A single-domain antibody against cd16a and uses thereof

Abstract

This invention belongs to the field of immunology and relates to a single-domain antibody against CD16a and its uses. The single-domain antibody is composed of heavy chains, including heavy chain CDR1 shown in any one of SEQ ID NO:11-SEQ ID NO:13, heavy chain CDR2 shown in any one of SEQ ID NO:14-SEQ ID NO:17, and heavy chain CDR3 shown in any one of SEQ ID NO:18-21. Compared with the prior art, the beneficial effects of this invention are: this invention uses bioengineering technology to screen for single-domain antibodies specifically targeting CD16a, exhibiting good antibody affinity.

Description

Technical Field

[0001] This invention relates to a single-domain antibody (hereinafter abbreviated as "CD16a single-domain antibody") capable of specifically binding to CD16a, a pharmaceutical composition containing the single-domain antibody as an active ingredient, and its use in pharmaceutical therapy. Background Technology

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

[0003] Single-domain antibodies (SDAbs) are currently the smallest antibody molecules, with a molecular weight only 1 / 10 that of a complete antibody. In addition to possessing the antigenic reactivity of complete antibodies, SDAs also have unique functional characteristics, such as small molecular weight, high stability, good solubility, easy expression, weak immunogenicity, strong penetration, strong targeting, and low preparation cost. They almost perfectly overcome the shortcomings of traditional antibodies, such as long development cycles, low stability, and stringent storage conditions.

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

[0005] The purpose of this patent is to provide a single-domain antibody that can specifically bind to CD16a and its uses.

[0006] A first aspect of the present invention provides a single-domain antibody against CD16a, said single-domain antibody being composed of heavy chains, the heavy chains including heavy chain CDR1 shown in any one of SEQ ID NO:11-SEQ ID NO:13, heavy chain CDR2 shown in any one of SEQ ID NO:14-SEQ ID NO:17, and heavy chain CDR3 shown in any one of SEQ ID NO:18-SEQ ID NO:21.

[0007] Preferably, the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are 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 with sequences that have "at least 80% homology" or sequences that replace only one or a few amino acids; preferably "at least 85% homology", more preferably "at least 90% homology", even more preferably "at least 95% homology", and most preferably "at least 98% homology".

[0015] In one embodiment, in any one or more CDRs of the heavy chain CDR1, CDR2, and CDR3, one to five arbitrary amino acid residues may be substituted with their conserved amino acids. Specifically, in the heavy chain CDR1, one to five amino acid residues may be substituted with their conserved amino acids; in the heavy chain CDR2, one to five amino acid residues may be substituted with their conserved amino acids; and in the heavy chain CDR3, one to five amino acid residues may be substituted with their conserved amino acids.

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

[0017] In some embodiments, the inventive objective can also be achieved by substituting only one or a few amino acids compared to the aforementioned sequence, for example, by including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions. These variations include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, most preferably up to 5) at the C-terminus and / or N-terminus. In practice, 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, those skilled in the art can consider so-called “conserved” amino acid substitutions, in which case the substitution would preferably be a conserved amino acid substitution. A conserved amino acid can generally be described as an amino acid residue substituted by another amino acid residue having a similar chemical structure, and this substitution has little or no effect on the function, activity, or other biological properties of the polypeptide. The conserved amino acid substitutions are common in the art. For example, a conserved amino acid substitution is the substitution 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 non-charged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. The particularly preferred conserved 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. Furthermore, those skilled in the art will understand that the inventiveness of the single-domain antibody lies in the CDR1-3 regions, while the frame region sequences FR1-4 are not immutable, and the sequences of FR1-4 can adopt conserved sequence variants of the sequences disclosed in this invention.

[0018] The term "anti-CD16a single-domain antibody" in this invention includes not only complete single-domain antibodies but also fragments, derivatives, and analogs of said anti-CD16a single-domain antibodies. As used herein, the terms "fragment," "derivative," and "analyte" have the same meaning and refer to polypeptides that substantially retain the same biological function or activity as the antibodies of this invention. The polypeptide fragments, derivatives, or analogs of this invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with an Fc tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0019] In a preferred embodiment, the antibody sequence further includes a framework region FR; the framework region FR includes the amino acid sequences of FR1, FR2, FR3, and FR4; the amino acid sequences of the framework region FR are as follows:

[0020] The FR1 or a variant of FR1 shown in any one of SEQ ID NO:22-26, wherein the variant of FR1 contains a substitution of up to 5 amino acids in the FR1;

[0021] The FR2 or a variant thereof shown in any one of SEQ ID NO:27-29, wherein the variant thereof contains a substitution of up to 5 amino acids in the FR2;

[0022] FR3 or a variant of FR3 shown in any one of SEQ ID NO:30-34, wherein the variant of FR3 contains substitutions of up to 5 amino acids in the FR3;

[0023] The FR4 or a variant thereof shown in SEQ ID NO: 35, wherein the variant thereof contains substitutions of up to 5 amino acids.

[0024] A second aspect of the present invention is to provide an amino acid sequence of a single-domain antibody capable of binding CD16a, wherein the amino acid sequence of the single-domain antibody is as shown in SEQ ID NO: 1-5, or the single-domain antibody has at least 80% sequence homology with the amino acid sequence of SEQ ID NO: 1-5 and is capable of specifically binding CD16a protein, or the amino acid sequence of the single-domain antibody, compared with any one of SEQ ID NO: 1-5, has at least one amino acid residue in the FR1, FR2, FR3 or FR4 sequence substituted by a conserved 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%, and 100% sequence homology with an amino acid sequence selected from SEQ ID NO: 1-5, and is capable of specifically binding to the CD16a protein.

[0026] A third aspect of the invention is to provide an Fc fusion antibody or humanized antibody that provides any of the aforementioned single-domain antibodies against CD16a.

