction.

[0123] Then, the pMECS vector and VHH fragment were 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 about 1×10 9 At the same time, the correct insertion rate of the library in the target fragment was detected by colony PCR identification.

[0124] 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%.

[0125] Example 3: Screening of single domain antibodies against CD16a protein:

[0126] 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 phages. The next day, the phages were precipitated with PEG / NaCl and the amplified phages were collected by centrifugation.

[0127] Diluted in 100 mM NaHCO, pH 8.3 3 500 μg of CD16a protein was coupled to the ELISA plate and placed at 4°C overnight. A negative control well (culture medium control) was set up at the same time. On the second day, 200 μL of 3% skim milk was added and the plate was blocked at room temperature for 2 h. After the blocking, 100 μl of amplified phage library (approximately 2×10 11 After 1 hour of incubation, the cells were washed 15 times with PBS + 0.05% Tween-20 to remove unbound phages.

[0128] The 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 h to produce and collect phages for the next round of screening. The same screening process was repeated for one round to gradually achieve enrichment.

[0129] When the enrichment multiple reaches more than 10 times, the enrichment effect is as follows Figure 1 shown.

[0130] Figure 1 In the figure, P / N = the number of monoclonal bacteria grown after the phages eluted from the positive wells in bio-panning infected with TG1 bacteria / the number of monoclonal bacteria grown after the phages eluted from the positive wells infected with TG1 bacteria. This parameter will gradually increase after enrichment occurs; I / E = the total amount of phages added to the positive wells in each round of bio-panning / the total amount of phages eluted from the positive wells in each round of bio-panning. This parameter will gradually approach 1 after enrichment occurs.

[0131] Example 4: Screening of specific positive clones against CD16a using phage ELISA:

[0132] According to the screening method in Example 3, three rounds of screening were performed for the single domain antibody against CD16a protein, and the phage enrichment factor of the anti-CD16a protein reached more than 10. After the screening, 384 single colonies were selected from the positive clones obtained by the screening and inoculated into 96 deep-well plates in 2×TY medium containing 100 μg / mL ampicillin, and a blank control was set. After culturing at 37° C. to the logarithmic phase, IPTG with a final concentration of 1 mM was added, and cultured at 28° C. overnight.

[0133] The crude antibody was obtained by osmotic swelling method; CD16a recombinant protein was released into 100 mM NaHCO at pH 8.3. 3 100 μg of protein was coated in an ELISA plate at 4°C overnight. 100 μL of the crude antibody extract was transferred to the ELISA plate with the antigen added and incubated at room temperature for 1 hour; unbound antibody was washed with PBST, and 100 μL of Mouse Anti-HA tag Antibody (HRP) (mouse anti-HA horseradish peroxidase labeled antibody, ThermoFisher) diluted 1:2000 was added and incubated at room temperature for 1 hour; unbound antibody was washed with PBST, and horseradish peroxidase colorimetric solution was added. After reacting at 37°C for 15 minutes, stop solution was added, and the absorbance value was read at a wavelength of 450 nm on the microplate reader.

[0134] When the OD value of the sample well is more than 5 times that of the control well, it is determined as a positive clone well; the bacteria in the positive clone well are transferred and shaken in LB medium containing 100 μg / mL ampicillin for plasmid extraction and sequencing.

[0135] According to the sequence alignment software VectorNTI, the gene sequences of each clone were analyzed. Clones with the same CDR1, CDR2, and CDR3 sequences were regarded as the same clone, while clones with different sequences were regarded as different clones. Finally, single-domain antibodies specifically targeting the CD16a protein were obtained.

[0136] The amino acid sequence of its antibody has the structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, constituting the entire VHH. The obtained single-domain antibody recombinant plasmid can be expressed in the prokaryotic system, and finally single-domain antibody protein is obtained.

[0137] Among them, the single-domain antibody clones screened from the antigen CD16a-F176 include 15E2 and 4B5; the single-domain antibody clones screened from the antigen CD16a-V176 include 14A3, 23D1, and 13G6.

[0138] Other single-domain antibody clones (sequences not shown): 18E4, 10B6, 8G8, 15A7, 24C6W were also screened through the antigens CD16a-F176 and CD16a-V176.

