A single-domain antibody against Nkp46 and its uses

Abstract

This invention belongs to the field of immunology and relates to a single-domain antibody against Nkp46 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:14, heavy chain CDR2 shown in any one of SEQ ID NO:16-SEQ ID NO:19, and heavy chain CDR3 shown in any one of SEQ ID NO:21-23. 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 Nkp46, exhibiting good antibody affinity.

Description

Technical Field

[0001] This invention relates to a single-domain antibody (hereinafter abbreviated as "Nkp46 single-domain antibody") capable of specifically binding to Nkp46, a pharmaceutical composition containing the single-domain antibody as an active ingredient, and its use in pharmaceutical therapy. Background Technology

[0002] NK cell activity is regulated by complex mechanisms involving activation and inhibition signals. Several distinct NK-specific receptors have been identified, playing crucial roles in NK cell-mediated recognition and killing of HLA-I class defective target cells. Natural cytotoxic receptors (NCRs) refer to a class of activated receptor proteins specifically expressed in NK cells, along with the genes expressing them. Examples of NCRs include NKp30, NKp44, and NKp46.

[0003] Nkp46 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.

[0004] 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, sdAbs 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.

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

[0006] The purpose of this patent is to provide a single-domain antibody that can specifically bind to Nkp46 and its uses.

[0007] A first aspect of the present invention provides a single-domain antibody against Nkp46, 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:14, heavy chain CDR2 shown in any one of SEQ ID NO:16-SEQ ID NO:19, and heavy chain CDR3 shown in any one of SEQ ID NO:21-SEQ ID NO:23.

[0008] Preferably, the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are one of the following (1)-(4):

[0009] (1) CDR1 shown in SEQ ID NO: 13, CDR2 shown in SEQ ID NO: 16, and CDR3 shown in SEQ ID NO: 23;

[0010] (2) CDR1 shown in SEQ ID NO: 12, CDR2 shown in SEQ ID NO: 18, and CDR3 shown in SEQ ID NO: 22;

[0011] (3) CDR1 shown in SEQ ID NO: 11, CDR2 shown in SEQ ID NO: 19, and CDR3 shown in SEQ ID NO: 21;

[0012] (4) CDR1 shown in SEQ ID NO: 14, CDR2 shown in SEQ ID NO: 17, and CDR3 shown in SEQ ID NO: 23;

[0013] The above CDR combinations (1)-(4) correspond to the single-domain antibodies 2B11, 7F10, 5B10 and 1H2 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-electrolyte 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 "single-domain antibody against Nkp46" in this invention includes not only complete single-domain antibodies but also fragments, derivatives, and analogs of such anti-Nkp46 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] FR1 or a variant of FR1 shown in any of SEQ ID NO: 25-27, wherein the variant of FR1 contains substitutions of up to 5 amino acids in the FR1;

[0021] The FR2 or a variant thereof shown in any one of SEQ ID NO:29-32, wherein the variant thereof contains a substitution of up to 5 amino acids in the FR2;

[0022] FR3 or a variant thereof shown in any one of SEQ ID NO:34-37, wherein the variant thereof contains a substitution of up to 5 amino acids in the FR3;

[0023] The FR4 or a variant thereof shown in SEQ ID NO: 39, wherein the variant thereof contains substitutions of up to 5 amino acids.

[0024] A second aspect of the invention is to provide an amino acid sequence of a single-domain antibody capable of binding Nkp46, wherein the amino acid sequences of the single-domain antibody are as shown in SEQ ID NO: 1-4, or the single-domain antibody has at least 80% sequence homology with the amino acid sequences of SEQ ID NO: 1-4 and is capable of specifically binding Nkp46 protein, or the amino acid sequence of the single-domain antibody, compared with any one of SEQ ID NO: 1-4, 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-Nkp46 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: 1-4, and is capable of specifically binding to the Nkp46 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 Nkp46.

