CN120025440A

CN120025440A - A single domain antibody against Nkp46 and its use

Info

Publication number: CN120025440A
Application number: CN202311557266.1A
Authority: CN
Inventors: 苏志鹏; 汪洋; 谢维
Original assignee: Nanjing Rongjiekang Biotechnology Co ltd
Current assignee: Nanjing Rongjiekang Biotechnology Co ltd
Priority date: 2023-11-21
Filing date: 2023-11-21
Publication date: 2025-05-23
Also published as: WO2025108080A1

Abstract

The present invention belongs to the field of immunology and relates to a single domain antibody against Nkp46 and its use. The single domain antibody is composed of a heavy chain, and the heavy chain includes a heavy chain CDR1 shown in any one of SEQ ID NO:11-SEQ ID NO: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:21-23. Compared with the prior art, the beneficial effect of the present invention is that the present invention uses biological genetic engineering technology to screen out a single domain antibody specific for Nkp46, and the antibody affinity is good.

Description

Anti Nkp single domain antibody and application thereof

Technical Field

The present invention relates to a single domain antibody capable of specifically binding to Nkp (hereinafter, abbreviated as "Nkp single domain antibody"), a pharmaceutical composition containing the single domain antibody as an active ingredient, and a pharmaceutical therapeutic use thereof.

Background

NK cell activity is regulated by complex mechanisms involving activation and inhibition signals. Several different NK-specific receptors have been identified that play an important role in NK cell-mediated recognition and killing of HLAI-like defective target cells. Natural Cytotoxic Receptor (NCR) refers to a class of activated receptor proteins specifically expressed in NK cells, as well as genes expressing them. Examples of NCR include NKp30, NKp44, and NKp46.

Nkp 46A low affinity and dominant class of activated transmembrane receptors expressed on NK cells, macrophages and mast cells belong to the immunoglobulin superfamily of transmembrane receptor members. On NK cells, the α chain of fcyriiia undergoes signal transduction in combination with an Immunoreceptor Tyrosine Activation Motif (ITAM) containing fceriγ chain and/or a T Cell Receptor (TCR)/CD 3 ζ chain.

Single domain antibodies or single domain antibodies (singledomain antibody, sdabs) are the smallest antibody molecules available today, with a molecular weight of 1/10 of that of the intact antibody. The single domain antibody has the antigen reactivity of the complete antibody, and also has some unique functional characteristics, such as small molecular weight, strong stability, good solubility, easy expression, weak immunogenicity, strong penetrability, strong targeting property, low preparation cost and the like, and almost perfectly overcomes the defects of long development period, lower stability, harsh preservation condition and the like of the traditional antibody.

Therefore, it is particularly necessary to study and develop a high affinity single domain (hereinafter also simply referred to as "single domain") Nkp antibody which has a small molecular weight, good tumor permeability, and can be used for free assembly into bispecific antibodies.

Disclosure of Invention

The invention of this patent aims to provide a single domain antibody capable of specifically binding to Nkp46 and its use.

In a first aspect the invention provides a single domain antibody against Nkp46, said single domain antibody is made up of a heavy chain comprising 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 NO. 21-SEQ ID NO. 23.

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

(1) CDR1 shown in SEQ ID NO. 13, CDR2 shown in SEQ ID NO. 16, CDR3 shown in SEQ ID NO. 23;

(2) CDR1 shown in SEQ ID NO. 12, CDR2 shown in SEQ ID NO. 18, CDR3 shown in SEQ ID NO. 22;

(3) CDR1 shown in SEQ ID NO. 11, CDR2 shown in SEQ ID NO. 19, CDR3 shown in SEQ ID NO. 21;

(4) CDR1 shown in SEQ ID NO. 14, CDR2 shown in SEQ ID NO. 17, CDR3 shown in SEQ ID NO. 23;

The above CDR combinations (1) - (4) correspond in sequence to single domain antibodies 2B11, 7F10, 5B10 and 1H 2.

All of the above sequences may be replaced with sequences having "at least 80% homology" 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".

In one embodiment, wherein any one to five of the amino acid residues in any one or more of the CDRs of heavy chain CDR1, CDR2 and CDR3 may be substituted with their conserved amino acids, respectively. Specifically, 1 to 5 amino acid residues in the heavy chain CDR1 can be replaced by the conserved amino acid, 1 to 5 amino acid residues in the heavy chain CDR2 can be replaced by the conserved amino acid, and 1 to 5 amino acid residues in the heavy chain CDR3 can be replaced by the conserved amino acid.

As used herein, the term "sequence homology" refers to the degree 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 with identical sequences have 100% homology.

In some embodiments, sequences that replace only one or a few amino acids, e.g., comprising 1,2, 3,4, 5,6, 7, 8, 9, or 10 conservative amino acid substitutions, as compared to the preceding sequences, may also achieve the object. Such variants include, but are not limited to, deletions, insertions and/or substitutions of one or more (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and the addition of one or more (typically less than 20, preferably less than 10, more preferably less than 5) amino acids at the C-terminus and/or N-terminus. In fact, the skilled person may consider so-called "conservative" amino acid substitutions, which in the case of substitution would preferably be conservative amino acid substitutions, in determining the degree of sequence homology between two amino acid sequences or in determining the CDR1, CDR2 and CDR3 combinations in a single domain antibody. The conserved amino acid, which may be generally described as an amino acid substitution of an amino acid residue with another amino acid residue having a similar chemical structure, has little or no effect on the function, activity, or other biological property of the polypeptide. Such conservative amino acid substitutions are common in the art, e.g., conservative amino acid substitutions are those in which one or a few amino acids in groups (a) - (d) are substituted for another or a few amino acids in the same group (a) a polar negatively charged residue and its uncharged amide Asp, asn, glu, gln, (b) a polar positively charged residue His, arg, lys, (c) an aromatic residue Phe, trp, tyr, and (d) an aliphatic nonpolar or weakly polar residue Ala, ser, thr, gly, pro, met, leu, ile, val, cys. Particularly preferred conservative amino acid substitutions are those wherein Asp is substituted by Glu, asn is substituted by Gln or His, glu is substituted by Asp, gln is substituted by Asn, his is substituted by Asn or Gln, arg is substituted by Lys, lys is substituted by Arg, gln, phe is substituted by Met, leu, tyr, trp is substituted by Tyr, tyr is substituted by Phe, trp, ala is substituted by Gly or Ser, ser is substituted by Thr, thr is substituted by Ser, gly is substituted by Ala or Pro, met is substituted by Leu, tyr or Ile, leu is substituted by Ile or Val, val is substituted by Ile or Leu, cys is substituted by Ser. In addition, those skilled in the art will recognize that the creativity of single domain antibodies is represented in the CDR1-3 regions, while the framework region sequences FR1-4 are not immutable, and that the sequences of FR1-4 may take the form of conservative sequence variants of the sequences disclosed herein.