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

[0028] A fifth aspect of the present invention is to provide a bispecific antibody or a multispecific antibody comprising a single-domain antibody according to any of the preceding claims, the single-domain antibody serving as a first antigen-binding portion specifically binding to CD16a.

[0029] In one embodiment, the bispecific antibody or multispecific antibody further includes a binding portion that specifically binds to 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 activator 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 activating 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 are available.

[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 besides CD16a and Nkp46.

[0033] In one implementation, 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, and 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 activating 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 to CD16a, a second antigen-binding portion that specifically binds to Nkp46, and a third antigen-binding portion that specifically binds to 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 to Nkp46 and the third antigen-binding portion that specifically binds to FOLR1 are VHH;

[0038] Preferably, the trispecific antibody does not fuse with Fc.

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

[0040] A sixth aspect of the present invention is to provide a nucleotide molecule encoding the aforementioned anti-CD16a single-domain antibody, the aforementioned Fc fusion antibody, or the aforementioned humanized antibody, wherein the nucleotide sequences are as shown in SEQ ID NO: 6-10, or the amino acid sequences encoded by the nucleotide sequences are the same as those 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 single-domain antibody against CD16a has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology with nucleotide sequences selected from SEQ ID NO: 6-10, and the single-domain antibody against CD16a encoded therefrom is capable of specifically binding to the CD16a protein.

[0042] A seventh aspect of the present invention is to provide a nucleotide molecule encoding the aforementioned bispecific antibody or multispecific antibody, the nucleotide sequence of which is shown in SEQ ID NO: 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 NO: 52-56, or has 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 or multispecific antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with the nucleotide sequences selected from SEQ ID NO: 52-56, and the bispecific or multispecific antibody encoded thereon is capable of specifically binding to CD16a, FOLR1, or Nkp46 proteins.

[0044] An eighth aspect of the present invention is to provide an expression vector comprising the aforementioned nucleotide molecule encoding a single-domain antibody, an Fc fusion antibody, or a humanized antibody against CD16a, or comprising the aforementioned nucleotide molecule encoding a bispecific antibody or a multispecific antibody.

[0045] In a preferred embodiment, the expression vector used can be RJK-V4-hFC (integrating a nucleotide molecule encoding a single-domain antibody or its Fc fusion antibody or humanized antibody against CD16a into RJK-V4-hFC through genetic engineering), and other general-purpose expression vectors can also be selected as needed.

[0046] A 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 thereof comprising the aforementioned. Preferably, the host cell is a bacterial cell, fungal cell, or mammalian cell.

[0047] In another preferred embodiment, the host cell includes prokaryotic or eukaryotic cells, 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 combinations thereof.

[0049] In another preferred embodiment, the prokaryotic cells are selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or combinations thereof.

[0050] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomiae, Trichoderma, or combinations thereof.

[0051] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of insect cells such as armyworms, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or combinations 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] A 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 shown in SEQ ID NO: 1-5, or a single-domain antibody having at least 80% homology with those in SEQ ID NO: 1-5, or a multi-epitope antibody, bispecific antibody, multispecific antibody, or multivalent antibody; for example, the multi-epitope antibody may consist of more than one sequence in SEQ ID NO: 1-5; the multivalent antibody may consist of one sequence in SEQ ID NO: 1-5 repeated a certain number of times; the multispecific antibody includes, but is not limited to, trispecific antibodies and tetraspecific antibodies; furthermore, the recombinant protein may be a fragment, derivative, or analog of the aforementioned antibody.

[0055] The eleventh aspect of the present invention is to provide a pharmaceutical composition comprising the aforementioned CD16a-binding single-domain antibody, the aforementioned bispecific antibody, or a multispecific antibody, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally 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 by approximately 2). The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intravenous, transdermal (direct application or patching to the affected area).

[0056] The pharmaceutical compositions of the present invention contain 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 solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. 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, comprising the aforementioned single-domain antibody for binding CD16a protein, the aforementioned bispecific antibody, or a 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, etc.

[0059] In a preferred embodiment, the kit includes an antibody that recognizes the CD16a protein, a lysis medium for dissolving the sample, and universal 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 second antibody and an enzyme or fluorescent or radiolabeled marker for detection, as well as a buffer solution.

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

[0062] In a thirteenth aspect of the present invention, a method for generating a single-domain antibody against CD16a is provided, comprising the steps of:

[0063] (a) The host cells described in the ninth aspect of the present invention are cultured under conditions suitable for the production of single-domain antibodies, thereby...

[0064] Obtain a culture containing the single-domain antibody against CD16a; and

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

[0066] (c) Optionally, purify and / or modify the CD16a single-domain antibody 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 medicament for treating a disease.

[0068] In a preferred embodiment, the disease is a variety of NK cell-mediated CD16a-related conditions.

[0069] In a preferred embodiment, various NK cell-mediated CD16a-related conditions 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 diseases (e.g., dry eye), ankylosing spondylitis, psoriatic arthritis, and cancers (e.g., multiple myeloma and breast cancer).