[0027] A 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 shown in SEQ ID NO: 1-4, or a single-domain antibody having at least 80% homology with those in SEQ ID NO: 1-4, 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: 1-4; the multivalent antibody may consist of one sequence in SEQ ID NO: 1-4 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 analogue of the aforementioned antibody.

[0028] A fifth aspect of the present invention is to provide a bispecific antibody or a multispecific antibody comprising any of the aforementioned single-domain antibodies, the single-domain antibody serving as a first antigen-binding portion that specifically binds to Nkp46.

[0029] In a preferred embodiment, the aforementioned bispecific or multispecific antibody further includes a binding portion that specifically binds to tumor antigens other than Nkp46.

[0030] 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.

[0031] In a preferred embodiment, the bispecific antibody includes 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.

[0032] It can be an Nkp46 / FOLR1 bispecific antibody, including a second antigen-binding moiety that specifically binds to FOLR1.

[0033] In a preferred embodiment, 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;

[0034] Preferably, the second antigen-binding portion that specifically binds to FOLR1 is VHH.

[0035] In a preferred embodiment, the amino acid sequences of the bispecific antibodies are shown in SEQ ID NO:40-43, respectively.

[0036] In a preferred embodiment, 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.

[0037] A sixth aspect of the present invention is to provide a nucleotide molecule encoding the aforementioned anti-Nkp46 single-domain antibody, the aforementioned Fc fusion antibody, or the aforementioned humanized antibody, wherein the nucleotide sequences are as shown in SEQ ID NO: 6-9, or the amino acid sequences encoded by the nucleotide sequences are the same as those encoded by any one of SEQ ID NO: 6-9, or have at least 95% sequence homology with any one of SEQ ID NO: 6-9.

[0038] In one embodiment, the nucleic acid molecule encoding the single-domain antibody against Nkp46 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: 6-9, and the single-domain antibody against Nkp46 encoded therein is capable of specifically binding to the Nkp46 protein.

[0039] A seventh aspect of the present invention provides a nucleotide molecule encoding the aforementioned bispecific antibody, wherein the nucleotide sequence is shown in any one of SEQ ID NO: 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 NO: 44-47.

[0040] An eighth aspect of the present invention is to provide an expression vector comprising a nucleotide molecule encoding a single-domain antibody, an Fc fusion antibody or a humanized antibody, a bispecific or multispecific antibody against Nkp46, wherein the nucleotide sequence encoding the single-domain antibody is as shown 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.

[0041] In a preferred embodiment, the expression vector used is RJK-V4-hFC (a nucleotide molecule encoding a single-domain antibody against Nkp46 or its Fc fusion antibody or humanized antibody is integrated into RJK-V4-hFC through genetic engineering). Other universal expression vectors may also be selected as needed.

[0042] A 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. Preferably, the host cell is a bacterial cell, fungal cell, or mammalian cell.

[0043] In another preferred embodiment, the host cell includes prokaryotic or eukaryotic cells, including bacteria and fungi.

[0044] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0045] 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.

[0046] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of: Pichia pastoris, Saccharomyces cerevisiae, and Schizosoma.

[0047] Trichoderma, or combinations thereof.

[0048] 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.

[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] A tenth aspect of the invention is to provide a pharmaceutical composition comprising the aforementioned single-domain antibody binding to Nkp46, the aforementioned bispecific antibody, or the aforementioned 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).

[0052] 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.

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

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

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

[0056] (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...

[0057] Obtain a culture containing the single-domain antibody against Nkp46; and

[0058] (b) Isolating or recovering the single-domain antibody against Nkp46 from the culture; and

[0059] (c) Optionally, purify and / or modify the single-domain antibody against Nkp46 obtained in step (b).

[0060] The fourteenth aspect of the present invention is the use of the aforementioned anti-Nkp46 single-domain antibody, or the aforementioned bispecific antibody, or multispecific antibody, or the aforementioned pharmaceutical composition in the preparation of a medicament for treating a disease.

[0061] In a preferred embodiment, the disease is a variety of NK cell-mediated Nkp46-related conditions.

