Nectin-4 single-domain antibody and use thereof

Abstract

An anti-Nectin-4 single-domain antibody and the use thereof. Provided are a single-domain antibody specifically targeting Nectin-4, and amino acid sequences of a framework region FR and a complementarity-determining region CDR of a VHH chain thereof. The anti-Nectin-4 single-domain antibody can bind to a Nectin-4 antigen. An antibody-drug conjugate and a multi-specific antibody targeting the Nectin-4 target prepared by means of using the single-domain antibody have an excellent activity and high therapeutic efficacy.

Description

Nectin-4 single-domain antibody and its use Technical Field

[0001] The present invention relates to the field of biomedicine or biopharmaceutical technology, and in particular to a nectin-4 single-domain antibody and uses thereof. Background Art

[0002] Nectin-4 / PVRL4 is a transmembrane cell adhesion molecule that activates the PI3K / AKT pathway, promotes tumor angiogenesis, and participates in tumor cell growth, dissemination, and migration. Nectin-4 was originally identified as a receptor for the measles virus, mediating viral entry into breast and colorectal cancer cells via endocytosis and serving as a primary vector for viral infection and transmission. Nectin-4 is overexpressed in various cancers, including gastric, breast, lung, urothelial, and colorectal cancers, and is a clinically validated tumor target.

[0003] Leveraging the biological properties of Nectin-4, antibody drugs developed against Nectin-4 have shown significant efficacy in cancer treatment. In the EV-301 clinical trial, Enfortumab Vedotin (Padcev), which targets Nectin-4, demonstrated significant therapeutic efficacy in the treatment of advanced urothelial carcinoma. Compared with the chemotherapy group, the risk of death was reduced by 30%, and overall survival, progression-free survival, and overall efficacy were significantly prolonged. This became the first FDA-approved ADC drug targeting Nectin-4. Furthermore, based on promising preclinical data, 9MW2821 and BT8009 are undergoing Phase I / II clinical trials in patients with advanced solid tumors.

[0004] It is well known that the binding site barrier is a key obstacle hindering the development of ADCs. Relevant studies have shown that proteins with smaller molecular weights can penetrate tissues more deeply and bind more evenly to targets, resulting in higher diffusion rates and uniform tissue distribution. Camel-derived single-domain antibodies are the smallest naturally occurring antigen-binding fragments with stable structures and biological properties, and can be rapidly produced and conveniently engineered. Their heavy chain variable domains are highly homologous to human antibodies, making them easy to humanize and with minimal immunogenicity. In addition, their size and affinity kinetics can affect the targeting and penetration of solid tumors. Single-domain antibodies have been proven in many preclinical studies for the development of therapeutic drugs.

[0005] However, there is currently no research data on the use of single-domain antibodies targeting nectin-4 for the treatment of multiple diseases. Therefore, there is an urgent need to develop new single-domain antibody drugs targeting nectin-4 with high therapeutic effects. Summary of the Invention

[0006] The object of the present invention is to provide a single-domain antibody capable of specifically binding to nectin-4 and its use.

[0007] The first aspect of the present invention provides a complementary determining region (CDR) region of an anti-nectin-4 single domain antibody VHH chain, wherein the complementary determining region (CDR) region of the VHH chain is selected from the following group:

[0008] (1) CDR1 shown in SEQ ID NO. 17, CDR2 shown in SEQ ID NO. 18, and CDR3 shown in SEQ ID NO. 19; or

[0009] (2) CDR1 shown in SEQ ID NO. 9, CDR2 shown in SEQ ID NO. 10, and CDR3 shown in SEQ ID NO. 11; or,

[0010] (3) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3.

[0011] In another preferred embodiment, any one of the above amino acid sequences further includes a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one amino acid and can retain the nectin-4 binding affinity.

[0012] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-5 (such as 1-3, preferably 1-2, more preferably 1).

[0013] In another preferred embodiment, the derivative sequence that has been added, deleted, modified and / or substituted at least one amino acid and can retain the nectin-4 binding affinity is an amino acid sequence with a homology or sequence identity of at least 90%, preferably at least 95%, and more preferably at least 99%.

[0014] In another preferred embodiment, the antibody or antigen-binding fragment thereof is partially or fully humanized.

