Single-domain antibody against nkp46 and use thereof

Abstract

The present invention relates to a single-domain antibody against Nkp46 and the use thereof. The single-domain antibody is composed of a heavy chain, and the heavy chain comprises a heavy chain CDR1 as shown in any one of SEQ ID NO: 11-SEQ ID NO: 14, a heavy chain CDR2 as shown in any one of SEQ ID NO: 16-SEQ ID NO: 19, and a heavy chain CDR3 as shown in any one of SEQ ID NOs: 21-23. The single-domain antibody specific to Nkp46 is screened out by means of a biological gene engineering technology, and the antibody has relatively good affinity.

Description

A single-domain antibody against Nkp46 and its use Technical Field

[0001] The present invention relates to a single-domain antibody capable of specifically binding to Nkp46 (hereinafter abbreviated as "Nkp46 single-domain antibody"), a pharmaceutical composition containing the single-domain antibody as an active ingredient, and pharmaceutical therapeutic uses thereof. Background Art

[0002] NK cell activity is regulated by a complex mechanism involving activation and inhibition signals. Several different NK specific receptors have been identified, which play an important role in the identification and killing of HLA class I defective target cells mediated by NK cells. Natural cytotoxicity receptors (NCRs) refer to a class of activating receptor proteins specifically expressed in NK cells, and the genes expressing them. Examples of NCRs include NKp30, NKp44, and NKp46.

[0003] NKp46 is a low-affinity, dominant activating transmembrane receptor expressed on NK cells, macrophages, and mast cells. It belongs to the immunoglobulin superfamily of transmembrane receptors. On NK cells, signal transduction occurs through the binding of the FcγRIIIA α chain to the immunoreceptor tyrosine-based activation motif (ITAM) containing the FcεRIγ chain and / or the T cell receptor (TCR) / CD3ζ chain.

[0004] Single-domain antibodies (sdAbs) are currently the smallest antibody molecules, with a molecular weight one-tenth that of intact antibodies. In addition to possessing the antigenic reactivity of intact antibodies, sdAbs also possess unique functional properties, such as small molecular weight, strong stability, good solubility, easy expression, weak immunogenicity, strong penetrability, strong targeting, and low production costs. These properties virtually overcome the drawbacks of traditional antibodies, such as long development cycles, low stability, and demanding storage conditions.

[0005] Therefore, it is particularly necessary to research and develop a high-affinity single-domain (hereinafter referred to as "single-domain") Nkp46 antibody with a small molecular weight, good tumor penetration, and the ability to be freely assembled into a bispecific antibody.

[0006] Summary of the Invention

[0007] The invention purpose of this patent is to provide a single-domain antibody that can specifically bind to Nkp46 and its use.

[0008] The first aspect of the present invention provides a single-domain antibody against Nkp46, which is composed of a heavy chain, and the heavy chain includes a heavy chain CDR1 shown in any one of SEQ ID NO: 11 to SEQ ID NO: 14, a heavy chain CDR2 shown in any one of SEQ ID NO: 16 to SEQ ID NO: 19, and a heavy chain CDR3 shown in any one of SEQ ID NO: 21 to SEQ ID NO: 23.

[0009] Preferably, the amino acid sequence of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 is one of the following (1)-(4):

[0010] (1) CDR1 shown in SEQ ID NO: 13, CDR2 shown in SEQ ID NO: 16, and CDR3 shown in SEQ ID NO: 23;

[0011] (2) CDR1 shown in SEQ ID NO: 12, CDR2 shown in SEQ ID NO: 18, and CDR3 shown in SEQ ID NO: 22;

[0012] (3) CDR1 shown in SEQ ID NO: 11, CDR2 shown in SEQ ID NO: 19, and CDR3 shown in SEQ ID NO: 21;

[0013] (4) CDR1 shown in SEQ ID NO: 14, CDR2 shown in SEQ ID NO: 17, and CDR3 shown in SEQ ID NO: 23;

[0014] The above CDR combinations (1)-(4) correspond to the single-domain antibodies 2B11, 7F10, 5B10 and 1H2, respectively.

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

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

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

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

[0019] The term "anti-Nkp46 single-domain antibody" as used herein includes not only intact single-domain antibodies, but also fragments, derivatives, and analogs of the anti-Nkp46 single-domain antibody. As used herein, the terms "fragment," "derivative," and "analog" have the same meaning and refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, where such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substituent group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence, or a sequence used to purify the polypeptide, or a proprotein sequence, or a fusion protein formed with an Fc tag). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

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

[0021] FR1 or a variant of FR1 as shown in any one of SEQ ID NOs: 25-27, wherein the variant of FR1 comprises up to 5 amino acid substitutions in FR1;

[0022] FR2 or a variant of FR2 set forth in any one of SEQ ID NOs: 29-32, wherein the variant of FR2 comprises up to 5 amino acid substitutions in FR2;

[0023] FR3 or a variant of FR3 set forth in any one of SEQ ID NOs: 34-37, wherein the variant of FR3 comprises up to 5 amino acid substitutions in FR3;

[0024] FR4 or a variant of FR4 shown in SEQ ID NO: 39, wherein the variant of FR4 comprises a substitution of up to 5 amino acids in the FR4.

[0025] The second aspect of the present invention is to provide an amino acid sequence of a single-domain antibody capable of binding to Nkp46, wherein the amino acid sequences of the single-domain antibodies are shown in SEQ ID NOs: 1-4, respectively, or the single-domain antibodies have at least 80% sequence homology with the amino acid sequences of SEQ ID NOs: 1-4 and are capable of specifically binding to the Nkp46 protein, or the amino acid sequence of the single-domain antibody is compared with any one of SEQ ID NOs: 1-4, and at least one amino acid residue in the FR1, FR2, FR3 or FR4 sequence is substituted by a conservative amino acid.

[0026] In one embodiment, the anti-Nkp46 single domain antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with an amino acid sequence selected from SEQ ID NOs: 1-4, and is capable of specifically binding to the Nkp46 protein.

[0027] The third aspect of the present invention is to provide an Fc fusion antibody or a humanized antibody of any of the aforementioned anti-Nkp46 single-domain antibodies.