[0062] In a preferred embodiment, the disease includes, but is not limited to, tumors, autoimmune diseases, metabolic-related diseases, and infectious diseases.

[0063] In a preferred embodiment, various NK cell-mediated Nkp46-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).

[0064] In a preferred embodiment, the tumor includes solid tumors and hematomas.

[0065] 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 cancer (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). Cancers related to the ovaries, fallopian tubes, peritoneum, vagina, vulva, penis, cervix, myometrium, and endometrium; thyroid cancer (e.g., follicular thyroid carcinoma); cancers related to the adrenal glands, prostate, skin, and adnexa (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 lymphoma such as diffuse large B-cell lymphoma). Cellular lymphomas (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) as well as 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 tumors, Sarcomas of 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 histiocytomas, 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); tumors of the eye and adnexa (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 patients to malignant tumors (e.g., xeroderma pigmentosum).

[0066] In a preferred embodiment, the diseases include, but are 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, hematologic disorders, and type I diabetes.

[0067] Beneficial effects

[0068] Compared with the prior art, the beneficial effects of the present invention are:

[0069] (1) The single-domain antibody of the present invention is specifically targeted at the Nkp46 protein with the correct spatial structure.

[0070] (2) The single-domain antibody against Nkp46 obtained in this invention has excellent antigen-binding ability and specificity, excellent ability to activate NK cells to release TNF-α, and can effectively mediate 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.

[0071] The ADCC effect mediated by the bispecific antibody Nkp46 / FOLR1 (Nkp46 / FOLR1 is only one implementation method; Nkp46 can also be combined with other tumor surface antigen targets) significantly enhanced the ADCC effect compared to the ADCC effect mediated by the FOLR1 monoclonal antibody. Attached Figure Description

[0072] 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.

[0073] Figure 1 The enrichment status of the library for screening antibodies targeting Nkp46 in Example 3;

[0074] Figure 2 The antibody-antigen binding dose-response curve determination diagram (1H2) in Example 12;

[0075] Figure 3 This is the antibody-antigen binding dose-response curve determination diagram (2B11) from Example 12.

[0076] Figure 4 This is the antibody-antigen binding dose-response curve determination diagram (7F10) from Example 12.

[0077] Figure 5This is the antibody-antigen binding dose-response curve determination diagram (5B10) from Example 12.

[0078] Figure 6 The antibody-stimulated NK cell release TNF-α assay in Example 14 (Tab1, Alemtuzumab, hIgG).

[0079] Figure 7 The antibody-stimulated NK cell release TNF-α experiment in Example 14 (5B10, 1H2, 7F10).

[0080] Figure 8 This refers to the antibody-stimulated NK cell release of TNF-α experiment in Example 14 (2B11).

[0081] Figure 9 This is a single-domain antibody-mediated ADCC effect (Tab1, 4F4).

[0082] Figure 10 The ADCC effect mediated by Nkp46 / FOLR1 (4F4-1H2, 4F4-2B11, 4F4-5B10 and 4F4-7F10).

[0083] Figure 11 This is a schematic diagram of the structure of the bispecific antibody Nkp46 / FOLR1. Detailed Implementation

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] In this invention, sequences with high homology to the CDR1-3 sequences disclosed herein can also yield single-domain antibodies against Nkp46. 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: 1-4 can achieve the purpose of the invention.

[0091] 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: 1-4, 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 peptide. 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-electrolyte 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.

[0092] The preferred host cell of the present invention is a bacterial cell, a fungal cell, or a mammalian cell.

[0093] 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.

[0094] The above technical solution will now be broken down and explained in detail, and described with specific embodiments:

[0095] Example 1: Preparation of recombinant extracellular domain protein of human Nkp46:

[0096] 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:

[0097] (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.

[0098] (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.