[0015] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0016] The second aspect of the present invention provides a VHH chain of an anti-nectin-4 single-domain antibody, wherein the VHH chain comprises a framework region FR and the complementarity determining region CDR according to the first aspect of the present invention.

[0017] In another preferred embodiment, the framework region FR includes:

[0018] (a) FR1 shown in SEQ ID NO: 20, FR2 shown in SEQ ID NO: 21, FR3 shown in SEQ ID NO: 22, and FR4 shown in SEQ ID NO: 23;

[0019] (b) FR1 shown in SEQ ID NO: 12, FR2 shown in SEQ ID NO: 13, FR3 shown in SEQ ID NO: 14, and FR4 shown in SEQ ID NO: 15;

[0020] (c) FR1 shown in SEQ ID NO: 4, FR2 shown in SEQ ID NO: 5, FR3 shown in SEQ ID NO: 6, and FR4 shown in SEQ ID NO: 7;

[0021] (d) FR1 set forth in SEQ ID NO:25, FR2 set forth in SEQ ID NO:30, FR3 set forth in SEQ ID NO:22, and FR4 set forth in SEQ ID NO:31;

[0022] (e) FR1 shown in SEQ ID NO: 28, FR2 shown in SEQ ID NO: 13, FR3 shown in SEQ ID NO: 14, and FR4 shown in SEQ ID NO: 26; or

[0023] (f) FR1 shown in SEQ ID NO: 25, FR2 shown in SEQ ID NO: 5, FR3 shown in SEQ ID NO: 6, and FR4 shown in SEQ ID NO: 26.

[0024] In another preferred example, the VHH chain sequence of the anti-nectin-4 single-domain antibody is shown in SEQ ID NO: 8, 16, 24, 27, 29 or 32.

[0025] The third aspect of the present invention provides an anti-nectin-4 single-domain antibody, which is a single-domain antibody targeting the nectin-4 epitope and has the VHH chain described in the second aspect of the present invention.

[0026] In another preferred embodiment, the anti-nectin-4 single-domain antibody includes a monomer, a bivalent body (bivalent antibody), a trivalent body (trivalent antibody) and / or a multivalent body (multivalent antibody).

[0027] In another preferred embodiment, the anti-nectin-4 single-domain antibody comprises one or more VHH chains having an amino acid sequence as shown in SEQ ID NO: 8, 16, 24, 27, 29 or 32.

[0028] In another preferred example, the VHH chain sequence of the anti-nectin-4 single-domain antibody is shown in SEQ ID NO: 8, 16, 24, 27, 29 or 32.

[0029] In another preferred example, the anti-nectin-4 single-domain antibody comprises two VHH chains having the amino acid sequences shown in SEQ ID NO: 8, 16, 24, 27, 29 and / or 32.

[0030] In another preferred example, the anti-nectin-4 single domain antibody has a VHH chain with an amino acid sequence as shown in SEQ ID NO: 8, 16, 24, 27, 29 and / or 32.

[0031] In another preferred embodiment, the two VHH chains having the amino acid sequences shown in SEQ ID NO: 8, 16, 24, 27, 29 and / or 32 are connected via a linker.

[0032] In another preferred embodiment, the linker is a peptide linker.

[0033] In another preferred embodiment, the peptide linker has 1-50 amino acids, preferably 1-20 amino acids.

[0034] In another preferred embodiment, the linker has a structure of (GGGGS)n, wherein n is a positive integer of 1-5.

[0035] In another preferred example, the sequence of the connecting peptide is GGGGSGGGGSGGGGSGGGGS.

[0036] In another preferred example, the anti-nectin-4 single domain antibody has an amino acid sequence as shown in SEQ ID NO: 33, 34 or 35.

[0037] A fourth aspect of the present invention provides an anti-nectin-4 single-domain antibody Fc fusion protein, wherein the structure of the fusion protein from N-terminus to C-terminus is as shown in Formula Ia or Ib:

[0038] ALB(Ia);

[0039] BLA(Ib);

[0040] in,

[0041] A is the anti-nectin-4 single domain antibody described in the third aspect of the present invention;

[0042] B is the Fc fragment of IgG; and

[0043] L is no or flexible joint.

[0044] In another preferred embodiment, the flexible linker is a peptide linker.

[0045] In another preferred embodiment, the peptide linker has 1-50 amino acids, preferably 1-20 amino acids.

[0046] In another preferred embodiment, the IgG Fc fragment includes the human IgG Fc fragment.