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

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

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

[0073] In a preferred embodiment, the tumor includes, but is not limited to, tumors of epithelial origin (adenomas and various types of carcinoma, including adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, and other cancers); bladder cancer and urinary tract cancer; breast cancer; gastrointestinal cancers (including esophageal cancer, stomach cancer, small bowel cancer, colon cancer, rectal cancer, and anal cancer); liver cancer (hepatocellular carcinoma); gallbladder and biliary system cancers, exocrine pancreatic cancers, kidney-related cancers; 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 cavity cancer, laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, tonsil cancer, salivary gland cancer, nasal cavity cancer, and paranasal sinus cancer); ovarian, fallopian tube, peritoneum, vagina, vulva, penis, cervix, myometrium, and endometrial-related cancers. Symptoms: thyroid cancer (e.g., follicular thyroid carcinoma); adrenal, prostate, skin, and adnexal cancers (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevus); hematologic malignancies (i.e., leukemia, lymphoma) and precancerous hematologic disorders and marginal malignancies, including hematologic malignancies and lymphoid spectrum 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 lymphoma, mantle cell lymphoma, T-cell lymphoma and leukemia, natural killer NK-cell lymphoma, Hodgkin's lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma). Multiple myeloma and post-transplant lymphoproliferative disorders; hematologic malignancies and myeloid-related diseases (e.g., acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), eosinophilic syndrome, myeloproliferative disorders such as polycythemia vera, essential thrombocythemia and essential myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome and promyelocytic leukemia); mesenchymal tumors, such as soft tissue, bone or cartilage sarcomas, 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 tumors. Histiocytoma and dermatofibrosarcoma protuberans; tumors of the central or peripheral nervous system (e.g., astrocytoma, glioma and glioblastoma, meningioma, ependymoma, pineal tumor and schwannoma); 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., teratoma, seminoma, dysgerminoma, hydatidiform mole and choriocarcinoma); pediatric and embryonic tumors (e.g., medulloblastoma, neuroblastoma, nephroblastoma and primitive neuroectodermal tumors); or congenital or other syndromes that predispose a patient to malignancy (e.g., xeroderma pigmentosum).

[0074] In a preferred embodiment, the diseases include, but are 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, clear cell renal 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 leukemia, and viral infections.

[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 specifically targeted at the CD16a protein with the correct spatial structure.

[0078] (2) The single-domain antibody obtained by the present invention has a flexible expression system. It can be expressed in a prokaryotic system or in a eukaryotic system of yeast cells or mammalian cells. Moreover, its expression cost in the prokaryotic expression system is low, which can reduce the later production cost.

[0079] (3) The single-domain antibody obtained by the present invention has simple multi-combination form modification. It can be obtained by simple tandem through genetic engineering to obtain multivalent and multispecific antibodies. Moreover, its immune heterogeneity is very low and it will not produce a strong immune response without humanization modification.

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

[0081] (5) The single-domain antibody against CD16a obtained in this invention has strong antigen-binding ability and specificity, and excellent redirection killing ability, which can effectively mediate the ADCC effect. This single-domain antibody can be combined with other antigen-binding parts to form bispecific or multispecific antibodies, and can also be used as part of a chimeric antigen receptor (CAR), or assembled into any other form of antibody.

[0082] The trispecific antibody FOLR1 / CD16a / Nkp46 (FOLR1 / CD16a / Nkp46 is only one implementation method; CD16a can also be combined with other tumor surface antigen targets) prepared on the basis of CD16a single domain antibody still exhibits surprisingly excellent ADCC effect without the addition of immunoglobulin Fc. Attached Figure Description

[0083] To more clearly illustrate the technical solution of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0084] Figure 1 The image shows the library enrichment results for screening CD16a-targeted antibodies in Example 3, with the left side showing the library enrichment results for screening CD16a-V176-targeted antibodies and the right side showing the library enrichment results for screening CD16a-F176-targeted antibodies.

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

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

[0087] Figure 4 The binding dose-response curves (15E2, 13G6) of CD16a-hFc antibody and human CD16a (V176) in Example 12 are shown.

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

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

[0090] Figure 7 The binding dose-response curves (15E2, 13G6) of the CD16a-hFc antibody and human CD16a (F176) in Example 12 are shown.

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

[0092] Figure 9 The binding dose-response curve (23D1) of the CD16a-hFc antibody and human CD16a (F176) in Example 12;

[0093] Figure 10 The results of the RKA assay for CD16a-hFc antibody (4B5);

[0094] Figure 11 The results of the RKA assay for CD16a-hFc antibody (15E2);

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

[0096] Figure 13 The results of the RKA assay for CD16a-hFc antibody (23D1);

[0097] Figure 14 The ADCC test results for CD16a-hFc are shown in Figure 4B5.

[0098] Figure 15 The ADCC test results for CD16a-hFc (15E2);

[0099] Figure 16 The ADCC test results for CD16a-hFc (13G6, 14A3);

[0100] Figure 17 The ADCC test results for CD16a-hFc (23D1);

[0101] Figure 18 The results of ADCC assay for the FOLR1 / Nkp46 / CD16a trispecific antibody;

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

[0103] Figure 22 The results of the ADCC assay for the FOLR1 / Nkp46 bispecific antibody are shown. Detailed Implementation

[0104] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0105] As used herein, “sdAb” (also referred to as nanobodies or VHH by the developer Ablynx) is well known to those skilled in the art. A single-domain antibody is an antibody whose complementarity-determining region is part of a single-domain polypeptide. Therefore, a single-domain antibody contains a single complementarity-determining region (a single CDR1, a single CDR2, and a single CDR3). Examples of single-domain antibodies include antibodies containing only heavy chains (which naturally do not contain 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 such as camels, dromedaries, llamas, and guanacos. Like complete antibodies, single-domain antibodies can selectively bind to specific antigens. Single-domain antibodies may contain only variable domains of the immunoglobulin chain, which have CDR1, CDR2, and CDR3, as well as a frame region.

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

[0108] As used in this article, the term "Fc fusion antibody" refers to a novel protein produced by fusing the Fc fragment of a target antibody with a biologically active functional protein molecule using genetic engineering techniques.

[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 by transplanting the complementarity-determining region (CDR1-3 sequence) of a target antibody into the variable region of a human antibody, or by mutating the target antibody according to the characteristics of the human antibody backbone region (FR1-4). Humanized antibodies can be produced using synthetic methods or site-directed mutagenesis methods.

[0110] In this invention, sequences with high homology to the CDR1-3 sequences disclosed herein can also yield 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" with the sequences in SEQ ID NO. 1-5 can achieve the purpose of the invention.