[0028] The fourth aspect of the present invention is to provide a recombinant protein comprising the aforementioned single-domain antibody against Nkp46. The recombinant protein may be a single-domain antibody as described in SEQ ID NOs: 1-4, or a single-domain antibody with at least 80% homology to SEQ ID NOs: 1-4, or a multi-epitope antibody, a bispecific antibody, a multispecific antibody, or a multivalent antibody. For example, the multi-epitope antibody may be composed of more than one sequence in SEQ ID NOs: 1-4; the multivalent antibody may be composed of one of the sequences in SEQ ID NOs: 1-4 repeated several times; the multispecific antibody includes but is not limited to a trispecific antibody and a tetraspecific antibody. In addition, the recombinant protein may be a fragment, derivative, or analog of the aforementioned antibody.

[0029] The fifth aspect of the present invention is to provide a bispecific antibody or a multispecific antibody comprising any of the aforementioned single-domain antibodies, wherein the single-domain antibody serves as a first antigen-binding portion that specifically binds to Nkp46.

[0030] In a preferred embodiment, the aforementioned bispecific antibody or multispecific antibody further comprises a binding portion that is specific for other tumor antigens other than Nkp46;

[0031] Other tumor antigens besides Nkp46 include FOLR1, CD123, BCMA, CD38, GPC3, B7H3, CD16, CD16a, CD20, IL-2R, IL-2Rβ, nectin-4, CD160, or any other tumor antigen.

[0032] In a preferred embodiment, the bispecific antibodies include Nkp46 / FOLR1, Nkp46 / CD123, Nkp46 / BCMA, Nkp46 / CD38, Nkp46 / GPC3, Nkp46 / B7H3, Nkp46 / CD16, Nkp46 / CD16a, Nkp46 / CD20, Nkp46 / IL-2R, Nkp46 / IL-2Rβ, Nkp46 / nectin-4, and Nkp46 / CD160 bispecific antibodies;

[0033] It may be an Nkp46 / FOLR1 bispecific antibody comprising a second antigen binding portion that specifically binds to FOLR1.

[0034] In a preferred embodiment, the second antigen-binding portion that specifically binds to FOLR1 comprises CDR1 shown in SEQ ID NO: 15, CDR2 shown in SEQ ID NO: 20, and CDR3 shown in SEQ ID NO: 24;

[0035] Preferably, the second antigen binding moiety that specifically binds FOLR1 is a VHH.

[0036] In a preferred embodiment, the amino acid sequences of the bispecific antibodies are shown as SEQ ID NOs: 40-43, respectively.

[0037] In a preferred embodiment, the multispecific antibody is a trispecific antibody, comprising a first antigen-binding portion that specifically binds Nkp46, a second antigen-binding portion that specifically binds FOLR1, and a third antigen-binding portion that specifically binds CD160 or CD16.

[0038] The sixth aspect of the present invention is to provide a nucleotide molecule encoding the aforementioned anti-Nkp46 single domain antibody or the aforementioned Fc fusion antibody or the aforementioned humanized antibody, whose nucleotide sequences are shown in SEQ ID NOs: 6-9, respectively, or the amino acid sequence encoded by the nucleotide sequence is the same as the amino acid sequence encoded by any one of SEQ ID NOs: 6-9, or has at least 95% sequence homology with any one of SEQ ID NOs: 6-9.

[0039] In one embodiment, the nucleic acid molecule encoding the anti-Nkp46 single-domain antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology with a nucleotide sequence selected from SEQ ID NOs: 6-9, and the anti-Nkp46 single-domain antibody encoded thereby can specifically bind to the Nkp46 protein.

[0040] The seventh aspect of the present invention provides a nucleotide molecule encoding the aforementioned bispecific antibody, whose nucleotide sequence is shown in any one of SEQ ID NOs: 44-47, 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: 44-47.

[0041] The eighth aspect of the present invention is to provide an expression vector comprising a nucleotide molecule encoding a single-domain antibody, Fc fusion antibody or humanized antibody, bispecific or multispecific antibody against Nkp46, wherein the nucleotide sequence encoding the single-domain antibody is respectively as described in SEQ ID NO: 6-9 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: 6-9.

[0042] In a preferred embodiment, the expression vector used is RJK-V4-hFC (a nucleotide molecule encoding an anti-Nkp46 single-domain antibody or its Fc fusion antibody or humanized antibody is integrated into RJK-V4-hFC by genetic engineering). Other general expression vectors can also be selected as needed.

[0043] The ninth aspect of the present invention is to provide a host cell capable of expressing the aforementioned anti-Nkp46 single-domain antibody, Fc fusion antibody, humanized antibody, bispecific 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.

[0044] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell, including bacteria and fungi.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of insect cells such as fall armyworm, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or a combination thereof.

[0049] In another preferred embodiment, the host cell is a suspension ExpiCHO-S cell.

[0050] In another preferred embodiment, the host cell is a suspension 293F cell.

[0051] The tenth aspect of the present invention is to provide a pharmaceutical composition comprising the aforementioned single-domain antibody, bispecific antibody, or multispecific antibody that binds to Nkp46, and a pharmaceutically acceptable carrier. Typically, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically determined based on the isoelectric point of the antibody (the pH of the aqueous carrier medium must deviate from the isoelectric point of the antibody by approximately 2). The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous, transdermal (direct application to the affected area or application of a plaster).

[0052] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the aforementioned single-domain antibody and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions.

[0053] The eleventh aspect of the present invention is to provide a drug for treating a disease, which comprises the aforementioned single-domain antibody for binding to the Nkp46 protein or the aforementioned bispecific antibody or multispecific antibody as an active ingredient.

[0054] The twelfth aspect of the present invention is to provide a kit for detecting Nkp46 levels, comprising the aforementioned anti-Nkp46 single-domain antibody, bispecific antibody, or multispecific antibody. In a preferred embodiment of the present invention, the kit further comprises a container, instructions for use, a buffer, and the like.

[0055] The thirteenth aspect of the present invention provides a method for producing a single domain antibody against Nkp46, comprising the steps of:

[0056] (a) culturing the host cell according to the ninth aspect of the present invention under conditions suitable for producing the single domain antibody, thereby obtaining a culture containing the anti-Nkp46 single domain antibody; and

[0057] (b) isolating or recovering the anti-Nkp46 single domain antibody from the culture; and

[0058] (c) Optionally, purifying and / or modifying the single domain antibody against Nkp46 obtained in step (b).

[0059] The fourteenth aspect of the present invention is to provide use of the aforementioned anti-Nkp46 single-domain antibody, the aforementioned bispecific antibody, the aforementioned multispecific antibody, or the aforementioned pharmaceutical composition in the preparation of a drug for treating a disease.

[0060] In a preferred embodiment, the disease is various NK cell-mediated disorders associated with Nkp46.