[0047] In another preferred embodiment, the peptide linker has a structure of (GGGGS)n, wherein n is a positive integer of 1-5.

[0048] In another preferred embodiment, the IgG Fc fragment includes the human IgG Fc fragment.

[0049] In another preferred embodiment, the IgG Fc fragment is selected from the following group: IgG1, IgG2, IgG3, IgG4 Fc fragment, or a combination thereof.

[0050] In another preferred embodiment, the Fc fragment of IgG is IgG4.

[0051] In another preferred embodiment, the fusion protein is a single-domain antibody Fc fusion protein targeting the nectin-4 epitope.

[0052] The fifth aspect of the present invention provides a multivalent antibody, which comprises the CDR region of the anti-nectin-4 single-domain antibody VHH chain according to the first aspect of the present invention, the VHH chain of the anti-nectin-4 single-domain antibody according to the second aspect of the present invention, the anti-nectin-4 single-domain antibody according to the third aspect of the present invention, and / or the fusion protein according to the fourth aspect of the present invention.

[0053] As used herein, "multivalent antibody" and "multispecific antibody" are used interchangeably.

[0054] In another preferred embodiment, the structure of the multivalent antibody from N-terminus to C-terminus is as shown in Formula Ic or Id:

[0055] Ab1-L-Ab2(Ic);

[0056] Ab2-L-Ab1(Id);

[0057] in,

[0058] Ab1 comprises the CDR region of the VHH chain of the anti-nectin-4 single-domain antibody according to the first aspect of the present invention, or the VHH chain of the anti-nectin-4 single-domain antibody according to the second aspect of the present invention, or is the anti-nectin-4 single-domain antibody according to the third aspect of the present invention;

[0059] Ab2 is another single domain antibody; and

[0060] L is no or flexible joint.

[0061] In another preferred embodiment, the flexible linker is a peptide linker.

[0062] In another preferred embodiment, the peptide linker has 1-50 amino acids, preferably 1-20 amino acids.

[0063] In another preferred embodiment, the Ab2 is an albumin single domain antibody.

[0064] In another preferred embodiment, the albumin comprises human albumin.

[0065] In another preferred embodiment, the peptide linker has a structure of (GGGGS)n, wherein n is a positive integer of 1-5.

[0066] In another preferred example, the amino acid sequence of the multivalent antibody is shown in SEQ ID NO: 36, 37 or 38.

[0067] The sixth aspect of the present invention provides a polynucleotide, which encodes a protein selected from the following group: the CDR region of the anti-nectin-4 single-domain antibody VHH chain of the first aspect of the present invention, the VHH chain of the anti-nectin-4 single-domain antibody of the second aspect of the present invention, the anti-nectin-4 single-domain antibody of the third aspect of the present invention, the fusion protein of the fourth aspect of the present invention, and / or the multivalent antibody of the fifth aspect of the present invention.

[0068] In another preferred embodiment, the polynucleotide comprises DNA or RNA.

[0069] The seventh aspect of the present invention provides an expression vector, which contains the polynucleotide described in the sixth aspect of the present invention.

[0070] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or a combination thereof.

[0071] Preferably, the expression vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0072] The eighth aspect of the present invention provides a host cell, which contains the expression vector described in the seventh aspect of the present invention, or the polynucleotide described in the sixth aspect of the present invention is integrated into its genome.

[0073] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0074] 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.

[0075] In another preferred embodiment, the prokaryotic cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or a combination thereof.

[0076] In another preferred embodiment, the eukaryotic cell is selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma, or a combination thereof.

[0077] In another preferred embodiment, the eukaryotic cells are selected from the following groups: insect cells such as fall armyworm, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or a combination thereof.

[0078] In another preferred embodiment, the host cell is preferably a mammalian cell, more preferably a HEK293 cell, a CHO cell, a BHK cell, a NSO cell or a COS cell.

[0079] In another preferred embodiment, the host cell is Pichia pastoris.

[0080] A ninth aspect of the present invention provides a method for producing an anti-nectin-4 single domain antibody or an Fc fusion protein thereof, comprising the steps of:

[0081] (a) culturing the host cell according to the eighth aspect of the present invention under conditions suitable for producing the single-domain antibody or Fc fusion protein thereof, thereby obtaining a culture containing the anti-nectin-4 single-domain antibody or Fc fusion protein thereof;

[0082] (b) isolating or recovering the anti-nectin-4 single domain antibody or Fc fusion protein thereof from the culture; and

[0083] (c) Optionally, purifying and / or modifying the anti-nectin-4 single domain antibody or Fc fusion protein thereof obtained in step (b).