[0111] In some embodiments, the inventive objective can also be achieved by replacing only one or a few amino acids compared to the sequences in SEQ ID NO. 1-5, for example, by including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions. In practice, 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, those skilled in the art can consider so-called “conserved” amino acid substitutions. In the case of substitution, the substitution will preferably be a conserved amino acid substitution, which can generally be described as an amino acid residue replaced by another amino acid residue having a similar chemical structure, and this substitution has little or no effect on the function, activity, or other biological properties of the polypeptide. The conserved amino acid substitutions are common in the art. For example, a conserved amino acid substitution is the substitution 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 non-charged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. The particularly preferred conserved 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. Furthermore, those skilled in the art will understand that the inventiveness of the single-domain antibody lies in the CDR1-3 regions, while the frame region sequences FR1-4 are not immutable, and the sequences of FR1-4 can adopt conserved sequence variants of the sequences disclosed in this invention.

[0112] The preferred host cell of the present invention is a bacterial cell, a fungal cell, or a mammalian cell.

[0113] This patent involves preparing a target protein and a truncated form of the target protein using genetic engineering technology. The obtained antigen protein is then used to immunize Bactrian camels in Alashan, Inner Mongolia. After multiple immunizations, peripheral blood lymphocytes or spleen cells of the camels are obtained. Through genetic engineering, the variable region coding sequence of the camel-derived antibody is recombined into a phage display vector. Specific antibodies against the antigen protein are screened using 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 will now be broken down and explained in detail, and described with specific embodiments:

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

[0116] The human recombinant extracellular domain protein used in this patent was expressed and purified by the company itself. The specific design scheme of the expression vector for the human recombinant CD16a protein is as follows:

[0117] (1) The coding sequence of CD16a was obtained by searching in NCBI. Its accession number is NM_000569.7, and the accession number of the amino acid sequence encoded by this sequence is NP_000560.6.

[0118] (2) The nucleotide sequence encoding amino acids 17-208 of CD16a (CD16a-V176, CD16a-F176) was cloned into the vector pcDNA3.4 using gene synthesis. The constructed vector was subjected to Sanger sequencing, and after comparison with the original sequence and confirmation that it was correct, the recombinant plasmid was extracted in large quantities, endotoxin was removed, and it was transfected into suspension 293F cells for expression and purification of the target protein. The purity reached more than 90%, which met the requirements for animal immunization.

[0119] The antigens consist of two types: one is amino acids 17-208 of CD16a, with amino acid 176 being F, abbreviated as CD16a-F176; the other is amino acids 17-208 of CD16a, with amino acid 176 being V, abbreviated as CD16a-V176. Both antigens were prepared and purified separately.

[0120] Example 2: Construction of a single-domain antibody library targeting the CD16a protein:

[0121] One 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 Alashan, Inner Mongolia, once a week for a total of seven consecutive immunizations. Except for the first immunization, the remaining six immunizations were performed using an equal volume of 1 mg of CD16a protein mixed with Freund's incomplete adjuvant. This immunization process was intended to concentrate the stimulation of camels to produce antibodies against CD16a protein. CD16a-V176 and CD16a-F176 were not mixed and were used to immunize animals separately to obtain antibodies.

[0122] After animal immunization, 150 mL of peripheral blood lymphocytes were collected from camels, and RNA was extracted from the cells. cDNA was synthesized using the extracted total RNA, and VHH (antibody heavy chain variable region) was amplified using nested PCR with the cDNA as a template.

[0123] Then, the pMECS vector and VHH fragment were digested with restriction endonucleases, and the digested fragments were ligated to the vector. The ligated fragments were electroporated into competent TG1 cells to construct a phage display library of CD16a protein, and the library size was determined to be approximately 1 × 10⁻⁶. 9 Meanwhile, the correct insertion rate of the target fragment in the library was detected by colony PCR.

[0124] The results showed that after PCR amplification of 30 colonies randomly selected from the library, 29 clones were able to amplify bands of the predicted size, and 1 clone amplified an incorrect band. Therefore, the correct insertion rate was 29 ÷ 30 × 100% ≈ 96.7%.

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

[0126] Take 200 μL of the recombinant TG1 cells from Example 2 and culture them in 2×TY medium. During the culture, add 40 μL of helper phage VCSM13 to infect the TG1 cells and culture them overnight to amplify the phage. The next day, precipitate the phage with PEG / NaCl and collect the amplified phage by centrifugation.

[0127] 500 μg of CD16a protein diluted in 100 mM pH 8.3 NaHCO3 was coupled onto an ELISA plate and incubated overnight at 4°C. A negative control well (culture medium control) was also included. The next day, 200 μL of 3% skim milk was added, and the plate was blocked at room temperature for 2 hours. After blocking, 100 μL of the amplified phage library (approximately 2 × 10⁻⁶) was added. 11 (1 phage particle), incubate at room temperature for 1 hour; after 1 hour, wash 15 times with PBS + 0.05% Tween-20 to remove unbound phage.

[0128] Phages that specifically bind to CD16a protein were dissociated using trypsin at a final concentration of 25 mg / mL and then used to infect E. 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 once to gradually enrich the cells.

[0129] When the enrichment factor reaches 10 times or more, the enrichment effect is as follows: Figure 1 As shown.

[0130] Figure 1 In this context, P / N = the number of monoclonal bacteria grown from phages eluted from positive wells in the biopanning process after infecting TG1 bacteria / the number of monoclonal bacteria grown from phages eluted from negative wells after infecting TG1 bacteria. This parameter gradually increases after enrichment occurs. I / E = the total number of phages added to positive wells in each round of the biopanning process / the total number of phages eluted from positive wells in each round of the biopanning process. This parameter gradually approaches 1 after enrichment occurs.

[0131] Example 4: Screening for CD16a-specific positive clones using phage enzyme-linked immunosorbent assay (ELISA):

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

[0133] Crude antibody was obtained using the osmotic burst method. CD16a recombinant protein was released into 100 mM NaHCO3 (pH 8.3), and 100 μg of protein was coated overnight at 4°C in an ELISA plate. 100 μL of the obtained crude antibody extract was transferred to an ELISA plate containing the antigen and incubated at room temperature for 1 h. Unbound antibody was washed away 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. The plate was incubated at room temperature for 1 h. Unbound antibody was washed away with PBST, and horseradish peroxidase chromogenic solution was added. The reaction was carried out at 37°C for 15 min, and then stop solution was added. The absorbance was read at 450 nm using an ELISA reader.