The meaning of "anti-Nkp-46 single domain antibody" in the present invention includes not only the whole single domain antibody but also fragments, derivatives and analogues of the single domain antibody of anti-Nkp 46. As used herein, the terms "fragment," "derivative," and "analog" are synonymous and refer to a polypeptide that retains substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with an Fc tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.

In a preferred embodiment, the antibody sequence further comprises a framework region FR comprising the amino acid sequences of FR1, FR2, FR3 and FR4, the amino acid sequences of the framework region FR being:

SEQ ID NO:25-27 or a variant of FR1 as set forth in any one of claims 25-27, said variant of FR1 comprising up to 5 amino acid substitutions in said FR 1;

29-32, or a variant of FR2 as set forth in any one of SEQ ID nos. 29-32, said variant of FR2 comprising up to 5 amino acid substitutions in said FR 2;

SEQ ID NO:34-37, or a variant of FR3 as set forth in any one of claims 34-37, said variant of FR3 comprising up to 5 amino acid substitutions in said FR 3;

FR4 shown in SEQ ID NO. 39 or a variant of FR4, said variant of FR4 comprising at most 5 amino acid substitutions in said FR 4.

In a second aspect of the invention, there is provided an amino acid sequence of a single domain antibody capable of binding Nkp to the protein 3946, said single domain antibody having an amino acid sequence as shown in SEQ ID NO. 1-4, or said single domain antibody having at least 80% sequence homology with the amino acid sequence of SEQ ID NO. 1-4 and being capable of specifically binding to the protein Nkp, or said single domain antibody having an amino acid sequence in which at least 1 amino acid residue in the FR1, FR2, FR3 or FR4 sequence is substituted with a conserved amino acid compared to any of SEQ ID NO. 1-4.

In one embodiment, the single domain antibody against Nkp has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-4 and is capable of specifically binding to Nkp46 protein.

A third aspect of the invention is to provide an Fc fusion antibody or a humanized antibody of any of the above anti-Nkp-46 single domain antibodies.

In a fourth aspect, the invention provides a recombinant protein comprising the aforementioned single domain antibody against Nkp. The recombinant protein can be a single domain antibody shown in SEQ ID NO. 1-4, a single domain antibody with at least 80% homology with SEQ ID NO. 1-4, a multi-epitope antibody, a bispecific antibody, a multi-specific antibody and a multivalent antibody, wherein the multi-epitope antibody can be composed of more than one sequence in SEQ ID NO. 1-4, the multivalent antibody can be composed of one sequence in SEQ ID NO. 1-4 repeatedly arranged for a plurality of times, the multi-specific antibody comprises but is not limited to a trispecific antibody and a tetraspecific antibody, and the recombinant protein can be a fragment, a derivative and an analogue of the antibody.

In a fifth aspect the invention provides a bispecific or multispecific antibody comprising a single domain antibody of any one of the preceding claims as a first antigen-binding portion which specifically binds Nkp.

In a preferred embodiment, the bispecific or multispecific antibodies described above further comprise binding moieties specific for a tumor antigen other than Nkp;

other tumor antigens other than Nkp46 include FOLR1, CD123, BCMA, CD38, GPC3, B7H3, CD16a, CD20, IL-2R, IL-2rβ, nectin-4, CD160, or any other tumor antigen.

In a preferred embodiment, the bispecific antibody comprises 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、Nkp46/CD160 bispecific antibodies;

May be Nkp/FOLR 1 bispecific antibodies comprising a second antigen-binding portion that specifically binds FOLR 1.

In a preferred embodiment, the second antigen binding portion that specifically binds FOLR1 comprises CDR1 as shown in SEQ ID No. 15, CDR2 as shown in SEQ ID No. 20, and CDR3 as shown in SEQ ID No. 24;

preferably, the second antigen binding portion that specifically binds FOLR1 is a VHH.

In a preferred embodiment, the amino acid sequences of the bispecific antibodies are shown in SEQ ID NOS.40-43, respectively.

In a preferred embodiment, the multispecific antibody is a trispecific antibody comprising a first antigen-binding portion that specifically binds Nkp, a second antigen-binding portion that specifically binds FOLR1, and further comprising a third antigen-binding portion that specifically binds CD160 or CD 16.

In a sixth aspect, the present invention provides a nucleotide molecule encoding the above-mentioned anti-Nkp 46 single domain antibody or the above-mentioned Fc fusion antibody or the above-mentioned humanized antibody, which has the nucleotide sequence shown in SEQ ID NO. 6-9, respectively, or the amino acid sequence encoded by the nucleotide sequence is identical to the amino acid sequence encoded by any one of SEQ ID NO. 6-9, or has at least 95% sequence homology with any one of SEQ ID NO. 6-9.

In one embodiment, the nucleic acid molecule encoding the anti-Nkp 46 single domain antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence homology to a nucleotide sequence selected from the group consisting of SEQ ID NOS: 6-9, and encodes a single domain antibody against Nkp capable of specifically binding to Nkp protein.

In a seventh aspect, the present invention provides a nucleotide molecule encoding the bispecific antibody, wherein 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 identical to the amino acid sequence encoded by any one of SEQ ID NOS 44-47.

In an eighth aspect, the invention provides an expression vector comprising a nucleotide molecule encoding an anti-Nkp single domain antibody, an Fc fusion antibody or a humanized antibody, a bispecific or multispecific antibody, wherein the nucleotide sequence encoding the single domain antibody is as set forth in SEQ ID NO. 6-9 or the nucleotide sequence encodes an amino acid sequence identical to the amino acid sequence encoded by any one of SEQ ID NO. 6-9, respectively.

In a preferred embodiment, the expression vector used is RJK-V4-hFC (the nucleotide molecules encoding the single domain antibody against Nkp46 or the Fc fusion antibody or humanized antibody thereof are integrated by genetic engineering means into RJK-V4-hFC), and other universal expression vectors may be selected as desired.

In a ninth aspect, the invention provides a host cell capable of expressing the above-described single domain antibody, fc fusion antibody or humanized antibody, bispecific or multispecific antibody of anti-Nkp, or comprising the above-described expression vector. Preferably the host cell is a bacterial cell, a fungal cell or a mammalian cell.

In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell, including bacteria, fungi.

In another preferred embodiment, the host cell is selected from the group consisting of E.coli, yeast cells, mammalian cells, phage, or combinations thereof.

In another preferred embodiment, the prokaryotic cell is selected from the group consisting of E.coli, bacillus subtilis, lactobacillus, streptomyces, proteus mirabilis, or a combination thereof.

In another preferred embodiment, the eukaryotic cell is selected from the group consisting of Pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, trichoderma, or a combination thereof.

In another preferred embodiment, the eukaryotic cell is selected from the group consisting of insect cells such as armyworm, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof.

