Anti-nkp46 single-domain antibody and use thereof

Abstract

Disclosed in the present invention are an anti-NKp46 single-domain antibody, an encoding sequence thereof, and a use thereof. Specifically, disclosed in the present invention is an anti-human NKp46 specific single-domain antibody. Further disclosed are an encoding sequence for encoding the single-domain antibody or a VHH chain thereof, a corresponding expression vector and host cell, and a method for producing the single-domain antibody of the present invention. Further disclosed is a multi-specific antibody containing the single-domain antibody of the present invention. The single-domain antibody of the present invention has high affinity and specificity, and can be used for NKp46 detection and targeted therapy.

Description

An anti-NKp46 single-domain antibody and its application Technical Field

[0001] The present invention relates to the technical field of biomedicine or biopharmaceuticals, and more particularly to an anti-NKp46 single-domain antibody and applications thereof. Background Art

[0002] NKp46, also known as NCR1 or CD335, is a natural cytotoxicity receptor (NCR) and one of the major activating receptors of NK cells. It has a molecular weight of 46 kDa and an extracellular domain composed of two C2-type Ig-like domains, belonging to the immunoglobulin (Ig) superfamily. NKp46 is primarily expressed on resting and activated NK cells, ILC1 (innate lymphoid cell 1), and a small number of lymphocytes. NKp46 is highly conserved in mammals and plays a role in NK cell lysis of autologous, allogeneic, or xenogeneic cells. NKp46 induces NK cell activation and direct killing of target cells in an HLA class 1-independent manner.

[0003] Single-domain antibodies (SDAs) are a new type of antibody molecule discovered in camelids by the research group of Belgian immunologist Hamers-Casterman. Compared to traditional antibodies, they have a smaller molecular weight, one-tenth the size of traditional IgG antibodies. Therefore, they have a simple structure, are easy to modify, have strong tissue penetration, and have low immunogenicity. Single-domain antibodies also lack an Fc domain and, when used as targeting molecules, do not mediate ADCC effects and cause cytotoxicity. Furthermore, they have the advantages of good stability, resistance to high temperatures and extreme pH environments, and low production costs. Therefore, single-domain antibodies are excellent targeting molecules with great value in targeted drug development.

[0004] However, there is currently a lack of single-domain antibodies targeting NKp46 in this field, and there are problems such as off-target and drug resistance after NK cells are activated.

[0005] Therefore, there is an urgent need in the art to develop new single-domain antibodies that can effectively target NKp46. Summary of the Invention

[0006] The present invention provides a new class of single-domain antibodies that can effectively target NKp46.

[0007] In a first aspect of the present invention, a single domain antibody against NKp46 is provided, wherein the single domain antibody has three complementarity determining regions (CDRs) derived from the VHH chain represented by the following amino acid sequences: SEQ ID NOs: 1-136;

[0008] The CDRs are CDR1, CDR2 and CDR3 determined by any one of the IMGT rules, Kabat rules, Chothia rules, or AbM rules.

[0009] In another preferred embodiment, the CDR1, CDR2 and CDR3 are selected from any one of the following CDR sequence combinations (i.e., any one of K1 to K114):

[0010] In another preferred embodiment, the amino acid sequence of the single-domain antibody is any one of SEQ ID NOs: 1 to 19.

[0011] In another preferred embodiment, the single-domain antibody is a humanized antibody, which includes the sequence shown in any one of SEQ ID NOs: 303 to 313.

[0012] In another preferred embodiment, the CDR region of the single-domain antibody VHH chain comprises an amino acid sequence having at least 80%, preferably at least 90%, and more preferably at least 95% sequence similarity to any of the above sequences.

[0013] In another preferred embodiment, any one of the above amino acid sequences further comprises a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain NKp46 binding affinity.

[0014] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-3, preferably 1-2, and more preferably 1.

[0015] In another preferred embodiment, the VHH chain of the single-domain antibody further includes a framework region (FR).

[0016] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0017] In another preferred embodiment, the framework region FR is of human, mouse, rabbit or camel origin.

[0018] In another preferred embodiment, the single-domain antibody binds to human, mouse or monkey NKp46.

[0019] In another preferred embodiment, the antibody is a heavy chain antibody, and the heavy chain antibody comprises heavy chain constant regions CH2 and CH3 (Fc segment).

[0020] In another preferred embodiment, the heavy chain constant region is derived from the Fc segment of IgG, preferably the Fc segment of human IgG.

[0021] In another preferred example, the VHH chain of the single-domain antibody targeting NKp46 has an amino acid sequence with an identity of ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% to the amino acid sequence shown in SEQ ID NO: 1-136.

[0022] In another preferred embodiment, the VHH chain of the anti-NKp46 single-domain antibody has one or more amino acid sequences as shown in SEQ ID NOs: 1 to 136.

[0023] In another preferred embodiment, the anti-NKp46 single-domain antibody includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and / or a multivalent body (multivalent antibody).

[0024] In another preferred embodiment, the amino acid sequence of the VHH chain of the single-domain antibody is selected from the following group: a sequence shown in any one of SEQ ID NOs: 1 to 136.

[0025] In another preferred embodiment, the single-domain antibody is a monovalent, bivalent, trivalent or tetravalent single-domain antibody.

[0026] In the second aspect of the present invention, a polynucleotide is provided, which encodes the anti-NKp46 single-domain antibody according to the first aspect of the present invention or the single-domain antibody fusion protein according to the third aspect.

[0027] In another preferred embodiment, the polynucleotide comprises DNA or RNA.

[0028] In the third aspect of the present invention, a single-domain antibody fusion protein is provided, wherein the fusion protein comprises the anti-NKP46 single-domain antibody according to the first aspect of the present invention as a first fusion element.

[0029] In another preferred embodiment, the fusion protein further comprises an additional fusion element fused with the first fusion element.

[0030] In another preferred embodiment, the additional fusion element includes: an antibody, a peptide, or a combination thereof.

[0031] In another preferred embodiment, the additional fusion element is an antibody targeting TAA.

[0032] In another preferred embodiment, the fusion protein is a bispecific or trispecific antibody.

[0033] The single-domain antibody fusion protein has a structure from N-terminus to C-terminus as shown in Formula I:

[0034] Z1-Z2-L-Z3 (I)

[0035] Where,

[0036] Z1 is the VHH chain of the anti-NKP46 single-domain antibody according to the first aspect of the present invention;

[0037] Z2 is the Fc segment of immunoglobulin;

[0038] L is the linker sequence;

[0039] Z3 is the immunomodulatory molecule.

[0040] In the fourth aspect of the present invention, an expression vector is provided, wherein the expression vector contains the polynucleotide described in the second aspect of the present invention.

[0041] In the fifth aspect of the present invention, a host cell is provided, wherein the host cell contains the expression vector described in the fourth aspect of the present invention, or the polynucleotide described in the second aspect of the present invention is integrated into its genome.

[0042] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0043] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or a combination thereof.

[0044] In a sixth aspect of the present invention, a method for producing an anti-NKp46 single domain antibody is provided, comprising the steps of:

[0045] (a) culturing the host cell according to the fifth aspect of the present invention under conditions suitable for producing the single-domain antibody, thereby obtaining a culture containing the anti-NKp46 single-domain antibody or the single-domain antibody fusion protein; and

[0046] (b) isolating or recovering the anti-NKp46 single-domain antibody or single-domain antibody fusion protein from the culture.

[0047] In another preferred embodiment, the anti-NKp46 single-domain antibody has an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 136.

[0048] In another preferred embodiment, the anti-NKp46 single-domain antibody has an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 19.

[0049] In a seventh aspect of the present invention, an immunoconjugate is provided, comprising:

[0050] (a) the anti-NKp46 single domain antibody according to the first aspect of the present invention; and

[0051] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an antisense oligonucleotide, a small interfering RNA, a microRNA, a nucleic acid aptamer, or an enzyme.

[0052] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0053] In another preferred embodiment, the drug is a cytotoxic drug.

[0054] In another preferred embodiment, the cytotoxic drug is selected from the following group: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or a combination thereof.

[0055] In another preferred embodiment, particularly useful examples of cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or a combination thereof.