[0084] The tenth aspect of the present invention provides an immunoconjugate, which comprises:

[0085] (a) the VHH chain of the anti-nectin-4 single domain antibody according to the second aspect of the present invention, the anti-nectin-4 single domain antibody according to the third aspect of the present invention, the fusion protein according to the fourth aspect of the present invention, or the multivalent antibody according to the fifth aspect of the present invention; and

[0086] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

[0087] In another preferred embodiment, the radioactive nuclides include:

[0088] (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or

[0089] (ii) therapeutic isotopes selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or a combination thereof.

[0090] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0091] In another preferred embodiment, the drug is a cytotoxic drug.

[0092] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of 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.

[0093] 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.

[0094] 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, chlortetracycline ... 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.

[0095] In another preferred embodiment, the coupling moiety is a detectable label.

[0096] In another preferred embodiment, the coupling portion is selected from the following group: 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.), 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)) or any form of nanoparticles.

[0097] In another preferred embodiment, the immunoconjugate contains: a multivalent (such as bivalent or tetravalent) VHH chain of the anti-nectin-4 single domain antibody as described in the second aspect of the present invention, the anti-nectin-4 single domain antibody as described in the third aspect of the present invention, the fusion protein as described in the fourth aspect of the present invention, or the multivalent antibody as described in the fifth aspect of the present invention.

[0098] In another preferred embodiment, the multivalency refers to the presence of multiple repeats of the VHH chain of the anti-nectin-4 single domain antibody described in the second aspect of the present invention, the anti-nectin-4 single domain antibody described in the third aspect of the present invention, the fusion protein described in the fourth aspect of the present invention, or the multivalent antibody described in the fifth aspect of the present invention in the amino acid sequence of the immunoconjugate.

[0099] The eleventh aspect of the present invention provides an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, wherein the structure of the antibody-drug conjugate is shown in Formula I: Ab-(JU)n (I)

[0100] Where,

[0101] Ab is anti-nectin-4 antibody;

[0102] U is the drug;

[0103] J is a chemical bond or linker;

[0104] n is 0 or a positive integer;

[0105] “-” represents a chemical bond, a linker, or a connector.

[0106] In another preferred embodiment, the antibody includes a monospecific antibody, a bispecific antibody, or a multispecific antibody (such as a trispecific antibody).

[0107] In another preferred embodiment, the antibodies include: monoclonal antibodies, single-chain antibodies (scFv), and nanobodies.

[0108] In another preferred embodiment, the antibody includes a monovalent, bivalent, or multivalent antibody.

[0109] In another preferred embodiment, the antibody comprises a multimeric antibody.

[0110] In another preferred embodiment, the antibody specifically binds to Nectin-4.

[0111] In another preferred embodiment, the antibody includes a nectin-4 monovalent antibody, a bivalent antibody, and / or a multivalent antibody.

[0112] In another preferred embodiment, the nectin-4 is human nectin-4 or non-human mammalian nectin-4 (such as mouse nectin-4).

[0113] In another preferred embodiment, the antibody is a human or non-human mammal antibody.

[0114] In another preferred embodiment, the non-human mammal is selected from the group consisting of camel, alpaca, mouse, and cynomolgus monkey.

[0115] In another preferred embodiment, the antibody is a nectin-4 antibody or an antibody derived therefrom.

[0116] In another preferred embodiment, the derivative antibody is a modified Nectin-4 antibody, including but not limited to linking the Nectin-4 antibody to an Fc fragment, human serum albumin, polyethylene glycol (PEG), to form a bivalent antibody and / or a multivalent antibody.

[0117] In another preferred embodiment, the antibodies include humanized antibodies, camel-derived antibodies, and chimeric antibodies.

[0118] In another preferred embodiment, any one of the above amino acid sequences further includes, optionally, a derivative sequence that has been subjected to addition, deletion, modification and / or substitution of at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid and can retain the ability to bind to nectin-4.

[0119] In another preferred example, the antibody sequence comprises an amino acid sequence having a sequence similarity of at least 90%, preferably at least 95%, more preferably at least 99% to SEQ ID NO: 8, 16, 24, 27, 29 and / or 32.