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

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

In a tenth aspect, the invention provides a pharmaceutical composition comprising the aforementioned single domain antibody that binds Nkp to 46, the aforementioned bispecific antibody or the aforementioned multispecific antibody, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally determined by the isoelectric point of the antibody (the pH of the aqueous carrier medium is required to deviate from and from about 2 from the isoelectric point of the antibody). The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to, intravenous, transdermal (directly applied or plastered to the affected area).

The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the foregoing single domain antibodies, together with a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions.

An eleventh aspect of the present invention provides a pharmaceutical agent for treating a disease, comprising the aforementioned single domain antibody for binding Nkp protein or the aforementioned bispecific or multispecific antibody as an active ingredient.

In a twelfth aspect, the invention provides a kit for detecting Nkp levels of 46, comprising the single domain antibody as described above against Nkp46 or the bispecific or multispecific antibody as described above. In a preferred embodiment of the invention, the kit further comprises a container, instructions for use, buffers, etc.

In a thirteenth aspect of the invention, there is provided a method of producing a single domain antibody against Nkp46, comprising the steps of:

(a) Culturing the host cell of the ninth aspect of the invention under conditions suitable for the production of a single domain antibody, thereby

Obtaining a culture comprising said anti-Nkp-specific antibody, and

(B) Isolating or recovering said anti-Nkp-binding domain antibody from said culture, and

(C) Optionally purifying and/or modifying the Nkp a 46 single domain antibody obtained in step (b).

In a fourteenth aspect the invention provides the use of a single domain antibody as hereinbefore defined against Nkp, or a bispecific or multispecific antibody as hereinbefore defined, or a pharmaceutical composition as hereinbefore defined, in the manufacture of a medicament for the treatment of a disease.

In a preferred embodiment, the disease is NK cell mediated various conditions associated with Nkp a 46.

In a preferred embodiment, the disease includes, but is not limited to, a tumor, an autoimmune disease, a metabolic-related disease, an infectious disease.

In a preferred embodiment, NK cell mediated various conditions associated with Nkp a 46 include, but are not limited to, rheumatoid Arthritis (RA), bone erosion, intraperitoneal abscesses, inflammatory bowel disease, allograft rejection, psoriasis, angiogenesis, atherosclerosis, asthma, multiple sclerosis, systemic Lupus Erythematosus (SLE), ocular surface conditions (e.g., dry eye), ankylosing spondylitis, psoriatic arthritis, cancers (e.g., multiple myeloma and breast cancer).

In a preferred embodiment, the tumor comprises a solid tumor, hematological tumor.

In a preferred embodiment, tumors include, but are not limited to, tumors of epithelial origin (adenomas and various types of cancers, including adenocarcinomas, squamous cell carcinomas, transitional cell carcinomas, and other carcinomas); bladder cancer and urinary tract cancer; breast cancer; gastrointestinal tract cancers (including esophageal, gastric, small intestinal, colon, rectal and anal cancers), liver (hepatocellular), gall bladder and biliary tract systems, exocrine pancreas, kidney-related cancers, lung cancers (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, broncheoalveolar and mesothelioma), head and neck cancers (e.g., tongue, oral, laryngeal, pharyngeal, nasopharyngeal, tonsil, salivary gland, nasal and paranasal sinus cancers), ovarian, fallopian tube, peritoneal, vaginal, vulva, penis, cervix, myometrium, endometrial-related cancers, thyroid cancers (e.g., thyroid follicular carcinoma), adrenal gland, prostate, skin and accessory-related cancers (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevi), hematologic and pre-and peripheral hematologic diseases including hematologic and lymphoid lineage-related conditions (e.g., acute lymphoblastic leukemia ALL, chronic lymphoblastic leukemia CLL, B cell lymphomas such as diffuse large B cell lymphoma, burk-cell lymphoma, schlemm, lymphoblastic, lymphomas of one cell, lymphomas of the like, lymphomas of the human significance, lymphomas of the human, lymphomas of the Zebra, lymphomas of the human, lymphomas of the cell type, lymphomas of the Zebra, and lymphomas Multiple myeloma and post-transplant lymphoproliferative diseases), hematological malignancies and myeloid-related diseases (e.g., acute myelogenous leukemia AML, chronic myelogenous leukemia CML, chronic myelomonocytic leukemia CMML, eosinophilic hyperplasia syndrome, myeloproliferative diseases such as polycythemia vera, primary 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 histiocytoma and fibrosarcoma of the carina-type skin, tumors of the central or peripheral nervous system (such as astrocytoma, glioma and glioblastoma, meningioma, ependymoma, pineal tumor and schwannoma), endocrine tumors (such as pituitary tumor, adrenal gland tumor, islet cell tumor, parathyroid tumor, carcinoid tumor and medullary thyroid carcinoma), ocular and accessory tumors (such as retinoblastoma), germ cell and trophoblastoma (such as teratoma, seminoma, asexual cell tumor, grape embryo and choriocarcinoma), pediatric and embryonal tumors (such as medulloblastoma, neuroblastoma, nephroblastoma and primitive neuroectodermal tumors), or congenital or other syndromes, patients are predisposed to malignancy (e.g., pigment dry skin disease).

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, hematological disorders, type I diabetes.

Advantageous effects

Compared with the prior art, the invention has the beneficial effects that:

(1) The single domain antibodies of the invention are specific for Nkp protein with the correct spatial structure.

(2) The single domain antibody aiming at Nkp-46, which is obtained by the invention, has excellent antigen binding capacity and specificity, has excellent capacity of activating NK cells to release TNFa, and can effectively mediate ADCC effect. The single domain antibodies, in combination with other antigen binding portions, form bispecific or multispecific antibodies, and may also be used as part of a Chimeric Antigen Receptor (CAR), or assembled into any other form of antibody.

The dual antibody Nkp/FOLR 1 mediated ADCC effect (Nkp/FOLR 1 is only used as an implementation mode, nkp and other tumor surface antigen targets can also be combined), compared with the FOLR1 monoclonal antibody mediated ADCC effect, the enhancement effect is remarkable.

Drawings

In order to more clearly illustrate the technical solution of the present application, the drawings that are needed in the embodiments will be briefly described below, and it will be obvious to those skilled in the art that other drawings can be obtained from these drawings without inventive effort.

FIG. 1 is a library enrichment profile of the targeting Nkp antibody screen of example 3;

FIG. 2 is a graph (1H 2) showing the measurement of the antibody-antigen binding response curve in example 12;

FIG. 3 is a graph (2B 11) showing the measurement of the antibody-antigen binding response curve in example 12;

FIG. 4 is a graph of the measurement of the antibody antigen binding response curve in example 12 (7F 10);

FIG. 5 is a graph (5B 10) showing the measurement of the antibody-antigen binding response curve in example 12;

FIG. 6 is an antibody stimulated NK releasing TNF-a assay (Tab 1, alemtuzumab, hIgG) in example 14;

FIG. 7 is an experiment (5B10,1H2,7F10) of the antibody-stimulated NK-release TNF-a in example 14;

FIG. 8 is an experiment (2B 11) of the antibody-stimulated NK-releasing TNF-a in example 14;

FIG. 9 shows single domain antibody mediated ADCC effects (Tab 1,4F 4);

FIG. 10 shows Nkp/FOLR 1 mediated ADCC effects (4F 4-1H2, 4F4-2B11, 4F4-5B10 and 4F4-7F 10);

FIG. 11 is a schematic diagram of the structure of bispecific antibody Nkp/FOLR 1.