[0056] In another preferred embodiment, the toxin is selected from the group consisting of auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansin, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxybenzoate, daptomycin, acetaminophen ... anthracnose dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, retstrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, a Sapaonaria officinalis inhibitor, a glucocorticoid, or a combination thereof.

[0057] In another preferred embodiment, the coupling moiety is a detectable label.

[0058] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antisense oligonucleotides, small interfering RNA, micro RNA, nucleic acid aptamers, antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles, etc.

[0059] In another preferred embodiment, the immunoconjugate comprises: a multivalent (e.g., bivalent) anti-NKp46 single-domain antibody according to the first aspect of the present invention. The multivalent nature means that the amino acid sequence of the immunoconjugate contains multiple repeats of the anti-NKp46 single-domain antibody according to the first aspect of the present invention.

[0060] In another preferred embodiment, the multivalency refers to the anti-NKp46 single domain antibody as described in the first aspect of the present invention, which contains multiple repeats in the amino acid sequence of the immunoconjugate.

[0061] In the eighth aspect of the present invention, there is provided a use of the anti-NKp46 single domain antibody as described in the first aspect of the present invention for preparing (a) a reagent for detecting NKP46 molecules; (b) a drug for treating tumors, autoimmune diseases or rare diseases.

[0062] In another preferred embodiment, the detection includes flow cytometry detection and cell immunofluorescence detection.

[0063] In the ninth aspect of the present invention, one or more uses of an anti-NKp46 single domain antibody are provided:

[0064] (i) for detecting human NKp46 molecules;

[0065] (ii) for flow cytometry;

[0066] (iii) for cell immunofluorescence detection;

[0067] (iv) for treating tumors;

[0068] (v) For tumor diagnosis.

[0069] In another preferred embodiment, the use is non-diagnostic and non-therapeutic.

[0070] In a tenth aspect of the present invention, a recombinant protein is provided, comprising:

[0071] (i) the sequence of the VHH chain of the anti-NKp46 single domain antibody according to the first aspect of the present invention;

[0072] (ii) optionally an Fc region; and

[0073] (iii) optionally a tag sequence to facilitate expression and / or purification.

[0074] In another preferred embodiment, the tag sequence includes a 6His tag and an HA tag.

[0075] In another preferred embodiment, the recombinant protein specifically binds to NKp46 protein.

[0076] In the eleventh aspect of the present invention, a multispecific antibody based on a compatible nucleic acid backbone is provided, wherein the multispecific antibody is a polymer formed by a complex of n monomers having a compatible nucleic acid backbone, where n is a positive integer of 2-8;

[0077] Wherein, the polymer includes the following monomers:

[0078] (a) a first monomer, comprising: (a1) a polypeptide element D1: an anti-NKp46 single domain antibody according to the first aspect of the present invention that binds to a first target protein NKp46; and (a2) a first backbone nucleic acid single strand;

[0079] (b) a second monomer comprising: (b1) a polypeptide element D2: an anti-CD16a antibody that binds to a second target protein CD16a; and (b2) a second backbone nucleic acid single strand; and

[0080] (c) a third monomer, comprising: (c1) a polypeptide element D3: an antibody that binds to a third target protein, wherein the third target protein is a tumor-associated antigen (TAA); and (c2) a third backbone nucleic acid single strand;

[0081] The polypeptide elements D1, D2 and D3 are covalently linked to the first, second and third backbone nucleic acid single strands, respectively.

[0082] In another preferred embodiment, in the polymer, the backbone nucleic acid single strand of each monomer forms a compatible double strand with the backbone nucleic acid single strand of other monomers through base complementarity, thereby forming a compatible nucleic acid backbone composite structure.

[0083] In another preferred embodiment, in the polymer, the backbone nucleic acid single strand of each monomer forms a mutually compatible double strand with the backbone nucleic acid single strands of the other two monomers through base complementarity.

[0084] In another preferred embodiment, the polymer further comprises the following monomers:

[0085] (d) a fourth monomer, wherein the fourth monomer comprises: (d1) a half-life extension module; and (d2) a fourth backbone nucleic acid single strand.

[0086] In another preferred embodiment, the half-life extension module is covalently linked to the fourth backbone nucleic acid single strand.

[0087] In another preferred embodiment, the half-life extension module is a protein or peptide with a long half-life.

[0088] In another preferred embodiment, the half-life extension module is selected from the following group: natural albumin, recombinant albumin, anti-albumin antibodies, nucleic acid aptamers that specifically bind to albumin, proteins and nucleic acid aptamers that directly bind to FcRn, or a combination thereof.

[0089] In another preferred embodiment, the anti-albumin antibody is selected from the following group: nanobodies, single-chain antibodies, Fab, monoclonal antibodies, or a combination thereof.

[0090] In another preferred embodiment, the half-life extension module is a protein selected from the following group: FcRn binding protein (such as serum albumin), or a protein that binds to FcRn binding protein (such as an antibody that specifically binds to serum albumin), or a combination thereof.

[0091] In another preferred embodiment, the TAA includes (but is not limited to) or one or more antigens selected from the following group: CD33, CD30, HER2, CD22, CD79b, Nectin-4, BCMA, EGFR, CD19, CLDN18.2, TROP2, c-Met, PSMA, Muc1, PDL1, ROR1, MSLN, TNF-α, CD25, ENPP3, Axl, CD20, ROR2, GPNMB, CEACAM6, CD138, CA6, FUT3, CD56, CD37, HER3, GPRC5D, STING, CEA, CD205, B7H4, CTLA4, RNF43, CDH3, DPEP3, 5T4, ITGB6, EFNA4, B7H3, CD228, Notch-3, CD46, CAIX, SLAMF6, ADAM9, GD3, TDGF1 VEGF, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, VISTA, WISP-1, WISP-2, WISP-3, or combinations thereof.

[0092] In another preferred embodiment, the polypeptide elements D2 and D3 are each independently selected from: a single domain antibody (VHH), a tandem single domain antibody, Fab, Fab', F(ab')2, TriFab, an Fv fragment, a fragment variable (Fv) heterodimer, a single chain Fv (scFv) fragment, a diabody, a bispecific T cell engager (BiTE) or a single domain fragment, preferably a single domain antibody (VHH).

[0093] In another preferred embodiment, the polypeptide element D2 is an anti-CD16a single domain antibody or scFv.

[0094] In another preferred embodiment, the polypeptide element D3 is an anti-CD30 single domain antibody or scFv.

[0095] In a twelfth aspect of the present invention, there is provided a pharmaceutical composition comprising:

[0096] (i) the anti-NKp46 single domain antibody according to the first aspect of the present invention, the single domain antibody fusion protein according to the third aspect of the present invention, the immunoconjugate according to the seventh aspect of the present invention, or the multispecific antibody according to the eleventh aspect of the present invention; and

[0097] (ii) a pharmaceutically acceptable carrier.

[0098] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0099] In the thirteenth aspect of the present invention, there is provided a use of the single domain antibody according to the first aspect of the present invention, the single domain antibody fusion protein according to the third aspect of the present invention, the immunoconjugate according to the seventh aspect of the present invention, or the multispecific antibody according to the eleventh aspect of the present invention, for preparing a medicament, reagent, detection plate or kit;

[0100] Wherein, the reagent, detection plate or kit is used for: detecting NKp46 protein in a sample;

[0101] The agent is used to treat or prevent tumors, autoimmune diseases or rare diseases that express NKp46 protein (ie, NKp46 positive).

[0102] In a fourteenth aspect of the present invention, a method for detecting NKp46 protein in a sample is provided, the method comprising the steps of:

[0103] (1) contacting a sample with the single domain antibody according to the first aspect of the present invention;

[0104] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of NKp46 protein in the sample.

[0105] In the fifteenth aspect of the present invention, a method for treating a disease is provided, comprising administering to a subject in need thereof the single domain antibody described in the first aspect of the present invention, the single domain antibody fusion protein as described in the third aspect of the present invention, or the immunoconjugate as described in the seventh aspect of the present invention, or the multispecific antibody as described in the eleventh aspect of the present invention.

[0106] In another preferred embodiment, the subject includes mammals, such as humans.

[0107] In the sixteenth aspect of the present invention, a NKp46 protein detection reagent is provided, wherein the detection reagent comprises:

[0108] (i) the anti-NKp46 single domain antibody of the first aspect of the present invention, the single domain antibody fusion protein of the third aspect of the present invention, the immunoconjugate of the seventh aspect of the present invention, or the recombinant protein of the eleventh aspect of the present invention; and

[0109] (ii) a carrier that is acceptable for detection.