[0099] Example 2: Construction of a single-domain antibody library targeting the Nkp46 protein:

[0100] One mg of the purified human recombinant Nkp46 protein obtained in Example 1 was mixed with an equal volume of Freund's complete adjuvant and used to immunize a Bactrian camel from Alashan, Inner Mongolia. The camel was immunized once a week for a total of 7 weeks. Except for the first immunization, the remaining six immunizations were performed by mixing one mg of Nkp46 protein with an equal volume of Freund's incomplete adjuvant. This immunization process was intended to concentrate the stimulation of the camel to produce antibodies against Nkp46 protein.

[0101] 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.

[0102] 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 the Nkp46 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.

[0103] 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%.

[0104] Example 3: Screening for single-domain antibodies against Nkp46 protein:

[0105] 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.

[0106] 500 μg of Nkp46 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.

[0107] Phages that specifically bind to Nkp46 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.

[0108] When the enrichment factor reaches 10 times or more, the enrichment effect is as follows: Figure 1 As shown.

[0109] Figure 1In 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.

[0110] Example 4: Screening for Nkp46-specific positive clones using phage enzyme-linked immunosorbent assay (ELISA):

[0111] Two rounds of screening were conducted on single-domain antibodies against the anti-Nkp46 protein according to the screening method in Example 3 above. The phage enrichment factor against the anti-Nkp46 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.

[0112] Crude antibody was obtained using the osmotic burst method. Nkp46 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 and 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.

[0113] 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 in order to extract plasmids and perform sequencing.

[0114] 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 Nkp46 protein was obtained.

[0115] 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, ultimately yielding single-domain antibody proteins (2B11, 7F10, 5B10, 1H2 and sequences not shown: 1G10, 3B11, 6H10, 8C5, 8B1; antibody clones with sequences not shown are...). Figures 2-5 , Figure 8 (Appeared in China).

[0116] Similarly, using the specific steps of Examples 1-4 (only the antigen was replaced with FOLR1), a single-domain antibody (VHH)-4F4 specifically targeting the FOLR1 protein was obtained.

[0117] The preparation process of the recombinant human FOLR1 extracellular domain protein is as follows:

[0118] 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:

[0119] (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.

[0120] (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.

[0121] 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 and nucleotide sequences of the single-domain antibodies are shown in Tables 8 and 9, respectively.

[0122] Table 1. CDR1 sequences of single-domain antibodies

[0123]

[0124] Table 2. CDR2 sequences of single-domain antibodies

[0125]

[0126] Table 3. CDR3 sequences of single-domain antibodies

[0127]

[0128] Table 4. FR1 sequences of single-domain antibodies

[0129]

[0130] Table 5. FR2 sequences of single-domain antibodies

[0131]

[0132] Table 6. FR3 sequences of single-domain antibodies

[0133]

[0134] Table 7. FR4 sequences of single-domain antibodies

[0135]

[0136] Table 8. Amino acid sequences of single-domain antibodies

[0137]

[0138] Table 9. Nucleic acid sequences of single-domain antibodies

[0139]

[0140] Example 5: Purification and expression of a specific single-domain antibody against Nkp46 protein in the host bacterium *Escherichia coli*.

[0141] 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.

[0142] 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.

[0143] Single-domain antibodies were purified by nickel column affinity chromatography.

[0144] Example 6: Construction of a eukaryotic expression vector for an Fc fusion antibody against a single domain antibody of anti-Nkp46

[0145] (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;

[0146] (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.

[0147] (3) Transform the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culture it for plasmid extraction, and remove endotoxins;

[0148] (4) The extracted plasmids were then sequenced and identified.

[0149] (5) After confirming that the recombinant vector is correct, prepare it for subsequent eukaryotic cell transfection and expression. After expressing the Fc protein of VHH by the method of Example 8 or 9, purify the above antibody by the method of Example 10.

[0150] Example 7: Construction of eukaryotic expression vector for anti-Nkp46 / FOLR1 bispecific antibody

[0151] (1) The gene sequences of the single-domain antibody against FOLR1 (named: 4F4) and the single-domain antibody against Nkp46 were synthesized into the vector RJK-V4-3 designed and modified by our company by sequence synthesis to obtain a recombinant eukaryotic expression vector (i.e., the nucleotide sequences of SEQ ID NO:44-47 were cloned into the vector respectively). The modification method of the vector is as described in Example 11.