Detailed Description

The present invention is described in further detail below with reference to examples to enable those skilled in the art to practice the same by referring to the description.

As used herein, a "single domain antibody" (sdAb, also called nanobody 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. Thus, a single domain antibody comprises a single complementarity determining region (single CDR1, single CDR2, and single CDR 3). Examples of single domain antibodies are heavy chain-only antibodies (which naturally do not comprise light chains), single domain antibodies derived from conventional antibodies, and engineered antibodies.

The single domain antibodies may be derived from any species including mice, humans, camels, llamas, goats, rabbits, and cattle. For example, naturally occurring VHH molecules may be derived from antibodies provided by camelidae species (e.g. camels, dromedaries, llamas and dromedaries). Like whole antibodies, single domain antibodies are capable of selectively binding to a particular antigen. A single domain antibody may contain only the variable domains of an immunoglobulin chain, which domains have CDR1, CDR2 and CDR3, as well as framework regions.

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

The term "humanized antibody" refers to an antibody obtained by fusing a heavy chain variable region of a target antibody (e.g., an animal antibody) with a constant region of a human antibody, or an antibody obtained by grafting complementarity determining regions (CDR 1 to CDR3 sequences) of a target antibody into a variable region of a human antibody, or an antibody obtained by subjecting a target antibody to amino acid mutation according to the characteristics of human antibody framework regions (FR 1 to FR 4). Humanized antibodies can be synthesized or site-directed mutagenesis.

In the present invention, a single domain antibody against Nkp can be obtained from a sequence having high sequence homology with the CDR1-3 disclosed in the present invention. In some embodiments, sequences having "at least 80% homology" with the sequences in SEQ ID NOS.1-4, or "at least 85% homology", "at least 90% homology", "at least 95% homology", "at least 98% homology" may be used for the purposes of the invention.

In some embodiments, sequences that replace only one or a few amino acids compared to the sequences in SEQ ID NOs 1-4, e.g., comprising 1,2, 3,4, 5, 6,7, 8,9 or 10 conservative amino acid substitutions, may also achieve the object. In fact, in determining the degree of sequence homology between two amino acid sequences or in determining the CDR1, CDR2 and CDR3 combinations in a single domain antibody, the skilled person may consider so-called "conservative" amino acid substitutions, which in the case of substitution will preferably be conservative amino acid substitutions, which may generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and which substitution has little or no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are common in the art, e.g., conservative amino acid substitutions are those in which one or a few amino acids in groups (a) - (d) are substituted for another or a few amino acids in the same group (a) a polar negatively charged residue and its uncharged amide Asp, asn, glu, gln, (b) a polar positively charged residue His, arg, lys, (c) an aromatic residue Phe, trp, tyr, and (d) an aliphatic nonpolar or weakly polar residue Ala, ser, thr, gly, pro, met, leu, ile, val, cys. Particularly preferred conservative amino acid substitutions are those wherein Asp is substituted by Glu, asn is substituted by Gln or His, glu is substituted by Asp, gln is substituted by Asn, his is substituted by Asn or Gln, arg is substituted by Lys, lys is substituted by Arg, gln, phe is substituted by Met, leu, tyr, trp is substituted by Tyr, tyr is substituted by Phe, trp, ala is substituted by Gly or Ser, ser is substituted by Thr, thr is substituted by Ser, gly is substituted by Ala or Pro, met is substituted by Leu, tyr or Ile, leu is substituted by Ile or Val, val is substituted by Ile or Leu, cys is substituted by Ser. In addition, those skilled in the art will recognize that the creativity of single domain antibodies is represented in the CDR1-3 regions, while the framework region sequences FR1-4 are not immutable, and that the sequences of FR1-4 may take the form of conservative sequence variants of the sequences disclosed herein.

Preferred host cells of the invention are bacterial cells, fungal cells or mammalian cells.

The preparation method comprises the steps of preparing target protein and a truncated form of the target protein through a genetic engineering technology, immunizing an inner Mongolian alashan alpaca with the obtained antigen protein, obtaining peripheral blood lymphocytes or spleen cells of the alpaca after multiple immunization, recombining a camel source antibody variable region coding sequence into a phage display carrier through a genetic engineering mode, screening out a specific antibody aiming at the antigen protein through the phage display technology, and further detecting the binding capacity of the specific antibody and the antigen and application of the specific antibody in treatment of autoimmune diseases.

The above technical solutions will now be described in detail by way of specific embodiments:

example 1 preparation of human Nkp46 recombinant ectodomain protein:

The human recombinant extracellular domain protein used in the patent is obtained by self-expression and purification of a company, and the design scheme of the expression vector of the human recombinant Nkp protein is as follows:

(1) The coding sequence for Nkp, which was identified as BC064806.1, was retrieved in NCBI and the resulting amino acid sequence was identified as AAH64806.1.

(2) The nucleotide sequence of the coding Nkp th 22-254 th amino acid is cloned into a vector pcDNA3.4 by using a gene synthesis mode. And (3) carrying out Sanger sequencing on the constructed vector, comparing the original sequences, carrying out mass extraction on the recombinant plasmid after confirming no errors, removing endotoxin, and carrying out target protein expression and purification on transfected suspension 293F cells, wherein the purity reaches more than 90%, and meets the animal immunization requirement.

Example 2 construction of a single domain antibody library against Nkp protein:

1mg of the recombinant Nkp protein of human origin obtained by purification in example 1 was mixed with an equal volume of Freund's complete adjuvant, immunized as an inner Mongolian Alexander bactrian camel, once a week, and a total of 7 continuous immunizations, except for the first immunization, were each immunized with 1mg of Nkp46 protein in equal volume with Freund's incomplete adjuvant, and the immunization was performed in order to concentrate the camel to produce antibodies against Nkp protein.

After the animal immunization is finished, 150mL of camel peripheral blood lymphocytes are extracted, and RNA of the cells is extracted. cDNA was synthesized using the extracted total RNA, and VHH (antibody heavy chain variable region) was amplified by a nested PCR reaction using the cDNA as a template.

Then, the vector pMECS and the VHH fragment are digested with restriction enzymes, respectively, and then the digested fragments and the vector are linked. The ligated fragments were electrotransformed into competent cells TG1, a phage display library of Nkp protein was constructed and the library capacity was determined, the library capacity being about 1X 10 ⁹, and the correct insertion rate of the library at the fragment of interest was detected by colony PCR identification.