[0110] In another preferred embodiment, the conjugated portion of the immunoconjugate is a diagnostic isotope.

[0111] In another preferred embodiment, the assay-acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0112] In another preferred embodiment, the detection reagent is one or more reagents selected from the following group: isotope tracers, contrast agents, flow cytometry detection reagents, cell immunofluorescence detection reagents, nanomagnetic particles and imaging agents.

[0113] In another preferred embodiment, the detection reagent is used for in vivo detection.

[0114] In another preferred embodiment, the dosage form of the detection reagent is liquid or powder (such as aqueous solution, injection, lyophilized powder, tablet, buccal preparation, inhaler).

[0115] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] FIG1 shows the protein sequence alignment results of NKp46 antigens of three species: human, cynomolgus macaque, and mouse.

[0117] Figure 2 shows the construction of an anti-NKp46 single-domain antibody yeast library: (a) library transformant counting plate; (b) library antibody sequence insertion rate detection; (c) library diversity amino acid sequence phylogenetic tree analysis.

[0118] Figure 3 shows the flow cytometry analysis of the NKp46 positive rate of the magnetic bead enriched library in one round: (left): magnetic bead enriched library; (middle) magnetic bead enriched library + anti-HA IgG-488; (right) magnetic bead enriched library + anti-HA IgG-488 + NKp46-650.

[0119] Figure 4 shows the ELISA assay for the binding ability of anti-NKp46 single domain antibodies to three species of NKp46 antigens: (A) binding activity to human NKp46 antigen; (B) binding activity to monkey NKp46 antigen; (C) binding activity to mouse NKp46 antigen.

[0120] FIG5 shows the flow cytometric assay for the binding ability of anti-NKp46 single domain antibodies to hNKp46 CHO-K1 cells.

[0121] Figure 6 shows the ELISA detection of the binding competition between three anti-NKp46 single-domain antibodies: (A) NKp46-209 was fixed, and the competition relationship between the other two antibodies and NKp46-209 was detected; (B) NKp46-645 was fixed, and the competition relationship between the other two antibodies and NKp46-645 was detected; (C) NKp46-681 was fixed, and the competition relationship between the other two antibodies and NKp46-681 was detected.

[0122] FIG7 shows SDS-PAGE electrophoresis gel images verifying the sample purity of three single-domain antibodies-PMO: (left) NKp46-209-PMO3; (center) NKp46-645-PMO3; (right) NKp46-681-PMO3.

[0123] Figure 8 shows a schematic diagram of the NKp46 / CD30 / CD16a trispecific antibody molecule and the purity verification of the assembled sample: (left) Schematic diagram of the trispecific antibody molecule (CD16a, CD30, and NKp46 in the figure represent their corresponding single-domain antibodies, respectively); (right) SDS-PAGE electrophoresis gel image verifying the purity of the trispecific antibody molecule.

[0124] FIG9 shows the validation of the anti-tumor activity of the NKp46 / CD30 / CD16a trispecific antibody.

[0125] FIG10 shows the ELISA assay for the binding ability of humanized anti-NKp46 VHH-Fc to human NKp46 antigen.

[0126] FIG11 shows the ELISA assay for the binding ability of humanized anti-NKp46 VHH-Fc to monkey NKp46 antigen.

[0127] FIG12 shows the ELISA assay for the binding ability of humanized anti-NKp46 VHH-Fc to mouse NKp46 antigen.

[0128] FIG13 shows the flow cytometric detection of the binding ability of humanized anti-NKp46 VHH-Fc to hNKp46 CHO-K1 cells. DETAILED DESCRIPTION

[0129] After extensive and in-depth research and extensive screening, the inventors have developed for the first time a class of single-domain antibodies and humanized antibodies that specifically target NKp46. In addition, the present invention also designs and constructs multispecific antibodies (such as NKp46 / CD16a / TAA multispecific antibodies) based on this antibody. Experimental results show that the single-domain antibodies and humanized antibodies obtained by the present invention have high specific affinity and binding activity to NKp46 and good stability; the single-domain antibodies of the present invention have cross-binding activity to human, mouse, and monkey species. Moreover, the anti-tumor effect of the trispecific antibody molecule containing the NKp46 single-domain antibody of the present invention is significantly better than the trispecific antibody molecule without the NKp46 single-domain antibody of the present invention. The present invention was completed on this basis.

[0130] the term

[0131] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0132] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0133] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0134] The term "fusion" refers to the connection of components directly by peptide bonds or with the help of connecting fragments, or non-covalent fusion through intermolecular interactions or covalent fusion through one or more disulfide bonds or chemical cross-linking. In a single peptide chain, fusion refers to the connection directly by peptide bonds or with the help of connecting fragments. "Multifunctional fusion protein" refers to a protein comprising two or more targeting domains (such as domains that bind to antigens and / or domains that bind to receptors) that can bind to two or more different epitopes (for example, two, three or more different epitopes). "Fusion position" refers to the position of a functional region or domain in a peptide chain, indicating the order in which the functional fragments on the peptide chain are connected.

[0135] As used herein, the terms "single-domain antibody of the present invention," "anti-NKP46 single-domain antibody of the present invention," "single-domain antibody targeting NKP46 of the present invention," "single-domain antibody of the present invention," and "NKP46 single-domain antibody of the present invention" are used interchangeably and refer to single-domain antibodies that specifically recognize and bind to NKP46 (including human NKP46). Particularly preferred are single-domain antibodies whose VHH chain amino acid sequences are as shown in any one of SEQ ID NOs.: 1-19.

[0136] As used herein, the terms "trispecific fusion protein", "trispecific antibody" and "trispecific antibody molecule of the present invention" are used interchangeably to refer to an antibody containing an anti-NKp46 single domain antibody, an anti-CD16a single domain antibody and an anti-TAA (such as CD30) single domain antibody.

[0137] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0138] As used herein, the terms "nanobody" and "single-domain antibody (VHH)" have the same meaning and refer to cloning the variable region of an antibody heavy chain to construct a single-domain antibody (VHH) consisting of only a single heavy chain variable region. It is the smallest antigen-binding fragment with complete function. Typically, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only a single heavy chain variable region.

[0139] As used herein, the term "variable" refers to certain parts of the variable region in an antibody that are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-pleated configuration and are connected by three CDRs that form a connecting loop, and in some cases can form a partial β-pleated structure. The CDRs in each chain are closely together through the FR region and form the antigen-binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0140] As known to those skilled in the art, immunoconjugates and fusion products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens bound to the anti-NKP46 protein antibodies or fragments thereof.

[0141] As used herein, the terms "heavy chain variable region" and "V H ” can be used interchangeably.

[0142] As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0143] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.

[0144] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.

[0145] In the present invention, the terms "antibody of the present invention," "protein of the present invention," or "polypeptide of the present invention" are used interchangeably to refer to polypeptides that specifically bind to NKP46 protein, such as proteins or polypeptides having a heavy chain variable region. These may or may not contain an initial methionine.

[0146] The present invention also provides other proteins or fusion expression products comprising the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) comprising a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.

[0147] Generally, an antibody's antigen-binding properties are described by three specific regions within the variable region of the heavy chain, known as the variable regions (CDRs). This region is divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a loop structure, spatially close to each other through the β-sheet formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antibody's antigen-binding site. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0148] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because they are at least partially involved in antigen binding. Thus, the present invention includes molecules having antibody heavy chain variable regions with CDRs that are 90% or more (preferably 95% or more, and most preferably 98% or more) homologous to the CDRs identified herein.

[0149] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0150] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. A polypeptide fragment, derivative, or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence, or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0151] The antibodies of the present invention refer to polypeptides that have NKP46 protein binding activity and include the aforementioned CDR regions. The term also encompasses variants of polypeptides that include the aforementioned CDR regions and have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more (generally 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, as well as the addition of one or more (generally within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. For example, substitutions with amino acids having similar or similar properties generally do not alter protein function. For another example, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter protein function. The term also encompasses active fragments and active derivatives of the antibodies of the present invention.