[0120] In another preferred embodiment, the drug is linked to the terminal amino group or side chain amino group of the heavy chain constant region or heavy chain variable domain of the anti-nectin-4 antibody.

[0121] In another preferred embodiment, the drug is linked to the sulfhydryl group of the anti-nectin-4 antibody.

[0122] In another preferred embodiment, the drug is site-specifically and / or randomly linked to the anti-nectin-4 antibody (ie, in Formula I, the U is site-specifically and / or randomly linked to Ab).

[0123] In another preferred embodiment, the U is site-specifically linked to Ab.

[0124] In another preferred embodiment, the chemical bond or linker includes GGC (glycine-glycine-cysteine), MPA-AEEA, MPA-AEEA-Val-Cit-PABC, or polyethylene glycol PEG.

[0125] In another preferred embodiment, the chemical bond or linker includes a derivative compound of MPA-AEEA, MPA-AEEA-Val-Cit-PABC, or polyethylene glycol PEG, including but not limited to replacement, modification or deletion of one or more groups based on each of them.

[0126] In another preferred embodiment, the degree of polymerization of the chemical bond is a positive integer greater than or equal to 1.

[0127] The twelfth aspect of the present invention provides the use of the VHH chain of the anti-nectin-4 single-domain antibody described in the second aspect of the present invention, the anti-nectin-4 single-domain antibody described in the third aspect of the present invention, or the fusion protein described in the fourth aspect of the present invention, or the multivalent antibody described in the fifth aspect of the present invention, for preparing a medicament for preventing or treating diseases or disorders associated with nectin-4 signaling.

[0128] The thirteenth aspect of the present invention provides a pharmaceutical composition, comprising:

[0129] (i) the complementarity determining region (CDR) of the anti-nectin-4 single domain antibody VHH chain according to the first aspect of the present invention, the VHH chain of the anti-nectin-4 single domain antibody according to the second aspect of the present invention, the anti-nectin-4 single domain antibody according to the third aspect of the present invention, or the fusion protein according to the fourth aspect of the present invention, or the multivalent antibody according to the fifth aspect of the present invention, or the immunoconjugate according to the tenth aspect of the present invention, or the antibody-drug conjugate according to the eleventh aspect of the present invention; and

[0130] (ii) a pharmaceutically acceptable carrier.

[0131] In another preferred embodiment, the conjugated moiety of the antibody-drug conjugate is a drug, a toxin, and / or a therapeutic isotope.

[0132] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0133] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug for preventing and / or treating diseases or conditions associated with nectin-4 signaling.

[0134] In another preferred embodiment, the diseases or conditions include but are not limited to: head and neck cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, colon cancer, reproductive system tumors, urinary system tumors, etc.

[0135] In another preferred embodiment, the head and neck cancer includes but is not limited to: head and neck squamous cell carcinoma, etc.

[0136] In another preferred embodiment, the pancreatic cancer includes but is not limited to metastatic pancreatic cancer, etc.

[0137] In another preferred embodiment, the reproductive system tumors include but are not limited to: endometrial papillary serous carcinoma (UPSC), cervical cancer, ovarian cancer, prostate cancer, penile squamous cell carcinoma, etc.

[0138] In another preferred embodiment, the urinary system tumors include but are not limited to urothelial cell carcinoma, bladder cancer, ureteral cancer, etc.

[0139] The fourteenth aspect of the present invention provides the use of one or more of the anti-nectin-4 single domain antibody according to the third aspect of the present invention, the fusion protein according to the fourth aspect of the present invention, and / or the multivalent antibody according to the fifth aspect of the present invention; for preparing

[0140] (a) A drug for preventing and / or treating a disease or condition associated with high expression of nectin-4;

[0141] (b) for detecting human nectin-4 molecules;

[0142] (c) for flow cytometry;

[0143] (d) used for cell immunofluorescence detection;

[0144] In another preferred embodiment, the use is diagnostic and / or non-diagnostic, and / or therapeutic and / or non-therapeutic.