The results showed that after PCR amplification of 30 randomly selected colonies from the library, 29 clones amplified bands of predicted size and 1 clone amplified incorrectly, so the correct insertion was 29.times.30.times.100%. Apprxeq.96.7%.

Example 3 screening of Single Domain antibodies against Nkp protein:

200. Mu.L of the recombinant TG1 cells of example 2 were cultured in 2 XTY medium, during which 40. Mu.L of helper phage VCSM13 was added to infect TG1 cells, and cultured overnight to amplify phage, the phage was precipitated the next day with PEG/NaCl, and the amplified phage was collected by centrifugation.

500. Mu.g of Nkp protein diluted in 100mM NaHCO ₃ at pH8.3 was coupled to the ELISA plate, left overnight at 4℃while negative control wells (medium control) were established, 200. Mu.L of 3% skim milk was added the next day, blocked at room temperature for 2h, 100. Mu.L of amplified phage library (approximately 2X 10 ¹¹ phage particles) was added after the end of blocking, and allowed to act at room temperature for 1h, and after 1h of action, washed 15 times with PBS+0.05% Tween-20 to wash out unbound phage.

The phage specifically binding to Nkp protein was dissociated with trypsin at a final concentration of 25mg/mL, and E.coli TG1 cells in logarithmic growth phase were infected, cultured at 37℃for 1h, phage were generated and collected for the next round of screening, and the same screening process was repeated for 1 round, and enrichment was gradually obtained.

When the enrichment multiple reaches more than 10 times, the enrichment effect is shown in figure 1.

In fig. 1, P/n=number of monoclonal bacteria grown after phage infection TG1 bacteria by biopanning positive Kong Xi/number of monoclonal bacteria grown after TG1 bacteria by phage infection TG1 bacteria by positive Kong Xi, which gradually increases after enrichment occurs, and I/e=total amount of phage added to positive wells per round of biopanning/total amount of phage removed from positive Kong Xi per round of biopanning, which gradually approaches 1 after enrichment occurs.

Example 4 screening of specific positive clones for Nkp-46 by phage enzyme-linked immunosorbent assay (ELISA):

screening was performed according to the screening method described in example 3 above for 2 rounds of screening against the single domain antibody against Nkp protein, the phage enrichment factor against Nkp protein was 10 or more, 384 single colonies were selected from positive clones obtained by screening after the end of screening, inoculated into 96-well plates containing 2×TY medium of 100. Mu.g/mL ampicillin, respectively, and a blank was set, and after 37℃to the logarithmic phase, IPTG was added at a final concentration of 1mM, and cultured overnight at 28 ℃.

The crude antibody was obtained by osmotic swelling, nkp46 recombinant proteins were released into 100mM NaHCO ₃, pH8.3, respectively, and 100. Mu.g of the protein was coated in an ELISA plate (ELISA plate) at 4℃overnight. Transferring 100 mu L of the obtained crude Antibody extract to an ELISA plate added with antigen, incubating for 1h at room temperature, washing away unbound Antibody by using PBST, adding 100 mu L of Mouse Anti-HA tag Anti-body (HRP) (Mouse Anti-HA horseradish peroxidase labeled Antibody diluted by 1:2000), incubating for 1h at room temperature, washing away unbound Antibody by using PBST, adding horseradish peroxidase chromogenic solution, reacting for 15min at 37 ℃, adding stop solution, and reading the absorption value at a wavelength of 450nm on an enzyme-labeled instrument.

When the OD value of the sample well is 5 times or more greater than that of the control well, positive clone wells are judged, and the positive clone wells are transferred to LB medium containing 100. Mu.g/mL ampicillin to extract plasmids and sequenced.

And analyzing the gene sequence of each clone strain according to sequence alignment software VectorNTI, and regarding strains with the same CDR1, CDR2 and CDR3 sequences as the same clone strain and strains with different sequences as different clone strains to finally obtain the single-domain antibody specific to Nkp protein.

The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 structure, which forms the whole VHH. The obtained single domain antibody recombinant plasmid can be expressed in a prokaryotic system, and finally single domain antibody proteins (2B 11, 7F10, 5B10, 1H2 and 1G10, 3B11, 6H10, 8C5 and 8B1 of which sequences are not shown are obtained, and antibody clones of which sequences are not shown are shown in FIGS. 2-5 and 8).

Similarly, using the specific procedure of examples 1-4 (only antigen replaced with FOLR 1), a single domain antibody (VHH) -4F4 specific for FOLR1 protein was obtained.

Wherein, the preparation process of the human FOLR1 recombinant extracellular domain protein is as follows:

The human recombinant extracellular domain protein used in the patent is obtained by self-expression and purification of a company, and the design scheme of an expression vector of the human recombinant FOLR1 protein is as follows:

(1) The coding sequence for FOLR1, which is identified as nm_000802.3, was retrieved in NCBI and the resulting amino acid sequence was identified as np_000793.1.

(2) The nucleotide sequence of the 25 th to 233 th amino acid of the coded FOLR1 is cloned into pcDNA3.4 by using a gene synthesis mode. And (3) carrying out Sanger sequencing on the constructed vector, comparing the original sequences, carrying out mass extraction on the recombinant plasmid after confirming no errors, removing endotoxin, and carrying out target protein expression and purification on transfected suspension 293F cells, wherein the purity reaches more than 90%, and meets the animal immunization requirement.

The CDR and FR sequences of the 4 single domain antibodies against Nkp and the 1 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.

TABLE 1 CDR1 sequences of single domain antibodies

Actual clone numbering	CDR1	SEQ ID
			Nkp46-2B11	GRNFDSYA	SEQ ID NO:11
Nkp46-7F10	GRTFSSYA	SEQ ID NO:12
			Nkp46-5B10	GTFFSYVA	SEQ ID NO:13
Nkp46-1H2	GTSSFSYVA	SEQ ID NO:14
			FOLR1-4F4	DGTYRRYC	SEQ ID NO:15

TABLE 2 CDR2 sequences of single domain antibodies

Actual clone numbering	CDR2	SEQ ID
			Nkp46-5B10	ISGDFTT	SEQ ID NO:16
Nkp46-1H2	ISGDSST	SEQ ID NO:17
			Nkp46-7F10	ISWSGDST	SEQ ID NO:18
Nkp46-2B11	ISWSGGST	SEQ ID NO:19
			FOLR1-4F4	IYTGDGTT	SEQ ID NO:20