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

[0135] The gene sequences of each clone were analyzed using the sequence alignment software VectorNTI. Clones with the same CDR1, CDR2 and CDR3 sequences were considered as the same clone, while clones with different sequences were considered as different clones. Finally, a single-domain antibody specifically targeting the CD16a protein was obtained.

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

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

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

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

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

[0141]

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

[0143]

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

[0145]

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

[0147] Actual clone number FR1 SEQ ID 15E2 QVQLVESGGGLVQAGDSLRLSCAAS SEQ ID NO:22 4B5 QVQLVESGGGLVQAGGSLRLSCAAS SEQ ID NO:23 23D1 QVQLVESGGGLVQAGGSLRLSCAVS SEQ ID NO:24 13G6 QVQLVESGGGLVQPGGSLRLSCAAS SEQ ID NO:25 14A3 QVQLVESGGGLVQPGGSLRLSCEVS SEQ ID NO:26

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

[0149]

[0150] Table 6. FR3 sequences of five single-domain antibodies

[0151]

[0152] Table 7. FR4 sequences of five single-domain antibodies

[0153]

[0154] Table 8. Amino acid sequences of five single-domain antibodies

[0155]

[0156] Table 9. Nucleic acid sequences of five single-domain antibodies

[0157]

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

[0159] Single-domain antibodies for FOLR1 and Nkp46 were obtained by screening according to the methods in Examples 1-4, namely FOLR1-4F4 single-domain antibody and Nkp46-7F10 single-domain antibody, respectively; the specific screening process is the same as that in Examples 1-4, except that the preparation process of the antigen is different.

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

[0161] The human recombinant extracellular domain protein used in this patent was expressed and purified by the company itself. The specific design scheme of the expression vector for the human recombinant Nkp46 protein is as follows:

[0162] (1) The coding sequence of Nkp46 was obtained by searching in NCBI. Its accession number is BC064806.1, and the accession number of the amino acid sequence encoded by this sequence is AAH64806.1.

[0163] (2) The nucleotide sequence encoding amino acids 22-254 of Nkp46 was cloned into the vector pcDNA3.4 using gene synthesis. The constructed vector was subjected to Sanger sequencing and compared with the original sequence. After confirming that there were no errors, the recombinant plasmid was extracted in large quantities, endotoxin was removed, and it was transfected into suspension 293F cells for expression and purification of the target protein. The purity reached more than 90%, which met the requirements for animal immunization.

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

[0165] The human recombinant extracellular domain protein used in this patent was expressed and purified by the company itself. The specific design scheme of the expression vector for the human recombinant FOLR1 protein is as follows:

[0166] (1) The coding sequence of FOLR1 was obtained by searching in NCBI. Its accession number is NM_000802.3, and the accession number of the amino acid sequence encoded by this sequence is NP_000793.1.

[0167] (2) The nucleotide sequence encoding amino acids 25-233 of FOLR1 was cloned into pcDNA3.4 using gene synthesis. The constructed vector was subjected to Sanger sequencing and compared with the original sequence. After confirming that there were no errors, the recombinant plasmid was extracted in large quantities, endotoxin was removed, and it was transfected into suspension 293F cells for expression and purification of the target protein. The purity reached more than 90%, which met the requirements for animal immunization.

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

[0169] The sequences of the screened Nkp46-7F10 single-domain antibodies are shown in Tables 13 and 14. Humanization was then performed on these antibodies, and the sequence of the humanized 7F10 is shown in Table 15. The modified region includes CDR3, as detailed in the underlined portion of Table 15.

[0170] Humanized 4F4 and humanized 7F10 were linked to form humanized FOLR1 / Nkp46, the sequence of which is shown in Table 16. The 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 the 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 and nucleic acid sequences 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 and nucleic acid sequences of humanized FOLR1 / Nkp46 bispecific antibodies.

[0184]

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

[0186] The plasmids (pMECS-VHH) of different clones obtained from sequencing analysis in Example 4 were electroporated into Escherichia coli HB2151 and plated on LB+amp+glucose culture plates containing ampicillin and glucose, and incubated overnight at 37°C. Single colonies were selected and inoculated into 5 mL of LB culture medium containing ampicillin and incubated overnight on a shaker at 37°C.

[0187] Inoculate 1 mL of overnight cultured bacteria into 330 mL of TB culture medium and incubate at 37°C in a shaker. When the OD600nm value reaches 0.6-0.9, add 1 M IPTG and incubate overnight at 28°C in a shaker. Centrifuge to collect E. coli and obtain crude antibody extract using the osmotic rupture method.

[0188] Single-domain antibodies were purified by nickel column affinity chromatography.

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

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

[0191] (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.

[0192] (3) Transform the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culture it for plasmid extraction, and remove endotoxins;

[0193] (4) The extracted plasmids were then sequenced and identified.

[0194] (5) After confirming the recombinant vector, prepare it for subsequent eukaryotic cell transfection and expression. After expressing the Fc protein of VHH using the method in Example 8 or 9, purify the antibody using the method in Example 10.