[0152] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0153] The present invention also provides other polypeptides, such as fusion proteins comprising single-domain antibodies or fragments thereof. In addition to substantially full-length polypeptides, the present invention also encompasses fragments of the single-domain antibodies of the invention. Typically, the fragment comprises at least about 50 contiguous amino acids of an antibody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

[0154] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides in which no more than 10, preferably no more than 8, more preferably no more than 5, and most preferably no more than 3 amino acids are replaced with amino acids having similar or similar properties, compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitutions according to Table 1.

[0155] Table 1

[0156] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies, fragments thereof, or fusion proteins thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand.

[0157] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0158] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0159] As used herein, the terms "antibody fragment," "antigen-binding fragment," "targeting domain," or "antigen-binding domain" are used to refer to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies. Regardless of the structure, antibody fragments bind to the same antigen recognized by the intact antibody. Examples of targeting domains of the present invention include, for example, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single-chain Fv (scFv) fragments, and single-domain fragments.

[0160] "Fv" fragment is the smallest fragment of an antibody containing a complete target recognition and binding site. This region is composed of a dimer (VH-VL dimer) of a heavy chain and a light chain variable domain in tight non-covalent binding. In this configuration, the three CDRs of each variable domain interact to define the target binding site on the surface of the VH-VL dimer. Typically, six CDRs confer target binding specificity to antibodies. However, in some cases, even a single variable domain (or only half of an Fv comprising three CDRs specific for a target) can have the ability to recognize and bind to a target, although its affinity is lower than that of the entire binding site.

[0161] "Single-chain Fv" or "scFv" antibody binding fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form a structure that is conducive to target binding.

[0162] A "single domain fragment" consists of a single VH or VL domain that exhibits sufficient affinity for coronavirus RBD. In a specific embodiment, the single domain fragment is camelized.

[0163] The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, wherein additional DNA segments can be connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Some vectors can replicate autonomously (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins) in the host cell introducing them. Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In addition, some vectors can instruct the expression of the gene operably connected thereto. Usually, expression vectors useful in recombinant DNA technology are usually in the form of plasmids.

[0164] As used herein, the term "antigen" or "target antigen" refers to a molecule or portion of a molecule that can be bound by an antibody or antibody-like binding protein. The term further refers to a molecule or portion of a molecule that can be used in an animal to produce an antibody that can bind to an epitope of the antigen. A target antigen may have one or more epitopes. For each target antigen recognized by an antibody or by an antibody-like binding protein, the antibody-like binding protein can compete with an intact antibody that recognizes the target antigen.

[0165] As used herein, the term "linker" refers to one or more amino acid residues inserted into an immunoglobulin domain that provide sufficient mobility for the domains of the light chain and heavy chain to fold into an exchange dual variable region immunoglobulin. Suitable examples of linkers include single glycine (Gly) or serine (Ser) residues. The identity and sequence of the amino acid residues in the linker can vary depending on the type of secondary structural element to be achieved in the linker. Preferred linkers can be (GS)n, (G3S)n, (G4S)n (n is selected from 1-6).

[0166] As used herein, a "variant" of an antibody, antibody fragment, or antibody domain refers to an antibody, antibody fragment, or antibody domain that: (1) has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the original antibody, antibody fragment, or antibody domain, and (2) specifically binds to the same target to which the original antibody, antibody fragment, or antibody domain specifically binds. It will be understood that where sequence identity is expressed in the form of "at least x% identical" or "at least x% identical," such embodiments include any and all numerical percentages equal to or above the lower limit. Furthermore, it will be understood that where an amino acid sequence is presented in this application, it should be construed as further disclosing or encompassing an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that amino acid sequence.

[0167] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0168] The full-length nucleotide sequence of the antibody of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis methods. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0169] Once the relevant sequence is obtained, recombinant methods can be used to obtain it in large quantities. This is typically accomplished by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) referred to in the present invention include biomolecules in isolated form.

[0170] Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into protein sequences of the present invention by chemical synthesis.

[0171] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0172] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 or Sf9; and animal cells such as CHO, COS7, and 293 cells.

[0173] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated using the CaCl2 method, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0174] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0175] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0176] The antibodies of the present invention may be used alone or in combination with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.

[0177] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0178] Therapeutic agents that can be combined or coupled with the antibodies of the present invention include but are not limited to: 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0179] Abbreviations

[0180] CDR: complementarity determining region

[0181] FR: amino acid residues in the antibody variable region excluding CDR residues

[0182] VH: antibody heavy chain variable region

[0183] VL: antibody light chain variable region

[0184] IgG: Immunoglobulin G

[0185] IMGT: numbering system based on the International Immunogenetics Information System initiated by Lefranc et al.

[0186] EC50: Half-maximal effect concentration, the concentration that can cause 50% of the maximum effect

[0187] ELISA: enzyme-linked immunosorbent assay

[0188] FACS: flow cytometry

[0189] PCR: polymerase chain reaction

[0190] HRP: horseradish peroxidase

[0191] ADCC: Antibody-dependent cell-mediated cytotoxicity

[0192] FBS: Fetal bovine serum

[0193] Immunoconjugates

[0194] The present invention also provides an immunoconjugate (ADC) based on the antibody of the present invention, preferably a single-domain antibody-drug conjugate (NDC).

[0195] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is coupled to the effector molecule, preferably chemically coupled. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, an agonist small molecule (STING, TLR7, TLR8, etc.), or a radionuclide.

[0196] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent may include any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent utilizing a carboxyl group may include any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (where the coupling site is an acylhydrazone).

[0197] Certain residues on antibodies (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (such as drugs, detection reagents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0198] Single-domain antibodies can be conjugated to drugs to form antibody-drug conjugates (NDCs). Typically, NDCs contain a linker positioned between the drug and the antibody. The linker can be a degradable or non-degradable linker. Degradable linkers typically readily degrade in the intracellular environment, for example, at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including linkers containing peptidyl groups that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidases. Peptide linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at a pH below 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug under conditions where the antibody is hydrolyzed by proteases.

[0199] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved via the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethylketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, where the counter ion is acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. Linkers may include, for example, maleimides attached to the antibody via thiosuccinimide.

[0200] The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, a linker connects the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group capable of forming a bond with the linker. In cases where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0201] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), and vinca alkaloids.

[0202] The immunoconjugate drug of the present invention may also be a radionuclide drug conjugate (RDC), which is composed of the antibody of the present invention conjugated to a radionuclide.

[0203] In the present invention, drug-linkers can be used to form NDCs in a single step. In other embodiments, bifunctional linker compounds can be used to form NDCs in a two-step or multi-step process. For example, a cysteine ​​residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, a functional group on the linker is reacted with a drug to form an NDC.

[0204] Typically, the functional group on the linker is selected to facilitate specific reaction with an appropriate reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Technology, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that, when a complementary pair of reactive functional groups is selected for selective reaction between the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0205] The present invention also provides a method for preparing NDC, which may further comprise: combining an antibody and a drug-linker compound under conditions sufficient to form an antibody conjugate (NDC).

[0206] In certain embodiments, the methods of the present invention comprise conjugating an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the present invention further comprise conjugating the antibody-linker conjugate to a drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody via the linker.

[0207] In some embodiments, the structure of an immunoconjugate, preferably a single domain antibody drug conjugate (NDC), is as shown below:

[0208] in:

[0209] nAb is the above-mentioned single-domain antibody targeting NKP46, heavy chain antibody targeting NKP46 or trispecific antibody,

[0210] LU is the linker / connector;

[0211] D is for medicine;

[0212] And the subscript p is a value selected from 1 to 10.

[0213] Composition

[0214] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising the above-mentioned antibody, active fragment thereof, or fusion protein thereof, and a pharmaceutically acceptable carrier. Typically, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.

[0215] The pharmaceutical composition of the present invention can be used to directly bind to NKP46 protein molecules, and thus can be used to treat tumors. In addition, other therapeutic agents can also be used simultaneously.

[0216] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned single-domain antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

[0217] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0218] Labeled single domain antibodies

[0219] In a preferred embodiment of the present invention, the single domain antibody carries a detectable label. More preferably, the label is selected from the group consisting of an isotope, a colloidal gold label, a colored label, or a fluorescent label.

[0220] Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, the anti-NKP46 single-domain antibody is labeled with colloidal gold to obtain a colloidal gold-labeled single-domain antibody.