TABLE 3 CDR3 sequences of single domain antibodies

TABLE 4 FR1 sequences of single domain antibodies

TABLE 5 FR2 sequences of single domain antibodies

Actual clone numbering	FR2	SEQ ID
			Nkp46-5B10	LAWYRQAPGKQRELVAG	SEQ ID NO:29
Nkp46-1H2	LGWYRQAPGKQRELVAG	SEQ ID NO:30
			Nkp46-2B11	MGWFRQAPGKEREFVAA	SEQ ID NO:31
Nkp46-7F10	MGWFRQAPGKEREFVAG	SEQ ID NO:32
			FOLR1-4F4	MGWFRQAPGKEREKVAA	SEQ ID NO:33

TABLE 6 FR3 sequences of single domain antibodies

TABLE 7 FR4 sequences of single domain antibodies

TABLE 8 amino acid sequences of single domain antibodies

Table 9 nucleic acid sequences of Single Domain antibodies

EXAMPLE 5 purification and expression of specific Single-domain antibodies to Nkp46 protein in E.coli

Plasmids (pMECS-VHH) of the different clones obtained by sequencing analysis in example 4 were electrotransformed into E.coli HB2151 and plated on LB+amp+glucose-containing culture plates, cultured overnight at 37℃and single colonies were selected and inoculated in 5mL ampicillin-containing LB medium and shake cultured overnight at 37 ℃.

Inoculating 1mL of overnight culture strain into 330mL of TB culture solution, performing shaking culture at 37 ℃ until the OD600nm value reaches 0.6-0.9, adding 1M IPTG, performing shaking culture at 28 ℃ overnight, centrifuging, collecting Escherichia coli, and obtaining an antibody crude extract by using a permeation swelling method;

the single domain antibody was purified by nickel column affinity chromatography.

EXAMPLE 6 construction of Fc fusion antibody eukaryotic expression vector of anti-Nkp Single-domain antibody

(1) Subcloning the target sequence obtained in example 4 into eukaryotic expression vector, and subjecting the antibody screened in example 4 to Sanger sequencing to obtain nucleotide sequence;

(2) Synthesizing the nucleotide sequence into a vector RJK-V4-hFC designed and modified by the company in a sequence synthesis mode to obtain a recombinant eukaryotic expression vector, wherein the modification method of the vector is as described in example 11;

(3) Converting the recombinant eukaryotic expression vector constructed in the step (2) into DH5 alpha escherichia coli, culturing to extract plasmids, and removing endotoxin;

(4) Sequencing and identifying the extracted plasmid;

(5) The recombinant vector after confirmation was prepared for subsequent eukaryotic cell transfection and expression, and the antibody was purified by the method of example 10 after the VHH Fc protein was expressed by the method of example 8 or 9.

EXAMPLE 7 construction of eukaryotic expression vectors for anti-Nkp/FOLR 1 bispecific antibodies

(1) The gene sequence of the anti-FOLR 1 single domain antibody (named 4F 4) and the gene sequence of the anti-Nkp single domain antibody are respectively synthesized into a vector RJK-V4-3 designed and modified by the company by a sequence synthesis mode to obtain a recombinant eukaryotic expression vector (namely, the nucleotide sequences of SEQ ID NOS: 44-47 are respectively cloned into the vector), and the modification method of the vector is as described in example 11;

(4) Sequencing and identifying the extracted plasmid;

(5) The recombinant vector after the determination was prepared for the subsequent eukaryotic cell transfection expression, the bispecific antibody was expressed by the method of example 8 or 9 and purified by the method of example 10, and the obtained diabodies were designated as 4F4-1H2 (FOLR 1 single domain antibody at amino acids 1-125, joint GGGGGGSGGGGGGGGGS at amino acids 126-140, nkp single domain antibody at amino acids 141-261), 4F4-2B11 (FOLR 1 single domain antibody at amino acids 1-125, joint GGGGSGGGGSGGGGS at amino acids 126-140, amino acids 141-270 are Nkp single domain antibodies at amino acids Nkp), 4F4-5B10 (FOLR 1 single domain antibody at amino acids 1-125, joint GGGGGSGGGGS at amino acids 126-140, amino acids 141-260 are Nkp single domain antibodies at amino acids), 4F4-7 (amino acids 1-125 are 35 amino acids at amino acids corresponding to amino acids of SEQ ID NO. 35-35:37 GGID NO shown in SEQ ID NO: 35), respectively.

The Nkp/FOLR 1 bispecific antibody structure is shown in fig. 11, in which FOLR1 VHH, nkp46 VHH, and Fc are linked in sequence.

The linkers in this specification are not limited to a particular sequence, and any other flexible or rigid linker known in the art for constructing engineered antibodies may be used. Nkp 46A-46 VHH and Fc may also be linked by a linker, such as GGGGSGGGGSGGGGS or any other linker.

Table 10 amino acid sequences of diabodies

Table 11 nucleic acid sequences of the diabodies

Example 8 expression of a Single Domain antibody against Nkp protein in suspension ExpiCHO-S cells

(1) Passaging and expanding ExpiCHO-S ^TM cells at 2.5X10 ⁵/mL cells 3 days prior to transfection, transferring the calculated desired cell volume to a 500mL shake flask containing fresh pre-warmed 120mL (final volume) ExpiCHO ^TM expression medium, bringing the cell concentration to about 4X 10 ⁶-6×10⁶ viable cells/mL;

(2) The day before transfection, expiCHO-S ^TM cells were diluted to 3.5X10 ⁶ viable cells/mL and allowed to incubate overnight;

(3) The day of transfection, cell density and percent viable cells were determined. The cell density should reach about 7X 10 ⁶-10×10⁶ viable cells/mL prior to transfection;

(4) Cells were diluted to 6×10 ⁶ viable cells/mL with fresh ExpiCHO ^TM expression medium pre-warmed to 37 ℃. The calculated desired cell volume was transferred to a 500mL shake flask containing fresh pre-warmed 100mL (final volume) ExpiCHO ^TM expression medium;

(5) Mixing ExpiFectamine ^TM CHO reagent gently upside down, diluting ExpiFectamine ^TM CHO reagent with 3.7mL OptiPRO ^TM medium, and stirring or mixing;

(6) Diluting plasmid DNA with refrigerated 4mL OptiPRO ^TM culture medium, and mixing;

(7) Incubating ExpiFectamine CHO/plasmid DNA (plasmid DNA is Fc fusion antibody eukaryotic expression vector of single domain antibody of anti Nkp prepared in example 6) complex for 1-5 min at room temperature, then adding gently to the prepared cell suspension, and gently agitating shake flask during addition;

(8) Shake culturing cells in 37 ℃ and 8% co ₂ in humidified air;

(9) 600ul ExpiFectamine ^TM CHO Enhancer and 24mL ExpiCHO feed were added on day 1 (18-22 hours post transfection).

(10) Supernatants were collected about 8 days after transfection (cell viability below 70%).