[0061] In a preferred embodiment, the disease includes but is not limited to tumors, autoimmune diseases, metabolism-related diseases, and infectious diseases.

[0062] In a preferred embodiment, various NK cell-mediated disorders associated with Nkp46 include, but are not limited to, rheumatoid arthritis (RA), bone erosion, intraperitoneal abscess, inflammatory bowel disease, allograft rejection, psoriasis, angiogenesis, atherosclerosis, asthma, multiple sclerosis, systemic lupus erythematosus (SLE), ocular surface disorders (e.g., dry eye), ankylosing spondylitis, psoriatic arthritis, cancer (e.g., multiple myeloma and breast cancer).

[0063] In a preferred embodiment, the tumor includes solid tumors and hematological tumors.

[0064] In a preferred embodiment, tumors include, but are not limited to, tumors of epithelial origin (adenomas and various types of carcinomas, including adenocarcinomas, squamous cell carcinomas, transitional cell carcinomas, and others); bladder and urinary tract cancers; breast cancers; gastrointestinal cancers (including esophageal cancer, stomach cancer, small intestine cancer, colon cancer, rectal cancer, and anal cancer); liver (hepatocellular carcinoma); cancers of the gallbladder and biliary system, exocrine pancreas, and kidney; lung cancers (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchioloalveolar carcinoma, and mesothelioma); head and neck cancers (e.g., tongue cancer, oral cancer, laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, tonsil cancer, salivary gland cancer, nasal cavity cancer, and paranasal sinus cancer); cancers of the ovary, fallopian tube, peritoneum, vagina, vulva, penis, cervix, myometrium, and endometrium; thyroid cancer (e.g., follicular thyroid carcinoma); adrenal, prostate, skin, and adnexal cancers (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthomas, dysplastic nevi); hematologic malignancies (i.e., leukemias, lymphomas) and precancerous and borderline malignant diseases, including hematologic malignancies and disorders of the lymphoid lineage (e.g., acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell lymphomas such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, T-cell lymphomas and leukemias, natural killer (NK) cell lymphomas, Hodgkin lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma); , multiple myeloma, and post-transplant lymphoproliferative disorders) and hematologic malignancies and myeloid-related disorders (e.g., acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), hypereosinophilic syndrome, myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome, and promyelocytic leukemia); tumors of mesenchymal origin, such as sarcomas of soft tissue, bone, or cartilage, such as osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant central or peripheral nervous system tumors (e.g., astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pinealomas, and schwannomas); endocrine tumors (e.g., pituitary tumors, adrenal tumors, pancreatic islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary thyroid carcinoma); ocular and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, seminoma, dysgerminoma, hydatidiform mole, and choriocarcinoma); pediatric and embryonal tumors (e.g., medulloblastoma, neuroblastoma, Wilms tumor, and primitive neuroectodermal tumor); or congenital or other syndromes that predispose patients to malignancy (e.g., xeroderma pigmentosum).

[0065] In a preferred embodiment, the disease includes but is not limited to multiple myeloma, diffuse large B-cell lymphoma, mantle cell lymphoma, marginal zone lymphoma, follicular lymphoma, acute myeloid leukemia, B-cell acute lymphoblastic leukemia, hepatocellular carcinoma, AL amyloidosis, myelodysplastic syndrome, blood disease, type I diabetes. Beneficial effects

[0066] Compared with the prior art, the present invention has the following beneficial effects:

[0067] (1) The single-domain antibody of the present invention is specific for the Nkp46 protein with a correct spatial structure.

[0068] (2) The single-domain antibody against Nkp46 obtained by the present invention has excellent antigen binding ability and specificity, has excellent ability to activate NK cells to release TNFa, and can effectively mediate ADCC effect. This single-domain antibody is combined with other antigen-binding moieties to form a bispecific or multispecific antibody, and can also be used as part of a chimeric antigen receptor (CAR) or assembled into any other form of an antibody.

[0069] The ADCC effect mediated by the bispecific antibody Nkp46 / FOLR1 prepared with it (Nkp46 / FOLR1 is only used as an implementation method, and Nkp46 can also be combined with other tumor surface antigen targets) is significantly enhanced compared to the ADCC effect mediated by the FOLR1 monoclonal antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] In order to more clearly illustrate the technical solution of the present application, the following is a brief introduction to the drawings required for use in the embodiments. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0071] Figure 1 shows the enrichment of the library for screening antibodies targeting Nkp46 in Example 3;

[0072] FIG2 is a graph showing the antibody-antigen binding dose-effect curve of Example 12 (1H2);

[0073] FIG3 is a graph showing the antibody-antigen binding dose-effect curve of Example 12 (2B11);

[0074] FIG4 is a graph showing the antibody-antigen binding dose-effect curve of Example 12 (7F10);

[0075] FIG5 is a graph showing the antibody-antigen binding dose-effect curve of Example 12 (5B10);

[0076] Figure 6 is an experiment showing the antibody-stimulated NK cell release of TNF-a in Example 14 (Tab1, Alemtuzumab, hIgG);

[0077] FIG7 is an experiment showing the antibody-stimulated NK cell release of TNF-a in Example 14 (5B10, 1H2, 7F10);

[0078] FIG8 is an experiment (2B11) showing the antibody-stimulated NK cell release of TNF-a in Example 14;

[0079] Figure 9 shows the ADCC effect mediated by single domain antibodies (Tab1, 4F4);

[0080] FIG10 shows the ADCC effect mediated by Nkp46 / FOLR1 (4F4-1H2, 4F4-2B11, 4F4-5B10, and 4F4-7F10);

[0081] Figure 11 is a schematic diagram of the structure of the bispecific antibody Nkp46 / FOLR1. DETAILED DESCRIPTION

[0082] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0083] As used herein, "single-domain antibodies" (sdAbs, also referred to as nanobodies or VHHs by Ablynx) are well known to those skilled in the art. Single-domain antibodies are antibodies whose complementary determining regions are part of a single domain polypeptide. Thus, single-domain antibodies comprise a single complementary determining region (a single CDR1, a single CDR2, and a single CDR3). Examples of single-domain antibodies are antibodies that have only heavy chains (which naturally do not contain light chains), single-domain antibodies derived from conventional antibodies, and engineered antibodies.