[0139] The CDR and FR sequences of the 5 single-domain antibodies are shown in Table 1-7, and the amino acid sequences and nucleotide sequences of the 5 single-domain antibodies are shown in Table 8 and Table 9 respectively.

[0140] Table 1 CDR1 sequences of 5 single-domain antibodies

[0141]

[0142] Table 2 CDR2 sequences of 5 single-domain antibodies

[0143]

[0144] Table 3 CDR3 sequences of 5 single-domain antibodies

[0145]

[0146] Table 4 FR1 sequences of 5 single-domain antibodies

[0147]

[0148] Table 5 FR2 sequences of 5 single-domain antibodies

[0149]

[0150] Table 6 FR3 sequences of 5 single domain antibodies

[0151]

[0152] Table 7 FR4 sequences of 5 single domain antibodies

[0153]

[0154] Table 8 Amino acid sequences of 5 single domain antibodies

[0155]

[0156] Table 9 Nucleic acid sequences of 5 single domain antibodies

[0157]

[0158] Example 5: Construction of humanized FOLR1 / Nkp46 bispecific antibody domain

[0159] 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 preparation process of the antigen was different.

[0160] Preparation of human Nkp46 recombinant extracellular domain protein:

[0161] The human recombinant extracellular domain protein used in this patent is obtained by the company's own expression and purification. The expression vector design scheme of human recombinant Nkp46 protein is as follows:

[0162] (1) The coding sequence of Nkp46 was retrieved from NCBI and its accession number is BC064806.1. The amino acid sequence generated by the sequence is accession number AAH64806.1.

[0163] (2) The nucleotide sequence encoding the 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 extracted in large quantities to remove endotoxins and transfected into suspended 293F cells for target protein expression and purification. The purity reached more than 90%, meeting the requirements for animal immunization.

[0164] Preparation of human FOLR1 recombinant extracellular domain protein:

[0165] The human recombinant extracellular domain protein used in this patent is obtained by the company's own expression and purification. The expression vector design scheme of the human recombinant FOLR1 protein is as follows:

[0166] (1) The coding sequence of FOLR1 was retrieved from NCBI and its accession number is NM_000802.3. The accession number of the amino acid sequence encoded by this sequence is NP_000793.1.

[0167] (2) The nucleotide sequence encoding amino acids 25 to 233 of FOLR1 was cloned into 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 extracted in large quantities to remove endotoxins and transfected into suspended 293F cells for target protein expression and purification. The purity reached more than 90%, meeting the requirements for animal immunization.

[0168] The sequences of the screened FOLR1-4F4 single domain antibodies are shown in Tables 10 and 11. On this basis, humanization was performed, and the sequence of 4F4 after humanization is shown in Table 12. The modified regions include FR1, CDR1, CDR2, CDR3, and FR4, as shown in the underlined parts in Table 12.

[0169] The sequences of the screened Nkp46-7F10 single domain antibodies are shown in Tables 13 and 14. On this basis, humanization was performed, and the sequence of 7F10 after humanization is shown in Table 15. The modified region includes CDR3, as shown in the underlined part in Table 15.

[0170] Humanized 4F4 and humanized 7F10 were connected to form humanized FOLR1 / Nkp46, and the sequence is shown in Table 16, wherein humanized 4F4 is shown as amino acids 1-125 of SEQ ID NO: 45, the linker between humanized 4F4 and humanized 7F10 is shown as amino acids 126-140 of SEQ ID NO: 45 (GGGGSGGGGSGGGGS), and humanized 7F10 is shown as amino acids 141-267 of SEQ ID NO: 45.