[0221] The anti-NKP46 single-domain antibody of the present invention has good specificity and high titer.

[0222] Multispecific antibodies

[0223] As used herein, the term "multispecific antibody" refers to a molecule comprising at least two targeting domains with different binding specificities, wherein at least one targeting domain specifically binds to an NK cell surface antigen. In some embodiments, a multispecific inhibitor is a polypeptide comprising a scaffold and two or more immunoglobulin antigen-binding domains targeting different antigens or epitopes. In certain embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody.

[0224] As used herein, the terms "trispecific antibody of the present invention," "trispecific fusion protein of the present invention," and "3Sbody" are used interchangeably herein and relate to molecules comprising three targeting domains with three different binding specificities. Each targeting domain is capable of specifically binding to a target molecule and, when bound to the target molecule, inhibits the biological function of the target molecule. In some embodiments, the trispecific antagonist is a polymer molecule having two or more peptides. In some embodiments, the targeting domain comprises an antigen-binding domain or CDR of an antibody. In some embodiments, the targeting domain comprises a ligand or a fragment thereof that specifically binds to a target protein.

[0225] It should be understood that the trispecific fusion proteins of the present invention are essentially trispecific antibodies, antibody molecules that can simultaneously and specifically bind to three antigens. Based on symmetry, trispecific antibodies can be divided into structurally symmetrical and asymmetrical molecules. Based on the number of binding sites, bispecific antibodies can be divided into bivalent, trivalent, tetravalent, and multivalent molecules. The two trispecific antibodies of the present invention are structurally asymmetrical trivalent trispecific antibodies, and each trispecific antibody is monovalent for each specific target.

[0226] Detection method

[0227] The present invention also relates to a method for detecting NKP46 protein. The method generally comprises the following steps: obtaining a cell and / or tissue sample; dissolving the sample in a medium; and detecting the level of NKP46 protein in the dissolved sample.

[0228] In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a sample containing cells in a cell storage medium.

[0229] Reagent test kit

[0230] The present invention also provides a kit containing the antibody (or fragment thereof) or detection plate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc.

[0231] The present invention also provides a detection kit for detecting NKP46 levels, which includes an antibody that recognizes the NKP46 protein, a lysis medium for dissolving the sample, and common reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. The detection kit can be an in vitro diagnostic device.

[0232] application

[0233] As described above, the single-domain antibodies of the present invention or multispecific fusion antibodies containing the single-domain antibodies of the present invention have broad biological and clinical applications, including in the diagnosis and treatment of NKP46-related diseases, basic medical research, biological research, and other fields. A preferred application is in clinical diagnosis and targeted therapy for NKP46.

[0234] The main advantages of the present invention include:

[0235] (a) The present invention provides a humanized single-domain antibody, which reduces the immunogenicity of the antibody and improves its in vivo safety.

[0236] (b) The single domain antibody of the present invention can be easily produced.

[0237] (c) The present invention provides single-domain antibodies with cross-species binding activity to humans, mice, and monkeys.

[0238] (d) The NKp46 / CD16a / TAA trispecific antibody designed based on the anti-NKp46 single-domain antibody of the present invention can effectively activate NK cells and effectively kill tumor cells; it has great potential for the development of anti-tumor drugs.

[0239] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0240] Example 1

[0241] Production of anti-NKp46 single domain antibodies induced by immunization of alpacas and detection of serum titers

[0242] The first immunization involved subcutaneous injection of 0.5 mg of human NKp46 antigen (ACRO, Cat#NC1-H5257) mixed with an equal volume of complete Freund's adjuvant (CFA) into healthy alpacas, stimulating their immune systems to produce corresponding anti-NKp46 antibodies. A second booster immunization involved subcutaneous injection of 0.25 mg of human NKp46 antigen mixed with an equal volume of incomplete Freund's adjuvant (IFA) into alpacas 20 days after the first immunization. Subsequently, third and fourth booster immunizations were administered at 20-day intervals, using the same antigen dosage and adjuvant as for the second immunization. Serum serum serum was collected for target antibody titers before immunization, and one week after the second, third, and fourth immunizations.

[0243] Serum titers were assessed using ELISA plates coated with immobilized NKp46 antigens from human (ACRO, Cat#NC1-H82F9) and cynomolgus macaque (ACRO, Cat#NC1-C82E5) species, respectively. Antibody titers against the antigens from different species were measured using an HRP-labeled goat anti-Alpaca secondary antibody. The results of serum titers from the second, third, and fourth immunizations are shown in Tables 1 to 3. Serum titers demonstrated that alpaca immunization with human NKp46 antigen produced high-titer anti-human NKp46 antibodies and moderate-titer anti-monkey NKp46 antibodies in serum. Sequence alignment of the extracellular regions of NKp46 antigens from human, cynomolgus macaque, and mouse revealed a sequence similarity of 87.29% between the human and monkey versions, and 62.71% between the human and mouse versions. The sequence alignment is shown in Figure 1. Alpaca serum antibody titers met expectations, and the peripheral blood from the fourth immunization was suitable for antibody library construction.

[0244] Table 1 - Alpaca secondary immune serum titer test

[0245] Table 2 - Alpaca three-immune serum titer test

[0246] Table 3 - Alpaca four-immune serum titer test

[0247] Example 2. Construction and screening of anti-NKp46 single domain antibody yeast library

[0248] 50 ml of peripheral blood from alpacas after four immunizations was collected to separate PBMC (Peripheral Blood Mononuclear Cell), and total RNA was extracted using RNAiso Plus reagent. TMII 1st Strand cDNA Synthesis Kit (Takara, Cat#6210A) was used to reverse transcribe 5 μg of total RNA into cDNA. The operation steps were referred to the kit instructions. Using cDNA as a template, 5 μl of 5-fold diluted cDNA was used for the first round of nested PCR. The PCR product fragment of about 750 bp was glued back as a template for the second round of nested PCR. After two rounds of nested PCR amplification, a single domain antibody fragment was obtained. After purification by the Cycle-Puer Kit, the PCR product was co-transfected into yeast competent cells together with the linearized vector fragment by electroporation, spread on a 200 mm SD-CAA plate, and the total volume of the plate was 4.5 mL. It was cultured at 30°C for 4 days. The reservoir capacity was calculated by taking 10 μL of the electroporated yeast cell suspension and gradient dilution and spreading it on a 90 mm plate. The total number of transformants measured by the plate counting method was 1.08 x 10 8 (Figure 2a). 48 single clones were randomly selected from the library plate for PCR identification, and the results showed that the insertion rate was 100% (Figure 2b). 60 positive PCR products were randomly selected for sequencing. After protein translation, the sequence was aligned with the amino acid sequence and a phylogenetic tree was drawn (Figure 2c). Based on the number of transformants in the library, the library insertion rate, and the diversity sequencing analysis results, the library capacity of the anti-NKp46 single domain antibody yeast library was 1.08 x 10 8 .

[0249] Antibody screening was performed using one round of magnetic bead enrichment followed by one round of flow cytometry. The first round of enrichment used 100 nM human NKp46 antigen and was performed by magnetic bead sorting. The yeast cell input was 1.38 x 10 10 cells. The second round of enrichment was performed on the magnetic bead enrichment library of the first round, using 100nM human NKp46 antigen. The proportion of double-positive cell populations was found to be 70.72% by flow sorting, and the double-positive monoclonal clone was sorted (Figure 3). Afterwards, the monoclonal clones were sequenced and sequence alignment analyzed, and the monoclonal cells corresponding to the unique sequences were further verified for binding activity to human, mouse, and monkey NKp46 antigens by flow cytometry. According to the sequence alignment and monoclonal flow cytometry binding activity verification analysis, a total of 136 human NKp46 antigen binding positive sequences were obtained (Table 4), involving a total of 71 CDR1, 34 CDR2 and 55 CDR3 sequences (using the IMGT numbering method)

[0250] Table 4. Sequences of human NKp46 antigen-binding positive single domain antibodies and their CDR sequences

[0251] Example 3. Construction of yeast expression strain for anti-NKp46 single domain antibody and protein expression and purification

[0252] The anti-NKp46 single-domain antibody gene sequence with an N-terminal his-tag was codon-optimized and constructed into the pPICZ alpha A plasmid. The plasmid was then linearized with Pem I enzyme. 5 μL of the linearized plasmid was added to 100 μL of competent cells and transformed into X33 Pichia pastoris cells using an electroporator (Biorad, MicroPulser) at 1.6 kV and 4 ms. The electroporated Pichia pastoris cells were restored with a mixed medium (YPD:Sorbitol = 1:1). Afterwards, 50 μL of the cells were plated onto YPD solid medium supplemented with 200, 400, 600, or 800 μg / mL zeocin (Invitrogen, Cat# R25001). Zeocin concentration gradient selection was performed to identify strains expressing high copies of the target gene.