[0084] Single domain antibodies can be derived from any species, including mice, humans, camels, llamas, goats, rabbits and cattle. For example, naturally occurring VHH molecules can be derived from antibodies provided by species of the Camelidae family (e.g., camels, dromedaries, llamas and guanacos). Like complete antibodies, single domain antibodies are capable of selectively binding to specific antigens. Single domain antibodies can contain only the variable domain of an immunoglobulin chain, which has CDR1, CDR2 and CDR3 and a framework region.

[0085] As used herein, the term "sequence homology" refers to the extent to which two (nucleotide or amino acid) sequences have identical residues at identical positions in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the entire length of the sequences being compared. Thus, two copies of identical sequences have 100% homology.

[0086] 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.

[0087] The term "humanized antibody" refers to an antibody obtained by fusing the heavy chain variable region of a target antibody (such as an animal antibody) with the constant region of a human antibody, or by transplanting the complementarity determining regions (CDR1-3 sequences) of a target antibody into the variable region of a human antibody, or by subjecting the target antibody to amino acid mutations based on the characteristics of the human antibody framework region (FR1-4). Humanized antibodies can be produced synthetically or by site-directed mutagenesis.

[0088] In the present invention, sequences with high homology to the CDR1-3 sequences disclosed herein can also be used to generate single-domain antibodies against Nkp46. In some embodiments, sequences with "at least 80% homology," or "at least 85% homology," "at least 90% homology," "at least 95% homology," or "at least 98% homology" to the sequences in SEQ ID NOs: 1-4 can also achieve the objectives of the invention.

[0089] In some embodiments, sequences that replace only one or a few amino acids compared to the sequences in SEQ ID NOs: 1-4, 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 skilled person may consider so-called "conservative" amino acid substitutions. In the case of substitutions, the substitution will preferably be a conservative amino acid substitution, which can generally be described as an amino acid substitution in which an amino acid residue is replaced by another amino acid residue with a similar chemical structure, and the substitution has little or substantially no effect on the function, activity or other biological properties of the polypeptide. The conservative amino acid substitutions are common in the art. For example, conservative amino acid substitutions are substitutions of one or a few amino acids in the following groups (a)-(d) by another or a few amino acids in the same group: (a) polar negatively charged residues and their uncharged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic non-polar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. Particularly preferred conservative amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg or Gln; Phe is replaced by Met, Leu, or Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe or Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr, or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser. In addition, those skilled in the art will appreciate that the creativity of single-domain antibodies is embodied in the CDR1-3 regions, while the framework region sequences FR1-4 are not immutable. The sequences of FR1-4 can be conservative sequence variants of the sequences disclosed in the present invention.

[0090] Preferred host cells of the present invention are bacterial cells, fungal cells or mammalian cells.

[0091] This patent uses genetic engineering technology to prepare target proteins and truncated forms of target proteins, and then immunizes the obtained antigen proteins into Bactrian camels in Alxa League, Inner Mongolia. After multiple immunizations, peripheral blood lymphocytes or spleen cells of the camels are obtained. Through genetic engineering, the camel-derived antibody variable region coding sequence is recombined into a phage display vector. Specific antibodies against the antigen protein are screened through phage display technology, and their ability to bind to the antigen and their application in the treatment of autoimmune diseases are further tested.

[0092] The above technical solution is now broken down into details and described in the form of specific embodiments:

[0093] Example 1: Preparation of human Nkp46 recombinant extracellular domain protein:

[0094] The human recombinant extracellular domain protein used in this patent was obtained through the company's own expression and purification. The expression vector design scheme for the human recombinant Nkp46 protein is as follows:

[0095] (1) The coding sequence of Nkp46 was retrieved from NCBI and its accession number is BC064806.1. The amino acid sequence generated by this sequence is accession number AAH64806.1.

[0096] (2) The nucleotide sequence encoding amino acids 22 to 254 of Nkp46 was cloned into the vector pcDNA3.4 by gene synthesis. The constructed vector was subjected to Sanger sequencing and compared with the original sequence. After confirmation, the recombinant plasmid was extensively extracted to remove endotoxins and transfected into suspension 293F cells for target protein expression and purification. The purity reached over 90%, meeting animal immunization requirements.

[0097] Example 2: Construction of a single domain antibody library against Nkp46 protein:

[0098] 1 mg of the human recombinant Nkp46 protein purified in Example 1 was mixed with an equal volume of Freund's complete adjuvant and used to immunize a Bactrian camel from Alxa, Inner Mongolia, once a week for a total of 7 consecutive immunizations. Except for the first immunization, the remaining six immunizations were performed using 1 mg of Nkp46 protein mixed with an equal volume of Freund's incomplete adjuvant. This immunization process is intended to stimulate the camel to produce antibodies against the Nkp46 protein.

[0099] After the animals were immunized, 150 mL of peripheral blood lymphocytes were collected and RNA was extracted from the cells. The extracted total RNA was used to synthesize cDNA, and the VHH (antibody heavy chain variable region) was amplified using the cDNA as a template through a nested PCR reaction.

[0100] The pMECS vector and VHH fragment were then digested with restriction endonucleases, and the digested fragments and vector were linked. The linked fragments were electroporated into competent cells TG1 to construct a phage display library of Nkp46 protein and determine the library capacity. The library capacity was approximately 1×10 9 At the same time, the correct insertion rate of the target fragment in the library was detected by colony PCR identification.

[0101] 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%.

[0102] Example 3: Screening of single domain antibodies against Nkp46 protein:

[0103] 200 μL of the recombinant TG1 cells in Example 2 were cultured in 2×TY medium, during which 40 μL of helper phage VCSM13 was added to infect the TG1 cells and cultured overnight to amplify the phage. The next day, the phage was precipitated with PEG / NaCl and the amplified phage was collected by centrifugation.

[0104] 500 μg of Nkp46 protein diluted in 100 mM NaHCO3 at pH 8.3 was coupled to an ELISA plate and placed at 4°C overnight. A negative control well (medium control) was also set up. The next day, 200 μL of 3% skim milk was added and the plate was blocked at room temperature for 2 h. After blocking, 100 μL of amplified phage library (approximately 2 × 10 11 phage particles) at room temperature for 1 hour; after 1 hour, the cells were washed 15 times with PBS + 0.05% Tween-20 to remove unbound phage.

[0105] Phages that specifically bound to the Nkp46 protein were dissociated using trypsin at a final concentration of 25 mg / mL and infected with Escherichia coli TG1 cells in the logarithmic growth phase. The cells were cultured at 37°C for 1 hour to produce and collect phages for the next round of screening. The same screening process was repeated for one round to gradually enrich the phages.