[0152] (3) Transform the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culture it for plasmid extraction, and remove endotoxins;

[0153] (4) The extracted plasmids were then sequenced and identified.

[0154] (5) After confirming the accuracy of the recombinant vector, prepare it for subsequent eukaryotic cell transfection and expression. After expressing the bispecific antibody according to the method in Example 8 or 9, purify the bispecific antibody according to the method in Example 10. The obtained bispecific antibodies are named as follows: 4F4-1H2 (amino acids 1-125 are FOLR1 single-domain antibody, amino acids 126-140 are linker GGGGSGGGGSGGGGS, amino acids 141-261 are Nkp46 single-domain antibody) and 4F4-2B11 (amino acids 1-125 are FOLR1 single-domain antibody, amino acids 126-140 are linker GGGGSG). The amino acid sequences of 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), and 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) are shown in SEQ ID NO:40-SEQ ID NO:43 (Table 10), and their corresponding nucleic acid sequences are shown in SEQ ID NO:44-SEQ ID NO:47 (Table 11).

[0155] The structure of the Nkp46 / FOLR1 bispecific antibody is as follows: Figure 11 As shown, FOLR1 VHH, Nkp46 VHH, and Fc are connected in sequence.

[0156] The adapters used in this specification are not limited to specific sequences; any other flexible or rigid adapters used in the prior art for constructing engineered antibodies may be applicable. Nkp46 VHH and Fc can also be connected via adapters, such as GGGGSGGGGSGGGGS or any other adapter.

[0157] Table 10. Amino acid sequences of the bispecific antibodies

[0158]

[0159] Table 11 Nucleic acid sequences of the bispecific antibodies

[0160]

[0161] Example 8: Expression of single-domain antibody against Nkp46 protein in suspension ExpiCHO-S cells

[0162] (1) Three days before transfection, use 2.5×10 5Passage and expand the culture of ExpiCHO-S™ cells at / mL, and transfer the calculated desired cell volume to a 500mL shake flask containing 120mL (final volume) of fresh, preheated ExpiCHO™ expression medium to achieve a cell concentration of approximately 4×10⁶ cells / mL. 6 -6×10 6 live cells / mL;

[0163] (2) One day before transfection, dilute ExpiCHO-S™ cells to a concentration of 3.5 × 10⁻⁶. 6 Live cells / mL, incubate cells overnight;

[0164] (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;

[0165] (4) Dilute the cells to 6 × 10⁶ using fresh ExpiCHO™ expression medium preheated to 37°C. 6 viable cells / mL. The calculated desired cell volume was transferred to a 500 mL shake flask containing 100 mL (final volume) of fresh, preheated ExpiCHO™ expression medium;

[0166] (5) Gently invert to mix the ExpiFectamine™CHO reagent, dilute the ExpiFectamine™CHO reagent with 3.7 mL of OptiPRO™ medium, and swirl or mix.

[0167] (6) Dilute the plasmid DNA with 4 mL of refrigerated OptiPRO™ medium and vortex to mix.

[0168] (7) Incubate the ExpiFectamine CHO / plasmid DNA (the plasmid DNA is the Fc fusion antibody eukaryotic expression vector of the anti-Nkp46 single-domain antibody prepared in Example 6) 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;

[0169] (8) Incubate the cells with shaking in humidified air at 37°C and 8% CO2;

[0170] (9) On day 1 after transfection (18-22 hours later), add 600ul ExpiFectamine™CHO Enhancer and 24mL ExpiCHO feed.

[0171] (10) Collect the supernatant about 8 days after transfection (when cell viability is less than 70%).