[0253] Monoclonal screening was performed using GMGY medium (Sangon, Cat#B540130) to culture the monoclonal strain at 30°C and 250 rpm. After obtaining sufficient bacteria, GMMY medium (Sangon, Cat#B540131) was used to induce secretion and expression of the target single-domain antibody at 20°C and 250 rpm. After 24 hours, 20 uL of supernatant was collected and the expression level of each colony was analyzed by gel electrophoresis. The strain with the highest expression level was selected for bacterial maintenance and protein expression production.

[0254] The protein was expressed and purified in large quantities as follows: 400 μL of bacterial culture was inoculated into 200 mL of BMGY medium and cultured for enrichment at 30°C and 250 rpm for 3 days. Subsequently, 80 mL of BMMY medium was replaced to induce expression, supplemented with 1% methanol every 24 hours. Secretory expression of the target single-domain antibody was induced at 20°C and 250 rpm for 3 days. After induction, the supernatant was collected by centrifugation at 12,000 rpm for 15 minutes in a high-speed refrigerated centrifuge. The protein was then purified using nickel affinity chromatography (Cytiva, Cat# 17092108). The nickel affinity column was equilibrated with binding buffer before the supernatant containing the target protein was passed through the column. The His-tagged protein was retained on the column by binding to nickel ions. Nonspecific binding to the column was then removed with a buffer containing 20 mM imidazole. Finally, the target protein was eluted with an elution buffer containing 250 mM imidazole.

[0255] Example 4. Analysis of the binding activity of anti-NKp46 single domain antibodies with human, monkey and mouse NKp46 recombinant proteins

[0256] Prepare biotinylated human NKp46-His (ACRO, Cat#NP6-H82H3), monkey NKp46-His (ACRO, Cat#NC1-C82E5), and mouse NKp46-His (ACRO, Cat#NC1-M82H5) antigen solutions in PBS to a final concentration of 1 μg / mL. Add 25 μl / well to a 384-well microtiter plate and coat overnight at 4°C. Wash three times with PBST (PBS + 0.05% Tween 20), add 50 μl / well of blocking solution (PBST + 3% BSA), and block at room temperature for 1 hour. Wash three times with PBST, add 25 μl / well of serially diluted single-domain antibody solution, and incubate at room temperature for 1 hour. The plates were washed three times with PBST, and 25 μl / well of a 1:5000 dilution of horseradish peroxidase-labeled rabbit anti-camelid VHH antibody (Genscript, Cat#A02016) was added and incubated at room temperature for 1 hour. The plates were washed three times with PBST, patted dry, and 25 μl / well of the chromogenic substrate TMB solution (Beyotime, Cat#P0209) was added. The plates were allowed to develop at room temperature for 5 to 30 minutes, followed by the addition of 25 μl / well of a color stop solution (Beyotime, Cat#P0215).

[0257] Absorbance was measured at 450 nm for each well using a multi-function microplate reader (Molecular Devices, SpectraMax i3x). Binding EC50 values ​​were calculated using a sigmoidal curve fit using a four-parameter equation using GraphPad Prism 9 software (Table 5). The results showed that all 19 candidate antibodies exhibited varying degrees of cross-species binding activity among human, monkey, and mouse (Figures 4A-4C).

[0258] Table 5. EC50 of single domain antibodies binding to human, monkey and mouse NKp46 proteins

[0259] Example 5. Analysis of the binding activity of anti-NKp46 single-domain antibodies to human NKp46 high-expressing cells

[0260] hNKp46 CHO-K1 cells were obtained by stably transfecting CHO-K1 cells with the pIRES-Neo3 vector expressing the human NKp46 gene (NM_004829.7). Cells in the logarithmic growth phase were collected, washed with flow cytometry buffer (PBS + 2% FBS), and the cell density was adjusted to 1×10 6 cells / mL, add 180 μl / well of the cell suspension to a 96-well U-bottom plate. Dilute the test sample stock solution with flow cytometry buffer to prepare a serial dilution of the antibody solution at 10* concentration. Add 20 μl of this solution to the cell suspension in the 96-well plate, vortex to mix, and incubate the 96-well plate at 4°C for 30 minutes. Centrifuge at 1000 rpm at 4°C for 5 minutes, discard the supernatant, wash the cells twice with flow cytometry buffer, then add 200 μl / well of a 1:1000 dilution of iFluor 647-conjugated rabbit anti-camelid VHH antibody (Genscript, Cat# A02019), vortex to mix, and incubate the 96-well plate at 4°C for 30 minutes. Centrifuge at 1000 rpm at 4°C for 5 minutes, discard the supernatant, wash the cells twice with flow cytometry buffer, and resuspend the cells in 200 μl / well of flow cytometry buffer. Measure the mean fluorescence intensity of each sample using a flow cytometer (BD, FACSCelesta). The binding EC50 values ​​were calculated using a sigmoidal 4-parameter equation fitted with GraphPad Prism 9 software.

[0261] The experimental results showed that all 19 candidate antibodies had varying degrees of cell binding ability ( FIG5 ).

[0262] Example 6. ELISA analysis of the binding competition relationship between three anti-NKp46 single-domain antibodies

[0263] NKp46-209, NKp46-645, and NKp46-681 single-domain antibody solutions were prepared in PBS to a final concentration of 1 μg / mL. 25 μl / well was added to a 384-well microtiter plate and coated overnight at 4°C. The plates were washed three times with PBST (PBS + 0.05% Tween 20). 50 μl / well of blocking buffer (PBST + 3% BSA) was added and blocked at room temperature for 1 hour. The plates were then washed three times with PBST. A serial dilution of the single-domain antibody solutions (lib052 was used as a non-competitive control) and a 2 nM biotinylated NKp46 antigen (ACRO, Cat#NP6-H82H3) solution were prepared in PBST buffer containing 1.5% BSA.

[0264] The single domain antibody gradient dilution solution was mixed with 2nM biotin-labeled NKp46 antigen solution and incubated at room temperature for 1 hour. In a 384-well plate with the blocking solution removed, 25ul / well of the single domain antibody antigen mixed solution was added and incubated at room temperature for 1 hour. Washed 3 times with PBST, 25ul / well of a 1:1000 diluted horseradish peroxidase-labeled streptavidin solution was added and incubated at room temperature for 1 hour. Washed 3 times with PBST, patted dry, and 25ul / well of the chromogenic substrate TMB solution (Beyotime, Cat#P0209) was added. The plate was developed at room temperature for 5 to 30 minutes, followed by the addition of 25ul / well of the chromogenic stop solution (Beyotime, Cat#P0215).

[0265] The absorbance of each well was measured at 450 nm using a multifunctional microplate reader (Molecular Devices, SpectraMax i3x), and the binding EC50 value was calculated using a sigmoidal curve 4-parameter equation fitted with GraphPad Prism 9 software.

[0266] The experimental results showed that the three candidate antibodies were in a competitive relationship with each other, and the three candidate binding epitopes were predicted to be similar or identical ( Figures 6A-6C ).

[0267] Example 7. Coupling of anti-NKp46 single domain antibodies to nucleic acids

[0268] The present invention uses a tetramer PMO nucleic acid framework, and the sequences of the four nucleic acid single strands are as follows (from 5' to 3'):

[0269] Chain 1 (PMO1):

[0270] 5'-CCTCAGTGACGTACTATGTGGCGGT-3'(SEQ ID NO:297)

[0271] Chain 2 (PMO2):

[0272] 5'-ACCGCCACATAGATACGAGACGCTG-3'(SEQ ID NO:298)

[0273] Chain 3 (PMO3):

[0274] 5'-CAGCGTCTCGTAAGGTTCCGGTGA-3'(SEQ ID NO:299)

[0275] Chain 4 (PMO4):

[0276] 5'-TCACACGGAACCAACGTCACTGAGG-3'(SEQ ID NO:300)

[0277] The 5' end of the nucleic acid chain is modified with an NH2 group for coupling with a single domain antibody.