[0171] Table 10 CDR and FR sequences of FOLR1 (4F4)

[0172]

[0173] Table 11 Amino acid sequence and nucleic acid sequence of FOLR1 (4F4)

[0174]

[0175] Table 12 CDR and FR sequences of FOLR1 (humanized 4F4)

[0176]

[0177] Table 13 CDR and FR sequences of Nkp46 (7F10)

[0178]

[0179] Table 14 Amino acid sequence of Nkp46 (7F10)

[0180]

[0181] Table 15 CDR and FR sequences of Nkp46 (humanized 7F10)

[0182]

[0183] Table 16 Amino acid sequence and nucleic acid sequence of humanized FOLR1 / Nkp46 dual antibody

[0184]

[0185] Example 6: Purification and expression of CD16a protein-specific single domain antibody in host bacteria Escherichia coli

[0186] The plasmids (pMECS-VHH) of different clones obtained by sequencing analysis in Example 4 were electrotransformed into Escherichia coli HB2151, and spread on LB+amp+glucose culture plates containing ampicillin and glucose, and cultured at 37°C overnight; a single colony was selected and inoculated into 5 mL of LB culture medium containing ampicillin, and cultured at 37°C in a shaking incubator overnight.

[0187] Inoculate 1 mL of overnight cultured bacteria into 330 mL of TB culture medium, culture at 37°C in a shaking incubator, add 1 M IPTG when the OD600nm value reaches 0.6-0.9, and culture overnight in a shaking incubator at 28°C; centrifuge to collect E. coli, and use the osmotic swelling method to obtain a crude antibody extract;

[0188] The single domain antibody was purified by nickel column affinity chromatography.

[0189] Example 7: Construction of a eukaryotic expression vector for Fc fusion antibody of an anti-CD16a single domain antibody

[0190] (1) Subcloning the target sequence obtained in Example 4 into a eukaryotic expression vector: The antibody screened in Example 4 was sequenced by Sanger sequencing to obtain its nucleotide sequence;

[0191] (2) synthesizing the above nucleotide sequence 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;

[0192] (3) transforming the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culturing for plasmid extraction, and removing endotoxin;

[0193] (4) Sequencing the extracted plasmid for further identification;

[0194] (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 antibody is purified by the method of Example 10.

[0195] Example 8: Expression of single domain antibodies against CD16a protein in suspension ExpiCHO-S cells

[0196] (1) 3 days before transfection, 2.5×10 5 / mL cell passaging and expansion of ExpiCHO-S TM Transfer the calculated volume of cells to a fresh 120 mL (final volume) of pre-warmed ExpiCHO TM The cell concentration was about 4 × 10 6 -6×10 6 Viable cells / mL;

[0197] (2) One day before transfection, place ExpiCHO-S TM The cells were diluted to a concentration of 3.5 × 10 6 viable cells / mL, cells were cultured overnight;

[0198] (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;

[0199] (4) Use fresh ExpiCHO preheated to 37°C TM Dilute the cells to 6 × 10 6 The calculated required volume of cells was transferred to a fresh 100 mL (final volume) of pre-warmed ExpiCHO TM in a 500 mL shake flask containing expression medium;

[0200] (5) Gently invert to mix ExpiFectamine TMCHO reagent, 3.7 mL OptiPRO TM Dilute ExpiFectamine in culture medium TM CHO reagent, swirl or mix;

[0201] (6) Use 4 mL of Refrigerated OptiPRO TM Dilute the plasmid DNA with culture medium and vortex to mix;

[0202] (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, and then gently add it to the prepared cell suspension, gently shaking the flask during the addition process;

[0203] (8) Incubate the cells at 37°C and 8% CO 2 , shake culture in humidified air;

[0204] (9) On the first day after transfection (18-22 hours later), add 600ul ExpiFectamine TM CHO Enhancer and 24mLExpiCHO feed.

[0205] (10) Collect the supernatant about 8 days after transfection (cell viability is less than 70%).

[0206] Example 9: Expression of single domain antibodies against CD16a protein in suspension 293F cells

[0207] Recombinant single domain antibody expression experimental process (taking 500mL shake flask as an example):

[0208] (1) 3 days before transfection, 2.5×10 5 / mL cell passage and expansion culture of 293F cells, the calculated required cell volume was transferred to a 500mL shake flask filled with fresh pre-warmed 120mL (final volume) of OPM-293CD05 Medium. The cell concentration reached about 2×10 6 -3×10 6 Viable cells / mL.

[0209] (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.