[0106] When the enrichment multiple reaches more than 10 times, the enrichment effect is shown in Figure 1.

[0107] In Figure 1, P / N = the number of monoclonal bacteria grown after phage eluted from the positive wells in bio-panning infected with TG1 bacteria / the number of monoclonal bacteria grown after phage eluted from the positive wells infected with TG1 bacteria. This parameter will gradually increase after enrichment occurs; I / E = the total amount of phage added to the positive wells in each round of bio-panning / the total amount of phage eluted from the positive wells in each round of bio-panning. This parameter will gradually approach 1 after enrichment occurs.

[0108] Example 4: Screening of specific positive clones against Nkp46 using phage enzyme-linked immunosorbent assay (ELISA):

[0109] Two rounds of screening were performed for single-domain antibodies against Nkp46 protein according to the screening method in Example 3 above. The phage enrichment factor of anti-Nkp46 protein reached 10 or more. After the screening, 384 single colonies were selected from the positive clones obtained by screening and inoculated into 96-deep-well plates in 2×TY medium containing 100 μg / mL ampicillin. A blank control was set up. After culturing at 37°C to the logarithmic phase, IPTG was added to a final concentration of 1 mM and cultured at 28°C overnight.

[0110] Crude antibody was obtained using the osmotic swelling method. Nkp46 recombinant protein was released into 100 mM NaHCO₃ (pH 8.3), and 100 μg of protein was coated on an ELISA plate overnight at 4°C. 100 μL of the crude antibody extract was transferred to the antigen-added ELISA plate and incubated at room temperature for 1 hour. Unbound antibody was washed with PBST, and 100 μL of a 1:2000 diluted Mouse Anti-HA tag Antibody (HRP) (Thermo Fisher) was added and incubated at room temperature for 1 hour. Unbound antibody was washed with PBST, and horseradish peroxidase colorimetric solution was added. After incubation at 37°C for 15 minutes, stop solution was added, and absorbance was read at 450 nm on a microplate reader.

[0111] When the OD value of the sample well is more than 5 times greater than that of the control well, it is determined to be a positive clone well; the bacteria in the positive clone well are transferred and shaken into LB medium containing 100 μg / mL ampicillin to extract the plasmid and perform sequencing.

[0112] The gene sequences of each clone were analyzed using the sequence alignment software VectorNTI. Strains with identical CDR1, CDR2, and CDR3 sequences were considered the same clone, while strains with different sequences were considered different clones. Finally, a single-domain antibody specific for the Nkp46 protein was obtained.

[0113] The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, which constitutes the entire VHH. The obtained single-domain antibody recombinant plasmid can be expressed in a prokaryotic system to ultimately obtain single-domain antibody proteins (2B11, 7F10, 5B10, 1H2, and 1G10, 3B11, 6H10, 8C5, 8B1 (not shown in the sequence); the antibody clones (not shown in the sequence) appear in Figures 2-5 and 8).

[0114] Similarly, the specific steps of Examples 1-4 were used (only the antigen was replaced with FOLR1) to obtain a single domain antibody (VHH)-4F4 specific for the FOLR1 protein.

[0115] The preparation process of the human FOLR1 recombinant extracellular domain protein is as follows:

[0116] The human recombinant extracellular domain protein used in this patent was obtained through the company's own expression and purification. The expression vector design scheme for the human recombinant FOLR1 protein is as follows:

[0117] (1) The coding sequence of FOLR1 was retrieved from NCBI and its accession number is NM_000802.3. The amino acid sequence generated by this sequence is accession number NP_000793.1.

[0118] (2) The nucleotide sequence encoding amino acids 25 to 233 of FOLR1 was cloned into pcDNA3.4 by gene synthesis. The constructed vector was Sanger sequenced and compared with the original sequence. After confirmation, the recombinant plasmid was extracted in large quantities to remove endotoxins and transfected into suspension 293F cells for target protein expression and purification. The purity reached over 90%, meeting animal immunization requirements.

[0119] The CDR and FR sequences of four single-domain antibodies against Nkp46 and one single-domain antibody against FOLR1 are shown in Tables 1-7, and the amino acid sequences and nucleotide sequences of the single-domain antibodies are shown in Tables 8 and 9, respectively.

[0120] Table 1 CDR1 sequences of single domain antibodies

[0121] Table 2 CDR2 sequences of single domain antibodies

[0122] Table 3 CDR3 sequences of single domain antibodies

[0123] Table 4 FR1 sequences of single domain antibodies

[0124] Table 5 FR2 sequences of single domain antibodies

[0125] Table 6 FR3 sequences of single domain antibodies

[0126] Table 7 FR4 sequences of single domain antibodies

[0127] Table 8 Amino acid sequences of single domain antibodies

[0128] Table 9 Nucleic acid sequences of single domain antibodies

[0129] Example 5: Purification and expression of a specific single domain antibody against Nkp46 protein in Escherichia coli

[0130] The plasmids (pMECS-VHH) of the different clones obtained by sequencing analysis in Example 4 were electroporated into Escherichia coli HB2151 and plated on LB+amp+glucose plates containing ampicillin and glucose, and cultured at 37°C overnight. Single colonies were selected and inoculated into 5 mL of LB culture medium containing ampicillin and cultured in a shaking incubator at 37°C overnight.

[0131] Inoculate 1 mL of overnight culture into 330 mL of TB medium and culture at 37°C with a shaker. When the OD600nm value reaches 0.6-0.9, add 1 M IPTG and culture overnight at 28°C with a shaker. Centrifuge to collect the E. coli and obtain a crude antibody extract using the osmotic swelling method.

[0132] The single domain antibody was purified by nickel column affinity chromatography.

[0133] Example 6: Construction of a eukaryotic expression vector for Fc fusion antibody of a single domain antibody against Nkp46

[0134] (1) Subcloning the target sequence obtained in Example 4 into a eukaryotic expression vector: The antibodies screened in Example 4 were subjected to Sanger sequencing to obtain their nucleotide sequences;

[0135] (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;

[0136] (3) transforming the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culturing and performing plasmid extraction to remove endotoxins;

[0137] (4) Sequencing the extracted plasmid;

[0138] (5) The confirmed recombinant vector is prepared for subsequent eukaryotic cell transfection and expression. The Fc protein of VHH is expressed by the method of Example 8 or 9, and the above-mentioned antibody is purified by the method of Example 10.