[0278] Dissolve the 5'-terminus NH2-modified PMO single chain in phosphate buffer (50 mM NaH2PO4, 150 mM NaCl, pH 7.4) to a final concentration of 1 mM. Dissolve SM(PEG)2 (linker molecule) powder in dimethyl sulfoxide (DMSO) to prepare a fresh 250 mM SM(PEG)2 stock solution. Add a 10- to 50-fold molar amount of SM(PEG)2 to the PMO single chain stock solution, mix rapidly, and react at room temperature for 30 minutes to 2 hours. After the reaction is complete, add 10% of the volume of 1M Tris-HCl (pH 7.0), mix, and incubate at room temperature for 20 minutes to terminate the reaction with excess SM(PEG)2. After incubation, remove unreacted SM(PEG)2 linker by acetone precipitation, and purify the SM(PEG)2-PMO conjugate for later use.

[0279] Cysteine ​​mutations were introduced into the carboxyl termini of NKp46-209, NKp46-645, and NKp46-681 single-domain antibodies for nucleic acid coupling. The single-domain antibody expression and purification methods were the same as in Example 3.

[0280] The single-domain antibody was dialyzed using a dialysis buffer containing a reducing agent (20mM Tris, 15mM NaCl, pH 7.4). During the dialysis process, the C-terminal sulfhydryl group was reduced, and impurities such as free -SH groups were removed. The reduced single-domain antibody was mixed with SM(PEG)2-PMO single chain in a molar ratio of 1:1-2, mixed evenly, and reacted at room temperature for 2 hours. Unreacted SM(PEG)2-PMO single chain was removed using a His-tag affinity column, and the single-domain antibody and single-domain antibody-PMO mixture were collected. The single-domain antibody and single-domain antibody-PMO conjugate were separated using Butyl4FF (Cytiva), and the final product was verified for sample purity by SDS-PAGE (Figure 7).

[0281] Single domain antibody-PMO conjugates targeting CD16a and CD30 were prepared respectively, using the same preparation methods as above.

[0282] The anti-CD16a single domain antibody sequence is as follows:

[0283] The anti-CD30 single domain antibody sequence is as follows:

[0284] The underlined part is the His tag.

[0285] Example 8. Assembly of NKp46 / CD30 / CD16a trispecific antibody

[0286] The trispecific antibody molecules are assembled by the PMO nucleic acid framework (Figure 8, left). In the multispecific molecule in Figure 8, left, CD16a, CD30, and NKp46 represent their corresponding single-domain antibodies.

[0287] This example describes the self-assembly of a trispecific antibody molecule, using molecule C as an example. The concentrations of PMO1, anti-CD16a single-domain antibody PMO2, NKp46-645-PMO3, and anti-CD30 single-domain antibody PMO4 were measured using a NanoDrop One (Thermo). Appropriate amounts of these components were preheated at 37°C for 5 minutes, then mixed at a molar ratio of 1:1:1:1 at 37°C and incubated for 1 minute to complete the self-assembly of molecule C.

[0288] The assembly method of molecules A, B, D, and E is the same as above. The required assembly modules are selected for assembly. After assembly, the homogeneity of each sample is verified by SDS-PAGE (Figure 8 right).

[0289] Example 9. Verification of the anti-tumor activity of the NKp46 / CD30 / CD16a trispecific antibody

[0290] CD30-expressing MJ cells (CTCC, Cat#CTCC-001-0685) were used as target cells and incubated with Calcein-AM reagent (Biyuntian, Cat#C2013L) at a dilution of 1:1000 at 37°C in the dark for 30 minutes. The cells were washed twice with PBS and resuspended in RPMI1640 medium (ThermoFisher, Cat#61870036) containing 10% FBS (ThermoFisher, Cat#10099141C). The cells were plated at a density of 40,000 cells / 80 μL / well in cell culture plates. A test substance group, a target cell spontaneous lysis group, a target cell maximum lysis group, and a culture medium control group were set up.

[0291] Prepare solutions of test substances A, B, C, D, and E at 10x the working concentration (1 μM) and add 20 μL of these solutions to the test substance groups, resulting in a test substance concentration of 2x the working concentration (200 nM). Add 20 μL of culture medium to the spontaneous target cell lysis group and 20 μL of 1% Triton X-100 solution to the maximum target cell lysis group. The culture medium control group, which contains no target cells, receives an equal volume of culture medium.

[0292] The cell culture plates were incubated at 37°C for 30 minutes. Human PBMCs (Rubai Biotechnology, Cat# PBMNC050C) were resuspended in RPMI1640 medium supplemented with 10% FBS and plated at a density of 400,000 cells / 100 μL / well in the test substance group, the target cell spontaneous lysis group, and the target cell maximum lysis group. An equal volume of culture medium was added to the culture medium control group. At this point, the test substance concentration was the final working concentration (100 nM). After gentle pipetting to mix, the cell culture plates were incubated at 37°C for 6 hours. The cell culture plates were centrifuged at 1000 rpm for 10 minutes at room temperature. 100 μL of the supernatant was transferred to a new black-bottomed clear plate. The fluorescence intensity of each well was read using a SpectraMax i3x multi-function microplate reader (Ex / Em = 490 nm / 515 nM).

[0293] Test article-mediated cytotoxicity (ADCC) was defined as the percentage of test article fluorescence intensity relative to the fluorescence intensity of the target cell maximal lysis group: ADCC (%) = (test article fluorescence intensity - target cell spontaneous lysis fluorescence intensity) / (target cell maximum lysis fluorescence intensity - target cell spontaneous lysis fluorescence intensity) * 100. Three replicate wells were used for the antibody test group, while six replicate wells were used for the spontaneous lysis and maximal lysis groups.

[0294] The experimental results showed that trispecific antibody drugs A, B, C, D, and E in the form of NAPPA (nucleic acid programmable protein array) can effectively mediate NK cell killing of MJ tumor cells, and the anti-tumor effects of trispecific antibody molecules C, D, and E containing anti-NKp46 single-domain antibodies are significantly better than those of trispecific antibody molecules A and B without anti-NKp46 single-domain antibodies (Figure 9).

[0295] Example 10. Humanization of anti-NKp46 single domain antibody

[0296] The humanization of the anti-NKp46 single-domain antibody was performed by aligning the parental sequence with the human Germline database to identify its CDR regions. Computer-assisted modeling was used to construct a 3D model of the non-humanized sequence and analyze key amino acid residues. The humanized template sequence with the highest match to the parental antibody sequence was identified. The parental CDR regions were then transplanted onto the humanized template sequence, and 11 sequences with a degree of humanization exceeding 95% were designed through backmutation (Table 6).

[0297] Table 6. Summary of humanized sequences of anti-NKp46 single domain antibodies

[0298] Example 11. Expression and purification of anti-NKp46 humanized VHH-Fc

[0299] The C-terminus of the humanized VHH sequence was fused to the human IgG1 Fc segment. After codon optimization, the fusion sequence was constructed into the pcDNA3.4 vector. The fusion expression plasmid was transiently transfected into Expi CHO cells for 7 days of expression. The expression medium was Expi CHO. TM Expression medium (Thermo fisher, Cat#A2910001), transfection kit is ExpiFectamine TM CHO Transfection Kit (Thermo Fisher, Cat# A29129). After the cells were expressed, the supernatant was centrifuged and filtered through a 0.22 μM filter membrane and then eluted with Protein A affinity filler (Cytiva, MabSelect SuRe TM ) Purification of humanized VHH-Fc protein.

[0300] Example 12. Stability Analysis of Anti-NKp46 Humanized VHH-Fc

[0301] The aggregation tendency of humanized VHH-Fc was analyzed by HPLC-SEC. The column used in the experiment was XBridge BEH SEC was performed using a 3.5 μm, 7.8 × 300 mm (Waters, 186007640) system with a flow rate of 0.8 mL / min and a detection wavelength of 280 nm. The results are shown in Table 7. The results indicate that all humanized antibodies exhibited 100% monomericity, indicating that the samples were relatively stable and did not readily form aggregates.