[0210] (3) Dilute the cells to 1×10 using pre-warmed OPM-293CD05 Medium. 6The calculated volume of cells was transferred to a 500 mL shake flask containing 100 mL (final volume) of fresh pre-warmed culture medium.

[0211] (4) Dilute PEI (1 mg / mL) reagent with 4 mL Opti-MEM medium, vortex or pipette to mix; dilute plasmid DNA (plasmid DNA is the Fc fusion antibody eukaryotic expression vector of the anti-CD16a single domain antibody prepared in Example 7) with 4 mL Opti-MEM medium, vortex to mix, and filter with a 0.22 um filter. Incubate at room temperature for 5 min.

[0212] (5) Add the diluted PEI reagent to the diluted DNA and mix 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 swirling the flask during the addition process.

[0213] (6) Incubate the cells at 37°C and 5% CO 2 , 120rpm shaking culture.

[0214] (7) Add 5 mL of OPM-CHO PFF05 feed at 24 h and 72 h after transfection.

[0215] (8) Collect the supernatant approximately 7 days after transfection (when cell viability is less than 70%).

[0216] Example 10: Purification of single domain antibodies against CD16a protein

[0217] (1) The protein expression supernatant obtained in Example 8 or 9 was filtered through a 0.45 μm disposable filter to remove insoluble impurities;

[0218] (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;

[0219] (3) The filtrate is passed through the Protein A prepacked column at a flow rate of 1 mL / min. In this step, the target protein in the filtrate will bind to the filler;

[0220] (4) washing the impurity proteins bound to the column with low-salt and high-salt buffers;

[0221] (5) Separate the target protein bound to the column using a low pH buffer;

[0222] (6) The eluate was quickly added with a Tris-HCl solution at pH 9.0 to neutralize it;

[0223] (7) The neutralized protein solution is dialyzed and then subjected to SDS-PAGE analysis to determine that the protein purity is above 95% and the concentration is above 0.5 mg / mL, and then stored at low temperature for future use.

[0224] Example 11: Construction of single domain antibody eukaryotic expression vector RJK-V4-hFC

[0225] The target vector RJK-V4-hFC, which is universal for nano-antibodies, is a modification of the company's commercial vector pCDNA3.4 (vector data link: https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / pcdna3_4_topo_ta_cloning_kit_man.pdf) based on Invitrogen, which was fused with the Fc segment in the heavy chain coding sequence of human IgG1. That is, the vector contains the hinge region (Hinge) CH2 and CH3 region of the IgG1 heavy chain. The specific modification plan is as follows:

[0226] (1) Select the restriction enzyme sites XbaI and AgeI on pcDNA3.4;

[0227] (2) introducing a multiple cloning site (MCS) and a 6×His tag at the 5′ and 3′ ends of the Fc fragment coding sequence respectively by overlapping PCR;

[0228] (3) amplifying the above fragment by PCR using a pair of primers with XbaI and AgeI restriction sites respectively;

[0229] (4) using restriction endonucleases XbaI and AgeI to digest pcDNA3.4 and the recombinant DNA fragments in (3), respectively;

[0230] (5) The digested vector and the inserted fragment are ligated with T4 ligase, and the ligated product is then transformed into Escherichia coli, amplified, and sequenced to obtain a recombinant plasmid.

[0231] Example 12: Antibody antigen binding dose-effect curve determination

[0232] This example was performed using a standard enzyme-linked immunosorbent assay (ELISA) procedure.

[0233] (1) Coat with 50 μL of 1 μg / mL human CD16a-V176 or human CD16a-F176 protein at 4°C overnight.

[0234] (2) Wash the plate; add 200 μL of 5% milk and block at 37°C for 2 h.

[0235] (3) Dilute VHH-Fc to 2ug / mL, and then dilute the antibody 5-fold in a gradient to a total of 8 concentration gradients. The VHH-Fc herein refers to the Fc fusion single domain antibody purified from Example 10.

[0236] (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.

[0237] (5) Wash the plate; add 50 μL of HRP-Goat anti hIgG secondary antibody and incubate at 37°C for 30 min.

[0238] (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 min in the dark.

[0239] (7) Add 50 μL of stop solution (1N HCl) and read the result with a microplate reader.