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

[0196] (1) Three days before transfection, use 2.5×10 5 / mL cell passage and expansion culture ExpiCHO-S TM Cells, the calculated desired cell volume, were transferred to 120 mL (final volume) of fresh, preheated ExpiCHO. TM In a 500 mL shake flask of expression medium, the cell concentration was increased to approximately 4 × 10⁻⁶ cells / mL. 6 -6×10 6 live cells / mL;

[0197] (2) One day before transfection, ExpiCHO-S TM Cells were diluted to a concentration of 3.5 × 10⁻⁶. 6 Live cells / mL, incubate cells overnight;

[0198] (3) On the day of transfection, measure cell density and percentage of viable cells. Cell density should reach approximately 7 × 10⁻⁶ cells / day before transfection. 6 -10×10 6 live cells / mL;

[0199] (4) Use fresh ExpiCHO preheated to 37°C TM The expression medium was used to dilute the cells to 6 × 10⁶. 6 viable cells / mL. The calculated desired cell volume was transferred to 100 mL (final volume) of fresh, preheated ExpiCHO. TM In a 500 mL shake flask of expression medium;

[0200] (5) Gently invert to mix ExpiFectamine TMCHO reagent, using 3.7 mL OptiPRO TM Culture medium for diluting ExpiFectamine TM CHO reagent, vortex or mix well;

[0201] (6) Use 4 mL of refrigerated OptiPRO TM Dilute the plasmid DNA in the 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 while gently shaking the flask during the addition process.

[0203] (8) The cells were cultured with shaking in humidified air at 37°C and 8% CO2.

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

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

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

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

[0208] (1) Three days before transfection, use 2.5×10 5 After passage and expansion of 293F cells at / mL, the calculated desired cell volume was transferred to a 500mL shake flask containing 120mL (final volume) of fresh, preheated OPM-293CD05 Medium to achieve a cell concentration of approximately 2×10⁻⁶. 6 -3×10 6 Live cells / mL.

[0209] (2) On the day of transfection, measure cell density and percentage of viable cells. Cell density should reach approximately 2 × 10⁻⁶ cells / day before transfection. 6 -3×10 6 Live cells / mL.

[0210] (3) Dilute the cells to 1×10⁻⁵ using preheated OPM-293CD05 Medium. 61 live cells / mL. Calculate the required cell volume and transfer it to a 500 mL shake flask containing 100 mL (final volume) of fresh, preheated culture medium.

[0211] (4) Dilute PEI (1 mg / mL) reagent with 4 mL Opti-MEM medium, and mix by swirling or pipetting; dilute plasmid DNA (the plasmid DNA is the eukaryotic expression vector of the Fc fusion antibody of the anti-CD16a single-domain antibody prepared in Example 7) with 4 mL Opt-MEM medium, mix by swirling, and filter with a 0.22 μm 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 while gently shaking the flask during the addition process.

[0213] (6) Culture the cells at 37°C, 5% CO2, and 120 rpm with shaking.

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

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

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

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

[0218] (2) The above filtrate was purified by affinity chromatography using a protein purifier. The agarose packing material coupled with Protein A was used to purify the filtrate by utilizing the ability of human Fc to bind to Protein A.

[0219] (3) Pass the filtrate through a pre-packed Protein A column at a flow rate of 1 mL / min. In this step, the target protein in the filtrate will bind to the packing material.

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

[0221] (5) Use a low pH buffer to systemically bind the target protein on the column;

[0222] (6) Quickly add the eluent to a Tris-HCl solution with a pH of 9.0 to neutralize it;

[0223] (7) After dialysis of the above-neutralized protein solution, perform SDS-PAGE analysis to confirm that the protein purity is above 95% and the concentration is above 0.5 mg / mL, and then store it at low temperature for later use.

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

[0225] The aforementioned universal target vector for nanobodies, RJK-V4-hFC, is a modified version of Invitrogen's commercial vector pCDNA3.4 (vector data link: https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / pcdna3_4_topo_ta_cloning_kit_man.pdf) by incorporating the Fc region of the human IgG1 heavy chain coding sequence. Specifically, this vector contains the CH2 and CH3 hinge regions of the IgG1 heavy chain. The specific modification scheme is as follows:

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

[0227] (2) Multiple cloning sites (MCS) and 6×His tags were introduced at the 5' and 3' ends of the Fc fragment coding sequence, respectively, by overlapping PCR.

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

[0229] (4) The recombinant DNA fragments in pcDNA3.4 and (3) were digested with restriction endonucleases XbaI and AgeI, respectively;

[0230] (5) The digested vector and the insert fragment were ligated with T4 ligase, and then the ligation product was transformed into E. coli, amplified, sequenced and verified to obtain the recombinant plasmid.

[0231] Example 12: Determination of Antibody-Antigen Binding Dose-Reaction Curve

[0232] This embodiment uses the standard enzyme-linked immunosorbent assay (ELISA) procedure.

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

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

[0235] (3) Dilute VHH-Fc to 2 μg / mL, and then perform a 5-fold serial dilution of the antibody to a total of 8 concentration gradients. Here, VHH-Fc refers to the Fc fusion single-domain antibody that was purified from Example 10.

[0236] (4) Wash the plate; add 50 μL of the single-domain antibody obtained by dilution in step (3), double replicates, 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 (wash several times); add 50 μL of pre-warmed solution. T MB, react at room temperature in the dark for 15 minutes.

[0239] (7) Add 50 μL of stop solution (1N HCl) and save the microplate reader reading.

[0240] (8) Plot the curve and calculate EC50, as follows: Figures 2-9 As shown, hIgG refers to the isotype control, an immunoglobulin molecule that does not bind to any target and is obtained through commercial purchase. Figure 2-5 These are the dose-response curves of binding between each single-domain antibody and human CD16a(V176). Figure 6-9 These are the dose-response curves of each single-domain antibody binding to human CD16a(F176).

[0241] Depend on Figure 2-9 It is evident that the single-domain antibodies of the present invention all exhibit 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, sequence from CN101583625B; Tab3, AFM13, sequence from CN110461357A; Tab4, AFM24, sequence from CN110461357A; and FOLR1-Tab1, farletuzumab.