[0139] Example 7: Construction of anti-Nkp46 / FOLR1 bispecific antibody eukaryotic expression vector

[0140] (1) The gene sequences of the anti-FOLR1 single-domain antibody (named: 4F4) and the anti-Nkp46 single-domain antibody were synthesized into the vector RJK-V4-3 designed and modified by our company to obtain recombinant eukaryotic expression vectors (i.e., the nucleotide sequences of SEQ ID NOs: 44-47 were cloned into the vectors, respectively). The modification method of the vectors is as described in Example 11;

[0141] (3) transforming the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culturing and performing plasmid extraction to remove endotoxins;

[0142] (4) Sequencing the extracted plasmid;

[0143] (5) The confirmed recombinant vector was prepared for subsequent eukaryotic cell transfection and expression. The bispecific antibody was expressed according to the method of Example 8 or 9 and purified by the method of Example 10. The obtained bispecific antibodies were named 4F4-1H2 (amino acids 1-125 were FOLR1 single domain antibody, amino acids 126-140 were linker GGGGSGGGGSGGGGS, and amino acids 141-261 were Nkp46 single domain antibody), 4F4-2B11 (amino acids 1-125 were FOLR1 single domain antibody, amino acids 126-140 were linker GGGGSGGGSGGGGS, and amino acids 141-261 were Nkp46 single domain antibody). GGGSGGGGS, amino acids 141-270 are Nkp46 single domain antibody), 4F4-5B10 (amino acids 1-125 are FOLR1 single domain antibody, amino acids 126-140 are linker GGGGSGGGGSGGGGS, amino acids 141-260 are Nkp46 single domain antibody), 4F4-7F10 (amino acids 1-125 are FOLR1 single domain antibody, amino acids 126-140 are linker GGGGSGGGGSGGGGS, amino acids 141-267 are Nkp46 single domain antibody), the amino acid sequences are shown in SEQ ID NO: 40 to SEQ ID NO: 43, respectively (Table 10), and the corresponding nucleic acid sequences are shown in SEQ ID NO: 44 to SEQ ID NO: 47, respectively (Table 11).

[0144] The structure of the Nkp46 / FOLR1 bispecific antibody is shown in Figure 11 , in which FOLR1 VHH, Nkp46 VHH and Fc are linked in sequence.

[0145] The linker in this specification is not limited to a specific sequence, and any other flexible or rigid linker used in the prior art for constructing engineered antibodies can be used. Nkp46 VHH and Fc can also be connected by a linker, such as GGGGSGGGGSGGGGS or any other linker.

[0146] Table 10 Amino acid sequences of bispecific antibodies

[0147] Table 11 Nucleic acid sequences of bispecific antibodies

[0148] Example 8: Expression of single domain antibodies against Nkp46 protein in suspension ExpiCHO-S cells

[0149] (1) 3 days before transfection, 2.5×10 5 / mL cell passaging and expansion culture of ExpiCHO-S TM The calculated volume of cells was transferred to a fresh, pre-warmed 120 mL (final volume) of ExpiCHO TM The cells were cultured in a 500 mL shake flask containing expression medium; the cell concentration reached approximately 4 × 10 6 -6×10 6 Viable cells / mL;

[0150] (2) One day before transfection, TM The cells were diluted to a concentration of 3.5 × 10 6 viable cells / mL, and cells were cultured overnight;

[0151] (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;

[0152] (4) Use fresh ExpiCHO preheated to 37℃ TM Dilute the cells to 6 × 10 6 The calculated required volume of cells was transferred to a fresh, pre-warmed 100 mL (final volume) of ExpiCHO TM in a 500 mL shake flask containing expression medium;

[0153] (5) Gently invert to mix ExpiFectamine TM CHO reagent, use 3.7mL OptiPRO TM Dilute ExpiFectamine in culture medium TM CHO reagent, swirl or mix;

[0154] (6) Use 4 mL of refrigerated OptiPRO TM Dilute the plasmid DNA in the culture medium and vortex to mix;

[0155] (7) Incubate the ExpiFectamine CHO / plasmid DNA (the plasmid DNA is the eukaryotic expression vector of the Fc fusion antibody of the single-domain antibody against Nkp46 prepared in Example 6) 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;

[0156] (8) The cells were cultured with shaking at 37°C, 8% CO2, and humidified air;

[0157] (9) On the first day after transfection (18-22 hours later), add 600ul of ExpiFectamine TM CHO Enhancer and 24mL ExpiCHO feed.

[0158] (10) Collect the supernatant approximately 8 days after transfection (cell viability is less than 70%).

[0159] Example 9: Expression of single-domain antibodies against Nkp46 protein in suspension 293F cells

[0160] Recombinant single domain antibody expression experimental process (taking 500mL shake flask as an example):

[0161] (1) 3 days before transfection, 2.5×10 5 For passage and expansion of 293F cells, the calculated volume of cells was transferred to a 500 mL shake flask containing 120 mL (final volume) of fresh pre-warmed OPM-293CD05 Medium. The cell concentration reached approximately 2 × 10 6 -3×10 6 Viable cells / mL.

[0162] (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.

[0163] (3) Dilute the cells to 1×10 6 The calculated volume of cells was transferred to a 500 mL shake flask containing 100 mL (final volume) of fresh pre-warmed culture medium.

[0164] (4) Dilute PEI (1 mg / mL) with 4 mL of Opti-MEM medium and mix thoroughly by vortexing or pipetting. Dilute plasmid DNA (the plasmid DNA is the eukaryotic expression vector for the Fc fusion antibody of the anti-Nkp46 single-domain antibody prepared in Example 6) with 4 mL of Opti-MEM medium, mix thoroughly by vortexing, and filter through a 0.22 μm filter. Incubate at room temperature for 5 min.

[0165] (5) Add the diluted PEI reagent to the diluted DNA and mix thoroughly by inverting. Incubate the PEI / plasmid DNA complex at room temperature for 15-20 minutes, then gently add it to the prepared cell suspension, gently shaking the flask during the addition process.

[0166] (6) The cells were cultured at 37°C, 5% CO2, and shaking at 120 rpm.

[0167] (7) Add 5 mL of OPM-CHO PFF05 feed at 24 h and 72 h after transfection.

[0168] (8) Collect the supernatant approximately 7 days after transfection (cell viability is less than 70%).

[0169] Example 10: Purification of single domain antibodies against Nkp46 protein

[0170] (1) The protein expression supernatant obtained in Example 8 or 9 was filtered through a 0.45 μm disposable filter to remove insoluble impurities;

[0171] (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;

[0172] (3) The filtrate is passed through a Protein A prepacked column at a flow rate of 1 mL / min. During this step, the target protein in the filtrate will bind to the filler.