[0240] (8) Draw the curve and calculate the EC50, such as Figure 2-Figure 9 As shown, hIgG refers to isotype control, an immunoglobulin molecule that does not bind to any target and is purchased commercially. Figure 2-5 is the binding dose-effect curve of each single domain antibody to human CD16a (V176), Figure 6-9 This is the binding dose-effect curve of each single domain antibody to human CD16a (F176).

[0241] Depend on Figure 2-9 It can be seen that the single domain antibodies of the present invention have good affinity and strong specificity for CD16a protein.

[0242] Example 13: Expression and purification of tool antibody (Tab)

[0243] 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.

[0244] The searched sequence was entrusted to General Biosystems (Anhui) Co., Ltd. for mammalian cell expression system codon optimization 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). According to the addition of 100 μg of plasmid (40 μg heavy chain + 60 μg light chain) per 100 mL of cells, PEI was used for transient expression in 293F cells (culture medium: FreeStyle 293Expression medium, Thermo, Cat#12338026+F-68, Thermo, Cat#24040032); 5% volume of 10% Peptone (Sigma, Cat#P0521-100G) was added 6 to 24 hours after transfection, and 8% CO 2 , cultured at 130 rpm for about 7-8 days; when the cell viability dropped to 50%, the expression supernatant was collected and purified using a Protein A (GE, Cat#17-5438-02) gravity column; after dialysis with PBS, the concentration was determined using Nanodrop, the purity was identified by SEC, and the binding capacity was verified by indirect ELISA;

[0245] 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%.

[0246] Example 14: Detection of the RKA effect of CD16a-hFc antibody

[0247] The purpose of this example is to perform RKA effect detection on Fc fusion single domain antibody specific for CD16a. The Fc fusion single domain antibody involved is purified from Example 10. The cells used are P815 cells. RKA refers to Redirected Killing Assay. The experimental steps are as follows:

[0248] (1) Collect P815 cells by centrifugation.

[0249] (2) Resuspend P815 cells in assay buffer (RPMI-1640 + 1% FBS) and adjust the cell density to 2×10 5 cells / mL.

[0250] (3) Place 50 μl of cell suspension into each well of a 96-well plate.

[0251] (4) Tab1 and the single domain antibody samples to be tested were diluted 10-fold in a serial dilution starting from 4×10 μg / ml.

[0252] (5) Add the gradient diluted antibody solution to the cell suspension and incubate for 0.5 h.

[0253] (6) Collect PBMCs by centrifugation and adjust the cell density to 2.5×10 6 cells / ml, and add 100 μl of PBMC cell suspension to each well.

[0254] (7) Incubate at 37°C, 5% CO2 for 15 hours.

[0255] (8) Centrifuge the cell plate at 2000 rpm for 3 min, take 50 μl of the supernatant and add it to a new 96-well plate.

[0256] (9) Add 50 μl of LDH detection reagent to each well and use FlexStation3 to detect OD492 and OD650.

[0257] According to the formula, target cell killing rate % = (sample-E / T) / (MAX-MIN);

[0258] 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.

[0259] The results of RKA effect detection 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.

[0260] Example 15: ADCC effect of CD16a-hFc antibody

[0261] The ADCC effect of the CD16a-hFc of the present invention was determined using a reporter gene method, and the steps are as follows:

[0262] (1) Add 25 μl of assay buffer to a 96-well plate;

[0263] (2) Tab, hIgG and VHH-hFc samples were prepared into a solution with a maximum concentration of 10 μg / mL and diluted 10-fold 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) by Example 10.

[0264] (3) adding the gradient diluted antibody solution into the cell culture wells according to the equal volume of the cell suspension;

[0265] (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 rate of 20,000 cells per well;

[0266] (5) After 6 h of incubation, cell killing was detected using the One-Glo kit and luminescence was read;

[0267] (6) Calculate the fold of induction = (sample-BG) / (E / T-BG)

[0268] 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, such as Figure 14-17 As shown. Figure 14-17 It was found that each CD16a-hFc antibody has an ADCC effect.