Example 9 expression of a Single Domain antibody against Nkp protein in suspension 293F cells

Recombinant single domain antibody expression experimental procedure (500 mL shake flask for example):

(1) 293F cells were passaged and expanded at 2.5X10 ⁵/mL 3 days prior to transfection, and the calculated required cell volume was transferred to 500mL shake flasks with fresh pre-warmed 120mL (final volume) OPM-293CD05 Medium. The cell concentration was brought to about 2X 10 ⁶-3×10⁶ viable cells/mL.

(2) The day of transfection, cell density and percent viable cells were determined. The cell density should reach about 2X 10 ⁶-3×10⁶ viable cells/mL prior to transfection.

(3) Cells were diluted to 1X 10 ⁶ viable cells/mL with pre-warmed OPM-293CD05 Medium. The calculated cell volume required was transferred to a 500mL shake flask containing fresh pre-warmed 100mL (final volume) of medium.

(4) PEI (1 mg/mL) reagent was diluted with 4mL of Opti-MEM medium, mixed by vortexing or blowing, plasmid DNA (plasmid DNA was the Fc fusion antibody eukaryotic expression vector of the anti-Nkp single domain antibody prepared in example 6) was diluted with 4mL of Opt-MEM medium, mixed by vortexing, and filtered with a 0.22um filter head. Incubate at room temperature for 5min.

(5) Diluted PEI reagent was added to the diluted DNA and mixed upside down. PEI/plasmid DNA complexes were incubated for 15-20 minutes at room temperature and then gently added to the prepared cell suspension, during which time the shake flask was gently swirled.

(6) Cells were shake cultured at 37℃with 5% CO ₂ at 120 rpm.

(7) 5ML OPM-CHO PFF05 feed was added 24h, 72h post transfection.

(8) Supernatants were collected about 7 days after transfection (cell viability below 70%).

Example 10 purification of Single-Domain antibodies against Nkp protein

(1) The protein expression supernatant obtained in example 8 or 9 was filtered with a disposable filter head of 0.45 μm to remove insoluble impurities;

(2) Purifying the filtrate by using a Protein purifier to perform affinity chromatography, and purifying by using agarose filler coupled with Protein A by utilizing the binding capacity of human Fc and Protein A;

(3) Passing the filtrate through a Protein A pre-packed column at a flow rate of 1 mL/min, wherein the target Protein in the filtrate is combined with the packing;

(4) Washing the column-bound impurity proteins with a low-salt and high-salt buffer;

(5) The target protein combined on the column is subjected to a system by using a low pH buffer solution;

(6) Rapidly adding the eluent into Tris-HCl solution with pH of 9.0 for neutralization;

(7) And (3) dialyzing the neutralized protein solution, performing SDS-PAGE analysis to determine that the protein purity is above 95%, and preserving the protein at a low temperature for later use after the concentration is above 0.5 mg/mL.

Example 11 construction of Single-Domain antibody eukaryotic expression vector RJK-V4-hFC

The mentioned nanobody universal targeting vector RJK-V4-hFC was engineered by the present company after fusion of the Fc segment in the heavy chain coding sequence of human IgG1 on the basis of the invitrogen commercial vector pCDNA3.4 (vector data link: https:// packages. Thermo-filter. Com/TFS-packages/LSG/manuals/pcdna 3_4_topo_ta_cloning_kit_man. Pdf), i.e.the vector contains the Hinge region (Hinge) CH2 and CH3 regions of the IgG1 heavy chain. The concrete improvement scheme is as follows:

(1) Selecting restriction enzyme cutting sites XbaI and AgeI on pcDNA3.4;

(2) Introducing multiple cloning sites (MCS, multiple Cloning Site) and a 6 XHis tag at the 5 'end and the 3' end of the coding sequence of the Fc fragment respectively by means of overlapping PCR;

(3) Amplifying the fragments by PCR using a pair of primers with XbaI and AgeI cleavage sites, respectively;

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

(5) And (3) connecting the digested vector and the inserted fragment under the action of T4 ligase, then converting the connection product into escherichia coli, amplifying, and checking by sequencing to obtain the recombinant plasmid.

Example 12 determination of antigen binding response of antibodies

This example was performed using standard enzyme-linked immunosorbent assay (ELISA) protocols.

(1) 50. Mu.L of 1. Mu.g/mL human Nkp protein was coated at 4℃overnight.

(2) Washing the plate, adding 200 μl of 5% milk, and sealing at 37deg.C for 2 hr.

(3) VHH was diluted to 2ug/mL and then the antibody was diluted 5-fold gradient for a total of 8 concentration gradients. The VHH herein refers to the prokaryotic-expressed single-domain antibody against Nkp protein prepared in example 5.

(4) Washing the plate, adding 50 mu L of the single-domain antibody obtained by dilution in the step (3), and incubating for 1h at 37 ℃ with two duplicate wells.

(5) Washing the plate, adding 50 mu L HRP-Goat anti hIgG secondary antibody, and incubating at 37 ℃ for 30min.

(6) Washing the plate (washing several times), adding 50 μl TMB for recovering normal temperature in advance, and reacting at normal temperature in dark place for 15min.

(7) Add 50. Mu.L of stop solution (1N HCl) and store the microplate reader reading.

(8) The EC50 was calculated by plotting a curve as shown in fig. 2-5, wherein igg designates a type control, immunoglobulin molecules that do not bind to any target, and are commercially available.

As shown in the figure, the single domain antibodies 1H2, 2B11, 5B10 and 7F10 of the invention have better affinity and stronger specificity to Nkp protein 46.

Example 13 expression and purification of Tool antibody (Tab) targeting human Nkp46

Tab1 used in examples 14 and 15 of the present invention was designated NKp46-3 antibody as described in FIG. 2D of U.S. Pat. No. 3, 11001629B2, and the searched sequence was subjected to codon optimization of mammalian cell expression system by general biosystems (Anhui) Inc., and cloned into pcDNA3.1 vector. After resistance selection, plasmid positive bacteria were selected for amplification and plasmids were extracted using a plasmid extraction kit (MACHEREY NAGEL, cat# 740412.50). According to the method, 100 mug of plasmid (40 mug heavy chain+60 mug light chain) is added to 100mL of cells, PEI is used for transient expression in 293F cells (culture medium: freeStyle 293Expression medium,Thermo,Cat#12338026+F-68, thermo, cat#24040032), 10% Peptone (Sigma, cat#P0521-100G) with 5% volume is added after 6-24 hours of transfection, 8% CO ₂ rpm is used for culturing for about 7-8 days, expression supernatant is collected when the cell viability is reduced to 50%, protein A (GE, cat#17-5438-02) is used for gravity column purification, after PBS dialysis, nanodrop is used for measuring concentration and SEC identification purity is used for indirect ELISA to verify binding capacity.

Tab1 obtained by the method has the concentration not less than 2mg/ml and the purity more than 95%.

In example 14, positive control Alemtuzumab (Alemtuzumab) was also used.