[0173] (4) washing the impurity proteins bound to the column with low-salt and high-salt buffers;

[0174] (5) Separate the target protein bound to the column with a low pH buffer;

[0175] (6) The eluate was quickly added to a pH 9.0 Tris-HCl solution for neutralization;

[0176] (7) The neutralized protein solution was dialyzed and analyzed by SDS-PAGE to confirm that the protein purity was above 95% and the concentration was above 0.5 mg / mL, and then stored at low temperature for future use.

[0177] Example 11: Construction of single-domain antibody eukaryotic expression vector RJK-V4-hFC

[0178] The target vector RJK-V4-hFC, commonly used for nanobodies, was modified by our company based on the Invitrogen commercial vector pCDNA3.4 (vector data link: https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / pcdna3_4_topo_ta_cloning_kit_man.pdf) by fusing the Fc region of the human IgG1 heavy chain coding sequence. This vector contains the hinge (CH2) and CH3 regions of the IgG1 heavy chain. The specific modification plan is as follows:

[0179] (1) Select the restriction enzyme sites XbaI and AgeI on pcDNA3.4;

[0180] (2) A multiple cloning site (MCS) and a 6×His tag were introduced at the 5′ and 3′ ends of the Fc fragment coding sequence, respectively, by overlapping PCR;

[0181] (3) amplifying the above fragment by PCR using a pair of primers with XbaI and AgeI restriction sites, respectively;

[0182] (4) using restriction endonucleases XbaI and AgeI to digest the recombinant DNA fragments in pcDNA3.4 and (3), respectively;

[0183] (5) The digested vector and the inserted fragment were ligated with T4 ligase, and the ligated product was transformed into Escherichia coli, amplified, and sequenced to obtain a recombinant plasmid.

[0184] Example 12: Antibody antigen binding dose-effect curve determination

[0185] This example was performed using a standard enzyme-linked immunosorbent assay (ELISA) procedure.

[0186] (1) Coat with 50 μL of 1 μg / mL human Nkp46 protein at 4°C overnight.

[0187] (2) Wash the plate; add 200 μL of 5% milk and block at 37°C for 2 h.

[0188] (3) Dilute the VHH to 2 μg / mL, and then dilute the antibody in a 5-fold gradient for a total of 8 concentration gradients. The VHH here refers to the prokaryotically expressed single-domain antibody against Nkp46 protein prepared in Example 5.

[0189] (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.

[0190] (5) Wash the plate; add 50 μL of HRP-Goat anti hIgG secondary antibody and incubate at 37°C for 30 min.

[0191] (6) Wash the plate several times; add 50 μL of TMB that has been restored to room temperature and react at room temperature for 15 minutes in the dark.

[0192] (7) Add 50 μL of stop solution (1N HCl) and read the result using a microplate reader.

[0193] (8) Draw the curve and calculate the EC50, as shown in Figure 2-5, where hIgG refers to the isotype control, an immunoglobulin molecule that does not bind to any target and is purchased commercially.

[0194] As can be seen from the figure, the single-domain antibodies 1H2, 2B11, 5B10, and 7F10 of the present invention all have good affinity and strong specificity for the Nkp46 protein.

[0195] Example 13: Expression and purification of tool antibody (Tab) targeting human Nkp46

[0196] Tab1 used in Examples 14 and 15 of the present invention is the antibody named NKp46-3 mentioned in Figure 2D of US11001629B2. The searched sequence was entrusted to Universal 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). 100 μg of plasmid (40 μg heavy chain + 60 μg light chain) was added per 100 mL of cells and transiently expressed using PEI in 293F cells (culture medium: FreeStyle 293 Expression medium, Thermo, Cat#12338026 + F-68, Thermo, Cat#24040032). 6 to 24 hours after transfection, 5% volume of 10% Peptone (Sigma, Cat#P0521-100G) was added and cultured at 8% CO2 and 130 rpm for approximately 7 to 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 against PBS, the concentration was determined using Nanodrop, the purity was identified using SEC, and the binding ability was verified by indirect ELISA.

[0197] The concentration of Tab1 obtained by this method is not less than 2 mg / ml and the purity is greater than 95%.

[0198] In addition, in Example 14, a positive control, Alemtuzumab, was also used.

[0199] Example 14: Antibody stimulates NK cells to produce TNFa

[0200] (1) Human PBMCs were isolated using a NK cell isolation kit (miltenyi, Cat: 130-050-401, Lot: 5220608838) to obtain primary NK cells;

[0201] (2) NK cells were centrifuged and resuspended in culture medium (containing 10 ng / ml 1L-2). 50 μL of cells were injected into the wells (1E5 cells / well). Tab1, Alemtuzumab, hlgG and the single domain antibody clones of the present invention (5B10, 7F10, 1H2, 2B11, and the single domain antibody clone 8B1 whose sequence is not shown) were added respectively. The set concentrations of each sample are shown in the table below.

[0202] (3) Incubate at 37°C for 24 hours.

[0203] (4) The cells were centrifuged, the supernatant was collected, and the TNF-a level was detected using an HTRF kit.

[0204] The concentration settings and results are shown in Tables 12-15:

[0205] Table 12 Alemtuzumab concentration settings

[0206] Table 13 Tab1 concentration settings

[0207] Table 14 Single domain antibody concentration settings

[0208] Table 15 hIgG concentration settings

[0209] The experimental results of antibody stimulation of NK cells to release TNFa are shown in Figures 6-8; it can be seen that NK cells can effectively respond to all single-domain antibodies of the present invention to produce TNFa.

[0210] Example 15: ADCC effect of Nkp46 / FOLR1 dual antibody

[0211] The ADCC effect of the Nkp46 / FOLR1 dual antibody of the present invention was determined using the LDH method, and the steps are as follows:

[0212] (1) SK-OV-3 cells at passage 3-4 after recovery were collected and plated into 96-well plates at 10,000 cells per well;

[0213] (2) Tab1 and the antibody sample VHH-hFc were prepared into a solution with a maximum concentration of 10 μg / mL and then diluted 10-fold to obtain 7 concentrations;

[0214] (3) Add the gradient diluted antibody solution to the cell culture wells in equal volumes of the cell suspension;

[0215] (4) For sample wells and E / T wells (antibody concentration is 0), collect PBMC cells and add 250,000 cells per well to the cell culture wells at twice the volume of the target cell suspension; for MAX wells, add lysis buffer twice the volume of the target cell suspension to each well; for MIN wells, add assay buffer twice the volume of the target cell suspension to each well;

[0216] (5) After 6 h of incubation, cell killing was detected using an LDH kit and the absorbance was read;

[0217] (6) Target cell killing rate % = (sample - E / T) / (MAX - MIN);

[0218] (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.