[0172] Example 9: Expression of single-domain antibody against Nkp46 protein in suspension 293F cells

[0173] Recombinant single-domain antibody expression experimental procedure (taking a 500mL shake flask as an example):

[0174] (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-293 CD05 Medium to achieve a cell concentration of approximately 2×10⁻⁶. 6 -3×10 6 Live cells / mL.

[0175] (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.

[0176] (3) Dilute the cells to 1×10⁻⁵ using preheated OPM-293 CD05 Medium. 6 1 live cells / mL. Calculate the required cell volume and transfer it to a 500mL shake flask containing 100mL (final volume) of fresh, preheated culture medium.

[0177] (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-Nkp46 single-domain antibody prepared in Example 6) with 4 mL Opt-MEM medium, mix by swirling, and filter with a 0.22 μm filter. Incubate at room temperature for 5 min.

[0178] (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, and then gently add it to the prepared cell suspension while gently shaking the flask during the addition process.

[0179] (6) Incubate the cells at 37°C, 5% CO2, and 120 rpm with shaking.

[0180] (7) Add 5 mL of OPM-CHO PFF05 feed at 24 h and 72 h after transfection.

[0181] (8) Collect the supernatant about 7 days after transfection (when cell viability is less than 70%).

[0182] Example 10: Purification of single-domain antibody against Nkp46 protein

[0183] (1) The protein expression supernatant obtained in Example 8 or 9 was filtered with a 0.45 μm disposable filter to remove insoluble impurities;

[0184] (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.

[0185] (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.

[0186] (4) Wash the impurity proteins bound to the column with low-salt and high-salt buffers;

[0187] (5) Use a low pH buffer to precipitate the target protein bound on the column;

[0188] (6) Quickly add the eluent to a Tris-HCl solution with a pH of 9.0 to neutralize it;

[0189] (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.

[0190] Example 11: Construction of the single-domain antibody eukaryotic expression vector RJK-V4-hFC

[0191] 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:

[0192] (1) Select the restriction enzyme sites XbaI and AgeI on pcDNA3.4;

[0193] (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.

[0194] (3) The above fragment was amplified by PCR using a pair of primers with XbaI and AgeI restriction sites respectively;

[0195] (4) The recombinant DNA fragments in pcDNA3.4 and (3) were digested with restriction endonucleases XbaI and AgeI, respectively;

[0196] (5) The digested vector and the insert fragment were ligated by T4 ligase, and the ligation product was then transformed into E. coli, amplified, sequenced and verified to obtain the recombinant plasmid.

[0197] Example 12: Determination of Antibody-Antigen Binding Dose-Reaction Curve

[0198] This embodiment uses the standard enzyme-linked immunosorbent assay (ELISA) procedure.

[0199] (1) Coat 50 μL of 1 μg / mL human Nkp46 protein and incubate overnight at 4°C.

[0200] (2) Wash the plate; add 200 μL of 5% milk and seal at 37°C for 2 hours.

[0201] (3) Dilute VHH to 2ug / mL, and then perform a 5-fold serial dilution of the antibody to a total of 8 concentration gradients. Here, VHH refers to the prokaryotic expression of the single-domain antibody against Nkp46 protein prepared in Example 5.

[0202] (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.

[0203] (5) Wash the plate; add 50 μL of HRP-Goat anti hIgG secondary antibody and incubate at 37°C for 30 min.

[0204] (6) Wash the plate (wash several times); add 50 μL of TMB that has been brought back to room temperature, and react at room temperature in the dark for 15 min.

[0205] (7) Add 50 μL of stop solution (1N HCl) and save the microplate reader reading.

[0206] (8) Plot the curve and calculate EC50, as follows: Figure 2-5 As shown, hIgG refers to the isotype control, an immunoglobulin molecule that does not bind to any target and is obtained through commercial purchase.

[0207] As shown in the figure, the single-domain antibodies 1H2, 2B11, 5B10, and 7F10 of the present invention all have good affinity and strong specificity for Nkp46 protein.