[0269] Example 16 Preparation of trispecific antibody FOLR1 / CD16a / Nkp46

[0270] The preparation process of trispecific antibodies includes the following steps:

[0271] (1) synthesizing the nucleotide sequence in Table 18 into pCDNA3.4 by sequence synthesis to obtain a recombinant eukaryotic expression vector;

[0272] (2) transforming the recombinant eukaryotic expression vector constructed in step (1) into DH5α Escherichia coli, culturing for plasmid extraction, and removing endotoxin;

[0273] (3) Sequencing the extracted plasmid for further identification;

[0274] (4) The confirmed recombinant vector is prepared for subsequent eukaryotic cell transfection and expression.

[0275] The trispecific antibody was assembled from FOLR1 / Nkp46 and anti-CD16a single domain antibody, with the structure as Fig.18 (FOLR1 VHH-linker-Nkp46 VHH-linker-CD16a VHH), the amino acid sequence is shown in SEQ ID NO: 47-51. The trispecific antibody is not additionally linked to Fc.

[0276] In SEQ ID NO:47-51, humanized 4F4 is shown as amino acids 1-125, the linker between humanized 4F4 and humanized 7F10 is shown as amino acids 126-140 (GGGGSGGGGSGGGGS), humanized 7F10 is shown as amino acids 141-267, and the linker between humanized 7F10 and CD16a single domain antibody is shown as amino acids 268-282 (GGGGSGGGGSGGGGS).

[0277] All linkers in this specification are not limited to specific sequences, and other linkers used to construct antibodies in the prior art can be used.

[0278] 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.

[0279] Table 17 Amino acid sequences of trispecific antibodies

[0280]

[0281] Table 18 Nucleic acid sequences of trispecific antibodies

[0282]

[0283]

[0284]

[0285] Example 17 ADCC effect of FOLR1 / Nkp46 / CD16a trispecific antibody

[0286] The ADCC effect of the trispecific antibody of the present invention is determined by the LDH method, and the steps are as follows:

[0287] (1) SK-OV-3 cells of passage 3-4 after recovery were collected and plated into 96-well plates at 10,000 cells per well;

[0288] (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;

[0289] (3) adding the gradient diluted antibody solution into the cell culture wells according to the equal volume of the cell suspension;

[0290] (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, 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;

[0291] (5) After 6 h of incubation, the cell killing was detected using an LDH kit and the absorbance was read;

[0292] (6) According to the formula, target cell killing rate % = (sample-E / T) / (MAX-MIN);

[0293] (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.

[0294] 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, such as Figure 19-22 As shown. Figure 19-22 It can be seen that each trispecific antibody has a better ADCC effect.

[0295] 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 solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A single domain antibody against CD16a, Features: The single domain antibody is composed of a heavy chain, and the heavy chain includes SEQ ID NO: 11-heavy chain CDR1 shown in any one of SEQ ID NO: 13, heavy chain shown in any one of SEQ ID NO: 14-SEQ ID NO: 17 CDR2 and the heavy chain CDR3 shown in any one of SEQ ID NO: 18 to SEQ ID NO:

21.

2. The single domain antibody of CD16a according to claim 1, Features: 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, Features: 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. A single domain antibody against CD16a, Features: The amino acid sequences of the single-domain antibodies are respectively shown as any one of SEQ ID NO: 1-5, or compared with any one of SEQ ID NO: 1-5, at least one amino acid residue in the 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 multispecific antibody, characterized in that it comprises the single-domain antibody according to any one of claims 1 to 4, and the single-domain antibody serves as a first antigen-binding portion specifically binding to CD16a.

8. The bispecific antibody or multispecific antibody according to claim 7, characterized in that it further comprises a binding portion specific for other tumor antigens other than CD16a; Preferably, other tumor antigens other than 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, It is 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, It is 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 shown in SEQ ID NO:58, CDR2 shown in SEQ ID NO:59, and CDR3 shown 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, It is 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, Features: 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 the bispecific antibody or multispecific antibody according to any one of claims 7 to 12, Features: 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, Features: 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, Features: 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, Features: 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. Medications used to treat diseases, Features: 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, Features: The disease includes a tumor.

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