[0244] The searched sequences were entrusted to General Biosystems (Anhui) Co., Ltd. for codon optimization in mammalian cell expression systems and cloned into the pcDNA3.1 vector. After antibiotic selection, plasmid-positive bacteria were selected for amplification, and plasmids were 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 the plasmid was transiently transfected into 293F cells (medium: FreeStyle 293Expression medium, Thermo, Cat#12338026+F-68, Thermo, Cat#24040032) using PEI. 6–24 hours after transfection, 5% volume of 10% Peptone (Sigma, Cat#P0521-100G) was added, and the cells were cultured at 130 rpm for approximately 7–8 days with 8% CO2. When cell viability decreased to 50%, the expression supernatant was collected and purified using a gravity column with Protein A (GE, Cat#17-5438-02). After dialysis with PBS, the concentration was determined using Nanodrop, purity was identified by SEC, and binding capacity was verified by indirect ELISA.

[0245] The Tab1, Tab3, Tab4, and FORL1-Tab1 obtained by this method have a concentration of not less than 2 mg / ml and a purity greater than 95%.

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

[0247] The purpose of this embodiment is to detect the Redirected Killing Assay (RKA) effect of an Fc fusion single-domain antibody specifically targeting CD16a. The Fc fusion single-domain antibody involved was purified from Example 10, and P815 cells were used. RKA refers to the 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 × 10⁻⁶ cells / cells. 5 Cells / mL

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

[0251] (4) Dilute Tab1 and the single-domain antibody sample to be tested 10-fold serially starting from 4×10μg / ml.

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

[0253] (6) Collect PBMCs by centrifugation and adjust the cell density to 2.5 × 10⁻⁶. 6 Cells / mL: Add 100 μl of PBMC cell suspension to each well.

[0254] (7) Incubate at 37℃ and 5% CO2 for 15 hours.

[0255] (8) Centrifuge the cell plate at 2000 rpm for 3 minutes, and add 50 μl of supernatant 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, the target cell killing rate % = (sample - E / T) / (MAX - MIN);

[0258] Based on the target cell killing rate and concentration, a four-parameter fitting was performed to calculate the EC50 concentration of each antibody-mediated ADCC action.

[0259] The results of the RKA effect detection are shown in Figures 10-13. It can be seen that the CD16a single-domain antibodies of this invention all have strong redirection killing ability.

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

[0261] The ADCC effect of CD16a-hFc in this 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) The Tab, hIgG and VHH-hFc samples were prepared into solutions with a maximum concentration of 10 μg / mL and then serially diluted 10 times 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.

[0264] (3) Add the serially diluted antibody solution to the cell culture wells in equal volumes to the cell suspension;

[0265] (4) For the sample wells and E / T wells (antibody concentration of 0), collect Jurkat-NFAT-luc-FcγRIIIa cells and add 20,000 cells per well to the cell culture wells;

[0266] (5) After incubation for 6 hours, 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] Based on the target cell killing rate and concentration, a four-parameter fitting was performed to calculate the EC50 concentration of each antibody-mediated ADCC effect, such as... Figure 14-17 As shown. From Figure 14-17 It can be seen that each CD16a-hFc antibody has an ADCC effect.

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

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

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

[0272] (2) The recombinant eukaryotic expression vector constructed in step (1) was transformed into DH5α Escherichia coli, and plasmids were extracted and endotoxins were removed.

[0273] (3) The extracted plasmids were then sequenced and identified.

[0274] (4) Prepare the recombinant vector after it has been confirmed to be correct for subsequent eukaryotic cell transfection and expression.

[0275] A trispecific antibody was assembled from FOLR1 / Nkp46 and anti-CD16a single-domain antibody, with the structure shown below. Figure 18 (FOLR1VHH-linker-Nkp46 VHH-linker-CD16a VHH), amino acid sequence as shown in SEQ ID NO:47-51. The trispecific antibody is not additionally linked to an Fc.

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

[0277] All adapters in this specification are not limited to specific sequences; other adapters used in the prior art for constructing antibodies may also 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 was determined using the LDH method, and the steps are as follows:

[0287] (1) Collect SK-OV-3 cells that have been revived and passaged for 3-4 generations and seed them into 96-well plates at a rate of 10,000 cells per well.

[0288] (2) Tab, hIgG, FOLR1 / Nkp46 and trispecific antibody were prepared into a solution with a maximum concentration of 10 μg / mL and then serially diluted 10-fold to obtain 7 concentrations.

[0289] (3) Add the serially diluted antibody solution to the cell culture wells in equal volumes to the cell suspension;

[0290] (4) For the sample wells and E / T wells (antibody concentration of 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 the MAX wells, add lysis buffer to each well at twice the volume of the target cell suspension; for the MIN wells, add test buffer to each well at twice the volume of the target cell suspension.

[0291] (5) After incubation for 6 hours, cell killing was detected using an LDH kit, and the absorbance was read.

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

[0293] (7) Based on the target cell killing rate and concentration, perform four-parameter fitting to calculate the EC50 concentration of each antibody-mediated ADCC action.

[0294] Based on the target cell killing rate and concentration, a four-parameter fitting was performed to calculate the EC50 concentration of each antibody-mediated ADCC effect, such as... Figure 19-22 As shown. From Figure 19-22 It can be seen that each of the three specific antibodies has a good ADCC effect.

[0295] The foregoing has shown and described 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 way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A single-domain antibody against CD16a, characterized in that: The single-domain antibody is composed of heavy chains, including heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3; The amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are as follows (1) or (2): (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:11, CDR2 shown in SEQ ID NO:16, and CDR3 shown in SEQ ID NO:

20.

2. The single-domain antibody against CD16a according to claim 1, characterized in that: The single-domain antibody further includes a frame region FR; the frame region FR includes the amino acid sequences of FR1, FR2, FR3, and FR4; the amino acid sequences of the frame region FR are as follows: FR1 or a variant thereof shown in SEQ ID NO: 24 or 23, wherein the variant thereof contains up to 5 amino acid substitutions in the FR1; FR2 or a variant thereof shown in SEQ ID NO: 28 or 29, wherein the variant thereof contains substitutions of up to 5 amino acids; FR3 or a variant thereof shown in SEQ ID NO: 33 or 31, wherein the variant thereof contains substitutions of up to 5 amino acids in the FR3; The FR4 or a variant thereof shown in SEQ ID NO:35, wherein the variant thereof contains substitutions of up to 5 amino acids.