[0208] Example 13: Expression and purification of a tool antibody (Tab) targeting human Nkp46

[0209] Tab1 used in embodiments 14 and 15 of this invention is US11001629B2 patent. Figure 2 The antibody named NKp46-3 involved in D had its sequence optimized for a mammalian cell expression system by Universal Biosystems (Anhui) Co., Ltd., and then cloned into the pcDNA3.1 vector. After antibiotic screening, plasmid-positive bacteria were selected for amplification, and plasmids were extracted using a plasmid extraction kit (Macherey Nagel, Cat#740412.50). Add 100 μg plasmid (40 μg heavy chain + 60 μg light chain) per 100 mL of cells and transiently transfect 293F cells (medium: FreeStyle 293 Expressionmedium, Thermo, Cat#12338026 + F-68, Thermo, Cat#24040032) using PEI. 6–24 hours after transfection, add 5% volume of 10% Peptone (Sigma, Cat#P0521-100G) and culture at 8% CO2 130 rpm for approximately 7–8 days. When cell viability drops to 50%, collect the expression supernatant and purify it using a gravity column with Protein A (GE, Cat#17-5438-02). After dialysis with PBS, determine the concentration using Nanodrop, identify purity using SEC, and verify binding ability using indirect ELISA.

[0210] The Tab1 obtained by this method has a concentration of not less than 2 mg / ml and a purity greater than 95%.

[0211] In addition, Alemtuzumab, a positive control, was used in Example 14.

[0212] Example 14: Antibody stimulation of NK cells to produce TNF-α

[0213] (1) Human PBMCs were sorted using an NK cell isolation kit (miltenyi, Cat:130-050-401, Lot:5220608838) to obtain primary NK cells;

[0214] (2) Centrifuge NK cells, resuspend the cells in culture medium (containing 10 ng / ml lL-2), take 50 uL of cells into wells (1E5 cells / well), and add Tab1, Alemtuzumab, hlgG and the single-domain antibody clones of the present invention (5B10, 7F10, 1H2, 2B11, and single-domain antibody clone 8B1 with no sequence shown). The concentration settings for each sample are shown in the table below.

[0215] (3) Incubate at 37℃ for 24 hours.

[0216] (4) Centrifuge the cells, collect the supernatant, and use the HTRF kit to detect TNF-α levels.

[0217] The concentration settings and results are shown in Tables 12-15:

[0218] Table 12 Alemumab Concentration Settings

[0219]

[0220] Table 13 Tab1 Concentration Settings

[0221]

[0222] Table 14 Single-domain antibody concentration settings

[0223]

[0224] Table 15 hIgG Concentration Settings

[0225]

[0226] The experimental results of antibody-stimulated NK cell release of TNF-α are as follows: Figure 6-8 As shown, NK cells can effectively respond to all single-domain antibodies of the present invention to produce TNF-α.

[0227] Example 15: ADCC effect of Nkp46 / FOLR1 bispecific antibody

[0228] The ADCC effect of the Nkp46 / FOLR1 bispecific antibody of this invention was determined using the LDH method, and the steps are as follows:

[0229] (1) Collect SK-OV-3 cells that have been passaged 3-4 times after resuscitation and seed them into 96-well plates at a density of 10,000 cells per well; (2) Prepare a solution of Tab1 and antibody sample VHH-hFc with a maximum concentration of 10 μg / mL and perform a 10-fold serial dilution to obtain 7 concentrations; (3) Add the serially diluted antibody solution to the cell culture wells at an equal volume to the cell suspension; (4) Collect PBMC cells for the sample wells and E / T wells (antibody concentration of 0) at a density of 250,000 cells per well. Add twice the volume of the target cell suspension to the cell culture wells; for MAX wells, add twice the volume of the target cell suspension lysis buffer to each well; for MIN wells, add twice the volume of the target cell suspension test buffer to each well; (5) after incubation for 6 hours, use the LDH kit to detect cell killing and read the absorbance; (6) according to the formula % target cell killing rate = (sample - E / T) / (MAX - MIN); (7) based on the target cell killing rate and concentration, perform four-parameter fitting and calculate the EC50 concentration of each antibody-mediated ADCC action.