Example 14 antibody stimulation of NK cells to produce TNFa

(1) Sorting human PBMC using NK cell separation kit (miltenyi, cat:130-050-401, lot: 5220608838) to obtain primary NK cells;

(2) NK cells were centrifuged, cells were resuspended in medium (containing 10ng/ml of lL-2), 50uL of cell wells (1E 5 cells/well) were taken, and Tab1, alemtuzumab, hlgG and the single domain antibody clone of the invention (5B 10, 7F10, 1H2, 2B11, and single domain antibody clone 8B1, not shown in the sequence) were added, respectively, and the set concentrations of each sample were as shown in the following Table.

(3) Incubate at 37 ℃ for 24h.

(4) Cells were centrifuged, the supernatant collected, and TNF-a levels were detected using HTRF kit.

The concentration settings and results are shown in tables 12-15:

table 12 alemtuzumab concentration settings

TABLE 13Tab1 concentration setting

Table 14 single domain antibody concentration settings

TABLE 15hIgG concentration settings

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

Example 15 ADCC Effect of Nkp46/FOLR1 double antibody

The ADCC effect of Nkp/FOLR 1 diabodies of the invention was measured by LDH method as follows:

(1) The SK-OV-3 cells of 3-4 generations after resuscitating are collected and spread into a 96-well plate according to 10000 holes;

(2) Preparing a solution with the highest concentration of 10 mug/mL from Tab1 and an antibody sample VHH-hFc, and carrying out 10-time gradient dilution to obtain 7 concentrations;

(3) Adding the antibody solution diluted in a gradient manner into a cell culture hole according to the equal volume of the cell suspension;

(4) For sample wells and E/T wells (antibody concentration 0), PBMC cells were collected and added to the cell culture wells at 250000 cells per well, twice the volume of the target cell suspension, for MAX wells, twice the volume of the target cell suspension, for MIN wells, twice the volume of the target cell suspension;

(5) After 6h incubation, detecting cell killing by using an LDH kit, and reading absorbance;

(6) Target cell killing% = (sample-E/T)/(MAX-MIN) according to the formula;

(7) Based on target cell killing rate and concentration, four parameter fits were performed to calculate EC50 concentrations for each antibody-mediated ADCC.

As shown in fig. 9-10. As can be seen from FIGS. 9 to 10, the bispecific antibodies (4F 4-1H2, 4F4-2H11, 4F4-5H10, 4F4-7F 10) have stronger ADCC effect than 4F4 and Tab 1.

The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be appreciated by persons skilled in the art that the above embodiments are not intended to limit the invention in any way, and that all technical solutions obtained by means of equivalent substitutions or equivalent transformations fall within the scope of the invention.

Claims

1. The anti-Nkp single domain antibody is characterized by comprising a heavy chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO:

Heavy chain CDR1 shown in any one of 11-SEQ ID NO. 14, heavy chain shown in any one of 16-SEQ ID NO. 19

CDR2 and SEQ ID NO:21-SEQ ID NO: 23.

2. The single domain antibody of Nkp, wherein the amino acid sequences of heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are one of the following (1) - (4):

(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 of anti-Nkp 46 of claim 1, further comprising a framework region FR comprising the amino acid sequences of FR1, FR2, FR3 and FR4, wherein the amino acid sequences of the framework region FR are:

4. An anti-Nkp single domain antibody is characterized in that the amino acid sequence of the single domain antibody is shown as any one of SEQ ID NO. 1-4, or at least 1 amino acid residue in the FR1, FR2, FR3 or FR4 sequence is substituted by a conservative amino acid compared with any one of SEQ ID NO. 1-4.

5. The Fc fusion antibody or humanized antibody of the single domain antibody of anti-Nkp-46 of any one of claims 1 to 4.

6. A recombinant protein comprising the anti-Nkp single domain antibody of any one of claims 1 to 4.

7. A bispecific or multispecific antibody, characterized in that it comprises a single domain antibody according to any one of claims 1 to 4 as a first antigen-binding portion which specifically binds Nkp.

8. A bispecific or multispecific antibody according to claim 7, further comprising a binding moiety specific for a tumor antigen other than Nkp;

preferably, other tumor antigens than Nkp46 include FOLR1, CD123, BCMA, CD38, GPC3, B7H3, 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, wherein the bispecific antibody comprises 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、Nkp46/CD160 bispecific antibody;

10. The bispecific antibody or multispecific antibody of claim 9, wherein the second antigen-binding portion that specifically binds FOLR1 comprises CDR1 as shown in SEQ ID No. 15, CDR2 as shown in SEQ ID No. 20, and CDR3 as shown in SEQ ID No. 24;

11. A bispecific antibody or multispecific antibody according to claim 10, wherein the amino acid sequences of the bispecific antibody 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 which specifically binds Nkp, a second antigen-binding portion which specifically binds FOLR1, and further comprising a third antigen-binding portion which specifically binds CD160 or CD 16.

13. A nucleotide molecule for coding the anti-Nkp single domain antibody as claimed in any one of claims 1 to 4, wherein the nucleotide sequence is shown in any one of SEQ ID NO. 6 to 9, or the amino acid sequence coded by the nucleotide sequence is the same as the amino acid sequence coded by any one of SEQ ID NO. 6 to 9.

14. A nucleotide molecule encoding the bispecific antibody of any one of claims 7 to 11, wherein the nucleotide sequence is shown in any one of SEQ ID NOS 44 to 47, or the amino acid sequence encoded by the nucleotide sequence is identical to the amino acid sequence encoded by any one of SEQ ID NOS 44 to 47.

15. An expression vector comprising a nucleotide molecule encoding the anti-Nkp single domain antibody of any one of claims 1 to 4 or the Fc fusion or humanized antibody of claim 5 or the bispecific or multispecific antibody of any one of claims 7 to 12 or the nucleotide molecule of claim 13 or 14.

16. A host cell capable of expressing the single domain antibody against Nkp or the Fc fusion antibody or humanized antibody according to claim 5 according to any one of claims 1 to 4 or the bispecific or multispecific antibody according to any one of claims 7 to 12 or comprising the expression vector according to claim 15.

17. A pharmaceutical composition comprising an anti-Nkp single domain antibody selected from the group consisting of the anti-bispecific antibody of any one of claims 1 to 4 or the bispecific antibody or multispecific antibody of any one of claims 7 to 12, and a pharmaceutically acceptable carrier.

18. Agent for the treatment of diseases, characterized in that it comprises as active ingredient the anti-Nkp single domain antibody of any one of claims 1 to 4 or the bispecific or multispecific antibody of any one of claims 7 to 12.

19. Use of the single domain antibody of anti Nkp as defined in any one of claims 1 to 4 or the bispecific or multispecific antibody of any one of claims 7 to 12 or the pharmaceutical composition of claim 17 in the manufacture of a medicament for the treatment of a disease.

20. The method of claim 19, wherein the disease comprises a tumor.