3. A single-domain antibody against CD16a, characterized in that: The amino acid sequences of the single-domain antibodies are shown in SEQ ID NO: 5 or 1, respectively.

4. The Fc fusion antibody or humanized antibody of the single-domain antibody against CD16a according to any one of claims 1 to 3.

5. A recombinant protein, characterized in that, The recombinant protein comprises the single-domain antibody against CD16a as described in any one of claims 1 to 3.

6. A bispecific antibody or a multispecific antibody, characterized in that, It comprises a single-domain antibody according to any one of claims 1 to 3, the single-domain antibody serving as a first antigen-binding portion that specifically binds to CD16a.

7. The bispecific or multispecific antibody according to claim 6, characterized in that, It also includes binding regions that specifically target tumor antigens other than CD16a; Other tumor antigens besides CD16a include FOLR1, Her2, DEC205, CLEC9A, CEACAM5, CTLA4, CD3, CD7, CD11c, CD19, CD20, CD22, CD40, CD44, CD206, EGFR, EGFRvIII, fibroblast activating protein (FAP), CA9, MMP-2, PD-L1, SIRPa, Trop2, GPC1, GPC3, cMET, BCMA, VEGFR, Cladin18.2, Nkp46, CD30, NKG2D, IL-2Rβ, CD123, TGF-β, CD38, IL-7, IL-8, FRα, NCR3, IL-15, Muc1, or IL-16.

8. The bispecific or multispecific antibody according to claim 6, characterized in that, The bispecific antibodies are 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, and CD206 / CD16a. EGFR / CD16a, EGFRvIII / CD16a, Fibroblast activating protein (FAP) / CD16a, CA9 / CD16a, MMP-2 / CD16a, PD-L1 / CD16a, SIRPa / CD16a, Trop2 / CD16a, GPC1 / CD16a, GPC3 / CD16a, cMET / CD16a, BCMA / CD16a, VEGFR / CD16a, Cladi Bispecific antibodies against n18.2 / CD16a, CD30 / CD16a, NKG2D / CD16a, IL-2Rβ / 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.

9. The bispecific antibody or multispecific antibody according to claim 6, characterized in that, The multispecific antibody mentioned above is a trispecific antibody; The aforementioned trispecific antibodies are 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, and CD206 / CD16a / Nkp46. EGFR / CD16a / Nkp46, EGFRvIII / CD16a / Nkp46, Fibroblast activating protein (FAP) / CD16a / Nkp46, CA9 / CD16a / Nkp46, MMP-2 / CD16a / Nkp46, PD-L1 / CD16a / Nkp46, SIRPa / CD16a / Nkp46, Trop2 / CD16a / Nkp46, GPC1 / CD16a / Nkp46, GPC3 / CD16a / Nkp46, cMET / CD16a / Nkp46, BCMA / CD16a / Nkp46, VEGFR / CD16a / Nkp46, Cladin18 Trispecific antibodies against .2 / CD16a / Nkp46, CD30 / CD16a / Nkp46, NKG2D / CD16a / Nkp46, IL-2Rβ / 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.

10. The bispecific antibody or multispecific antibody according to claim 6, characterized in that, The multispecific antibody is a trispecific antibody, consisting of 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 FOLR1. 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; 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.

11. The bispecific antibody or multispecific antibody according to claim 10, characterized in that, The second antigen-binding moiety that specifically binds to Nkp46 and the third antigen-binding moiety that specifically binds to FOLR1 are VHH.

12. The bispecific antibody or multispecific antibody according to claim 10, characterized in that, The trispecific antibody does not fuse with Fc.

13. 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 NO: 51 or 47, respectively.

14. A nucleotide molecule encoding a single-domain antibody against CD16a according to any one of claims 1-3, characterized in that: The nucleotide sequences are shown in SEQ ID NO: 10 or 6, respectively, or the amino acid sequence encoded by the nucleotide molecule is the same as the amino acid sequence encoded by SEQ ID NO: 10 or 6.

15. A nucleotide molecule encoding any one of the bispecific or multispecific antibodies of claims 6-13, characterized in that: The nucleotide sequences are shown in SEQ ID NO: 56 or 52, respectively, or the amino acid sequence encoded by the nucleotide molecule is the same as the amino acid sequence encoded by SEQ ID NO: 56 or 52.

16. An expression carrier, characterized in that: It comprises a nucleotide molecule encoding a single-domain antibody against CD16a as described in any one of claims 1 to 3, or an Fc fusion antibody or humanized antibody as described in claim 4, or a nucleotide molecule of a bispecific antibody or multispecific antibody as described in any one of claims 6 to 13, or a nucleotide molecule as described in claim 14 or claim 15.

17. A host cell, characterized in that: It can express the single-domain antibody against CD16a as described in any one of claims 1 to 3, or the Fc fusion antibody or humanized antibody as described in claim 4, or the bispecific antibody or multispecific antibody as described in any one of claims 6 to 13, or the expression vector as described in claim 16.

18. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises a single-domain antibody against CD16a selected from any one of claims 1 to 3 or a bispecific or multispecific antibody as described in any one of claims 6 to 13, and a pharmaceutically acceptable carrier.

19. A medicine for treating diseases, characterized in that: It comprises an anti-CD16a single-domain antibody of any one of claims 1 to 3 or a bispecific antibody or multispecific antibody of any one of claims 6 to 13 as an active ingredient.

20. The use of the trispecific antibody FOLR1 / CD16a / Nkp46 in the preparation of a medicament for treating a disease, characterized in that: The disease described is ovarian cancer; the trispecific antibody FOLR1 / CD16a / Nkp46 is composed of 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 FOLR1; the first antigen-binding portion that specifically binds to CD16a is a single-domain antibody as described in any one of claims 1 to 3.

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