[0302] Table 7. Summary of HPLC-SEC results of anti-NKp46 humanized VHH-Fc

[0303] Note: “ / ” represents none.

[0304] The thermal stability of humanized VHH-Fc was further analyzed using differential scanning fluorimetry (DSF). The instrument used was an ABI7500 Fast Real-Time PCR instrument. The experiment was performed using a melting curve in continuous mode, with a scanning temperature of 25°C to 99°C. The temperature corresponding to the first peak and valley of the derivative of the melting curve was determined as the denaturation temperature (T) of the protein. m1 The temperature corresponding to the second peak valley is determined as the denaturation temperature T of the protein. m2 The temperature corresponding to the third peak valley is determined as the denaturation temperature T of the protein. m3 , the test results are shown in Table 8.

[0305] The results showed that all humanized versions of T m1 The range is between 59℃-63℃, among which the T m1 The value is 60.91℃; T of VHH1, 2, 8, 10 m1 The value is higher than that of the parent; T m1 The value is the same as the parent; T of other humanized versions m1 The value is lower than that of the mother plant.

[0306] Table 8. Summary of DSF results of anti-NKp46 humanized VHH-Fc

[0307] Example 13. Analysis of Binding Activity of Humanized VHH-Fc to Human and Monkey NKp46 Proteins

[0308] The binding activity of humanized VHH-Fc to human, monkey, and mouse NKp46 proteins was analyzed by enzyme-linked immunosorbent assay (ELISA). Biotinylated human NKp46-His (ACRO, Cat#NP6-H82H3), monkey NKp46-His (ACRO, Cat#NC1-C82E5), and mouse NKp46-His (ACRO, Cat#NC1-M82H5) antigen solutions were prepared in PBS to a final concentration of 1 μg / mL. 25 μl / well was added to a 384-well ELISA plate and coated overnight at 4°C. The plates were washed three times with PBST (PBS + 0.05% Tween 20), and 50 μl / well of blocking solution (PBST + 3% BSA) was added and blocked at room temperature for 1 hour. The plates were washed once with PBST, and 25 μl / well of serially diluted VHH-Fc solution was added and incubated at room temperature for 1 hour. The cells were washed 3 times with PBST, and 25 μl / well of a 1:5000 diluted horseradish peroxidase-labeled goat anti-human IgG antibody (Yeasen, Cat#33501ES60) solution was added and incubated at room temperature for 1 hour. The cells were washed 3 times with PBST, patted dry, and 25 μl / well of a chromogenic substrate TMB solution (Beyotime, Cat#P0209) was added. The cells were allowed to develop for 5 to 30 minutes at room temperature, followed by addition of 25 μl / well of a chromogenic stop buffer (Beyotime, Cat#P0215). The absorbance at 450 nm was measured in each well using a multifunctional microplate reader (Molecular Devices, SpectraMax i3x). The EC50 values ​​(Table 11) were calculated using the sigmoid curve 4-parameter equation fitted using GraphPad Prism 9 software.

[0309] The experimental results showed that all humanized VHH-Fc versions bound to NKp46-His of human, monkey and mouse origin. Except for VHH11, the binding activities of the three genera of the other 10 humanized versions were comparable to those of the parent version (Figures 10-12).

[0310] The binding activity of humanized VHH-Fc to human NKp46 protein was analyzed by surface plasmon resonance (SPR). The instrument used was a biacore T200 (Cytiva). The test results are shown in Table 9. All humanized VHH-Fcs bound to human NKp46-His. With the exception of VHH11, the binding activity of the other 10 humanized versions was comparable to that of the parental version.

[0311] Table 9. SPR detection results of anti-NKp46 humanized VHH-Fc

[0312] Example 14. Analysis of Binding Activity of Humanized VHH-Fc to Human TfR Highly Expressing Cells

[0313] hNKp46 CHO-K1 cells in the logarithmic growth phase were collected, washed with flow cytometry buffer (PBS + 2% FBS) and the cell density was adjusted to 1×10 6 cells / mL, add 180 μl / well of the cell suspension to a 96-well U-bottom plate. Dilute the test sample stock solution with flow cytometry buffer to prepare a serial dilution of the antibody solution at a 10x concentration. Add 20 μl of this solution to the cell suspension in the 96-well plate, vortex to mix, and incubate the 96-well plate at 4°C for 30 minutes. Centrifuge at 1000 rpm at 4°C for 5 minutes, discard the supernatant, wash the cells twice with flow cytometry buffer, then add 200 μl / well of a 1:1000 dilution of FITC-conjugated mouse anti-human IgG antibody (Biolegend, Cat#410720), vortex to mix, and incubate the 96-well plate at 4°C for 30 minutes. Centrifuge at 1000 rpm at 4°C for 5 minutes, discard the supernatant, wash the cells twice with flow cytometry buffer, and resuspend the cells in 200 μl / well of flow cytometry buffer. Measure the mean fluorescence intensity of each sample using a flow cytometer (BD, FACSCelesta). The sigmoidal curve 4-parameter equation was fitted and the binding EC50 values ​​were calculated using GraphPad Prism 9 software (Table 10).

[0314] The experimental results showed that all humanized VHH-Fcs bound to hNKp46 CHO-K1 cells with binding activity comparable to that of the parent VHH (Figure 13).

[0315] Table 10. Summary of protein and cell binding EC50 of anti-NKp46 humanized VHH-Fc

[0316] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An anti-NKp46 single-domain antibody, characterized in that The single domain antibody has three complementary determining regions (CDRs) derived from the VHH chain represented by the following amino acid sequences: SEQ ID NOs: 1-136; The CDRs are CDR1, CDR2 and CDR3 determined by any one of the IMGT rules, Kabat rules, Chothia rules, or AbM rules.

2. The single domain antibody according to claim 1, wherein The CDR1, CDR2 and CDR3 are selected from any one of the following CDR sequence combinations:

3. A polynucleotide, characterized in that The polynucleotide encodes the anti-NKp46 single-domain antibody according to claim 1.

4. An expression vector, characterized in that The expression vector contains the polynucleotide according to claim 3.

5. A host cell, characterized in that The host cell contains the expression vector according to claim 4, or the polynucleotide according to claim 3 is integrated into its genome.

6. A method for producing an anti-NKp46 single domain antibody, characterized in that: Including steps: (a) culturing the host cell according to claim 5 under conditions suitable for producing the single domain antibody, thereby obtaining a culture containing the anti-NKp46 single domain antibody; as well as (b) isolating or recovering the anti-NKp46 single domain antibody from the culture.

7. An immunoconjugate, characterized in that The immunoconjugate contains: (a) the anti-NKp46 single domain antibody according to claim 1; and (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an antisense oligonucleotide, a small interfering RNA, a microRNA, a nucleic acid aptamer, or an enzyme.

8. The use of the anti-NKp46 single domain antibody according to claim 1, wherein For preparing (a) a reagent for detecting NKP46 molecules; or (b) a drug for treating tumors, autoimmune diseases or rare diseases.

9. A multispecific antibody based on a compatible nucleic acid backbone, characterized in that: The multispecific antibody is a polymer formed by a complex of n monomers having mutually compatible nucleic acid backbones, where n is a positive integer of 2-8; Wherein, the polymer includes the following monomers: (a) a first monomer, comprising: (a1) a polypeptide element D1: an anti-NKp46 single domain antibody according to claim 1 that binds to a first target protein NKp46; and (a2) a first backbone nucleic acid single strand; (b) a second monomer comprising: (b1) a polypeptide element D2: an anti-CD16a antibody that binds to a second target protein CD16a; and (b2) a second backbone nucleic acid single strand; and (c) a third monomer, comprising: (c1) a polypeptide element D3: an antibody that binds to a third target protein, wherein the third target protein is a tumor-associated antigen (TAA); and (c2) a third backbone nucleic acid single strand; The polypeptide elements D1, D2 and D3 are covalently linked to the first, second and third backbone nucleic acid single strands, respectively.

10. A pharmaceutical composition, characterized in that include: (i) the anti-NKp46 single domain antibody according to claim 1, the immunoconjugate according to claim 7, or the multispecific antibody according to claim 9; and (ii) a pharmaceutically acceptable carrier.

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