[0219] As shown in Figures 9 and 10 , it can be seen from Figures 9 and 10 that the bispecific antibodies (4F4-1H2, 4F4-2H11, 4F4-5H10, and 4F4-7F10) all have stronger ADCC effects than 4F4 and Tab1.

[0220] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the scope of protection of the present invention.

Claims

1. A single domain antibody against Nkp46, characterized in that: The single-domain antibody is composed of a heavy chain, which includes a heavy chain CDR1 shown in any one of SEQ ID NO:11-SEQ ID NO:14, a heavy chain CDR2 shown in any one of SEQ ID NO:16-SEQ ID NO:19, and a heavy chain CDR3 shown in any one of SEQ ID NO:21-SEQ ID NO:

23.

2. The single domain antibody of Nkp46 according to claim 1, characterized in that: The amino acid sequence of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 is one of the following (1)-(4): (1) CDR1 shown in SEQ ID NO: 13, CDR2 shown in SEQ ID NO: 16, and CDR3 shown in SEQ ID NO: 23; (2) CDR1 shown in SEQ ID NO: 12, CDR2 shown in SEQ ID NO: 18, and CDR3 shown in SEQ ID NO: 22; (3) CDR1 shown in SEQ ID NO:11, CDR2 shown in SEQ ID NO:19, and CDR3 shown in SEQ ID NO:21; (4) CDR1 shown in SEQ ID NO: 14, CDR2 shown in SEQ ID NO: 17, and CDR3 shown in SEQ ID NO: 23; 3. The single domain antibody against Nkp46 according to claim 1, characterized in that: The single domain antibody further comprises a framework region FR; the framework region FR comprises the amino acid sequences of FR1, FR2, FR3 and FR4; the amino acid sequences of the framework region FR are respectively: FR1 or a variant of FR1 as shown in any one of SEQ ID NOs: 25-27, 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: 29-32, 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:34-37, 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:39, wherein the variant of FR4 comprises a substitution of up to 5 amino acids in the FR4.

4. A single domain antibody against Nkp46, characterized in that: The amino acid sequence of the single-domain antibody is shown in any one of SEQ ID NOs: 1-4, or the amino acid sequence of the single-domain antibody is compared with any one of SEQ ID NOs: 1-4, and at least one amino acid residue in FR1, FR2, FR3 or FR4 sequence is replaced by a conservative amino acid. 5 . The Fc fusion antibody or humanized antibody of the anti-Nkp46 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-Nkp46 single domain antibody according to any one of claims 1 to 4.

7. A bispecific antibody or a multispecific antibody, characterized in that: It comprises the single domain antibody according to any one of claims 1 to 4 as a first antigen binding part that specifically binds to Nkp46.

8. A bispecific antibody or multispecific antibody according to claim 7, characterized in that: It also contains binding portions that are specific for other tumor antigens besides Nkp46; Preferably, other tumor antigens besides Nkp46 include FOLR1, CD123, BCMA, CD38, GPC3, B7H3, CD16, CD16a, CD20, IL-2R, IL-2Rβ, nectin-4, CD160 or any other tumor antigen.

9. A bispecific antibody or multispecific antibody according to claim 7, characterized in that: The bispecific antibodies include Nkp46 / FOLR1, Nkp46 / CD123, Nkp46 / BCMA, Nkp46 / CD38, Nkp46 / GPC3, Nkp46 / B7H3, Nkp46 / CD16, Nkp46 / CD16a, Nkp46 / CD20, Nkp46 / IL-2R, Nkp46 / IL-2Rβ, Nkp46 / nectin-4, and Nkp46 / CD160 bispecific antibodies; It may be a Nkp46 / FOLR1 bispecific antibody comprising a second antigen binding portion that specifically binds to FOLR1.

10. A bispecific antibody or multispecific antibody according to claim 9, characterized in that: The second antigen binding portion that specifically binds to FOLR1 includes CDR1 shown in SEQ ID NO: 15, CDR2 shown in SEQ ID NO: 20, and CDR3 shown in SEQ ID NO: 24; Preferably, the second antigen binding moiety that specifically binds FOLR1 is a VHH.

11. A bispecific antibody or multispecific antibody according to claim 10, characterized in that: The amino acid sequences of the bispecific antibodies are shown in SEQ ID NOs: 40-43, respectively.

12. A bispecific antibody or multispecific antibody according to claim 8, wherein the multispecific antibody is a trispecific antibody, comprising a first antigen-binding portion that specifically binds to Nkp46, a second antigen-binding portion that specifically binds to FOLR1, and a third antigen-binding portion that specifically binds to CD160 or CD16.

13. A nucleotide molecule encoding the anti-Nkp46 single domain antibody according to any one of claims 1 to 4, characterized in that: The nucleotide sequence is shown in any one of SEQ ID NOs: 6-9, 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: 6-9.

14. A nucleotide molecule encoding the bispecific antibody according to any one of claims 7 to 11, characterized in that: The nucleotide sequence is shown in any one of SEQ ID NOs: 44-47, 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: 44-47.

15. An expression vector, characterized in that: It comprises a nucleotide molecule encoding the anti-Nkp46 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 the bispecific antibody or multispecific antibody according to any one of claims 7 to 12, or the nucleotide molecule according to claim 13 or 14.

16. A host cell, characterized in that: It can express the single domain antibody against Nkp46 according to any one of claims 1 to 4, or the Fc fusion antibody or humanized antibody according to claim 5, or the bispecific antibody or multispecific antibody according to any one of claims 7 to 12, or it contains the expression vector according to claim 15.

17. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises an anti-Nkp46 single domain antibody selected from any one of claims 1 to 4 or a bispecific antibody or a multispecific antibody according to any one of claims 7 to 12, and a pharmaceutically acceptable carrier.

18. A medicament for treating a disease, characterized in that: It comprises the anti-Nkp46 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-Nkp46 single domain antibody according to any one of claims 1 to 4, the bispecific antibody or multispecific antibody according to any one of claims 7 to 12, or the pharmaceutical composition according to claim 17 in the preparation of a medicament for treating a disease.

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

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