[0230] like Figure 9-10 As shown. From Figures 9-10 It can be seen that the bispecific antibodies (4F4-1H2, 4F4-2H11, 4F4-5H10, 4F4-7F10) all have stronger ADCC effects compared with 4F4 and Tab1.

[0231] 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 Nkp46, 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: CDR1 shown in SEQ ID NO: 12, CDR2 shown in SEQ ID NO: 18, and CDR3 shown in SEQ ID NO:

22.

2. The single-domain antibody against Nkp46 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: The FR1 or a variant thereof shown in SEQ ID NO: 26, wherein the variant thereof contains substitutions of up to 5 amino acids; The FR2 or a variant thereof shown in SEQ ID NO: 32, wherein the variant thereof contains substitutions of up to 5 amino acids; The FR3 or a variant thereof shown in SEQ ID NO: 36, wherein the variant thereof contains substitutions of up to 5 amino acids; The FR4 or a variant thereof shown in SEQ ID NO:39, wherein the variant thereof contains substitutions of up to 5 amino acids.

3. A single-domain antibody against Nkp46, characterized in that: The amino acid sequence of the single-domain antibody is shown in SEQ ID NO:

4.

4. The Fc fusion antibody or humanized antibody of the single-domain antibody against Nkp46 according to any one of claims 1 to 3.

5. An Nkp46 / FOLR1 bispecific antibody, characterized in that, It comprises a first antigen-binding portion that specifically binds to Nkp46 and a second antigen-binding portion that specifically binds to FOLR1, wherein the first antigen-binding portion that specifically binds to Nkp46 is a single-domain antibody as described in any one of claims 1 to 3.

6. The Nkp46 / FOLR1 bispecific antibody according to claim 5, 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; The second antigen-binding site that specifically binds to FOLR1 is VHH.

7. The Nkp46 / FOLR1 bispecific antibody according to claim 6, characterized in that, The amino acid sequence of the bispecific antibody is shown in SEQ ID NO:

43.

8. A nucleotide molecule encoding a single-domain antibody against Nkp46 as described in any one of claims 1-3, characterized in that: Its nucleotide sequence is shown in SEQ ID NO: 9, or the amino acid sequence encoded by the nucleotide molecule is the same as the amino acid sequence encoded by SEQ ID NO:

9.

9. A nucleotide molecule encoding the Nkp46 / FOLR1 bispecific antibody according to any one of claims 5-7, characterized in that: Its nucleotide sequence is shown in SEQ ID NO: 47, or the amino acid sequence encoded by the nucleotide molecule is the same as the amino acid sequence encoded by SEQ ID NO:

47.

10. An expression vector, characterized in that: It comprises a nucleotide molecule encoding a single-domain antibody against Nkp46 as described in any one of claims 1 to 3, or an Fc fusion antibody or humanized antibody as described in claim 4, or an Nkp46 / FOLR1 bispecific antibody as described in any one of claims 5 to 7, or a nucleotide molecule as described in claim 8 or 9.

11. A host cell, characterized in that: It can express the single-domain antibody against Nkp46 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 Nkp46 / FOLR1 bispecific antibody as described in any one of claims 5 to 7, or the expression vector as described in claim 10.

12. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises a single-domain antibody against Nkp46 selected from any one of claims 1 to 3 or an Nkp46 / FOLR1 bispecific antibody as described in any one of claims 5 to 7, and a pharmaceutically acceptable carrier.

13. A medicine for treating diseases, characterized in that: It comprises an anti-Nkp46 single-domain antibody of any one of claims 1 to 3 or an Nkp46 / FOLR1 bispecific antibody of any one of claims 5 to 7 as an active ingredient.

14. The use of the Nkp46 / FOLR1 bispecific antibody according to any one of claims 5-7 in the preparation of a medicament for treating diseases, characterized in that: The disease in question is ovarian cancer.

Images