[0145] A fifteenth aspect of the present invention provides a recombinant protein, wherein the recombinant protein has:

[0146] (i) the VHH chain according to the second aspect of the present invention, the anti-nectin-4 single domain antibody according to the third aspect of the present invention, the fusion protein according to the fourth aspect of the present invention, and / or the multivalent antibody according to the fifth aspect of the present invention; and

[0147] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0148] In another preferred embodiment, the tag sequence includes an Fc tag, an HA tag and a 6His tag.

[0149] In another preferred embodiment, the recombinant protein specifically binds to Nectin-4 protein.

[0150] In a sixteenth aspect, the present invention provides a use of the VHH chain of the anti-nectin-4 single-domain antibody according to the second aspect of the present invention, the anti-nectin-4 single-domain antibody according to the third aspect of the present invention, the fusion protein according to the fourth aspect of the present invention, the multivalent antibody according to the fifth aspect of the present invention, the immunoconjugate according to the tenth aspect of the present invention, and / or the antibody-drug conjugate according to the eleventh aspect of the present invention, for preparing a medicament, a reagent, a detection plate, or a kit;

[0151] Wherein, the reagent, detection plate or kit is used for: detecting Nectin-4 protein in a sample;

[0152] The medicament is used for treating and / or preventing diseases or disorders associated with nectin-4 signaling.

[0153] In another preferred embodiment, the diseases or conditions include but are not limited to: gastric cancer, cervical cancer, breast cancer, lung cancer, prostate cancer, colon cancer, endometrial serous papillary carcinoma, ovarian cancer, etc.

[0154] In another preferred embodiment, the detection includes flow cytometry detection and cell immunofluorescence detection.

[0155] A seventeenth aspect of the present invention provides a method for detecting Nectin-4 protein in a sample, the method comprising the steps of:

[0156] (1) contacting a sample with the VHH chain of the second aspect of the present invention, the single domain antibody of the third aspect of the present invention, the fusion protein of the fourth aspect of the present invention, the multivalent antibody of the fifth aspect of the present invention, the immunoconjugate of the tenth aspect of the present invention, and / or the antibody-drug conjugate of the eleventh aspect of the present invention;

[0157] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of nectin-4 protein in the sample.

[0158] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

[0159] The eighteenth aspect of the present invention provides a Nectin-4 protein detection reagent, the detection reagent comprising:

[0160] (i) the VHH chain of the second aspect of the invention, the single domain antibody of the third aspect of the invention, the fusion protein of the fourth aspect of the invention, the multivalent antibody of the fifth aspect of the invention, the immunoconjugate of the tenth aspect of the invention, and / or the antibody-drug conjugate of the eleventh aspect of the invention; and

[0161] (ii) a carrier that is acceptable for detection.

[0162] In another preferred embodiment, the conjugated portion of the immunoconjugate is a diagnostic isotope.

[0163] In another preferred embodiment, the assay-acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0164] 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.

[0165] In another preferred embodiment, the detection reagent is used for in vivo detection.

[0166] 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).

[0167] The nineteenth aspect of the present invention provides a kit for detecting Nectin-4 protein, the kit comprising the immunoconjugate as described in the tenth aspect of the present invention, the antibody-drug conjugate as described in the eleventh aspect of the present invention and / or the detection reagent as described in the eighteenth aspect of the present invention, and instructions.

[0168] In another preferred embodiment, the instructions state that the kit is used for non-invasively detecting the expression of Nectin-4 in a subject.

[0169] The twentieth aspect of the present invention provides a use of the immunoconjugate according to the tenth aspect of the present invention and / or the antibody-drug conjugate according to the eleventh aspect of the present invention for preparing a contrast agent for detecting nectin-4 protein in vivo.

[0170] In another preferred embodiment, the detection is used for the diagnosis or prognosis of diseases or disorders associated with Nectin-4 protein.

[0171] The twenty-first aspect of the present invention provides a method for treating a disease or condition associated with nectin-4 signaling, comprising administering to a subject in need thereof the single-domain antibody described in the third aspect of the present invention, the fusion protein described in the fourth aspect of the present invention, the multivalent antibody described in the fifth aspect of the present invention, the immunoconjugate described in the tenth aspect of the present invention, and / or the antibody-drug conjugate described in the eleventh aspect of the present invention, or the pharmaceutical composition described in the thirteenth aspect of the present invention.

[0172] In another preferred embodiment, the subject includes humans or non-human mammals.

[0173] In another preferred embodiment, the non-human mammals include rodents (such as mice and rabbits) and non-human primates (such as monkeys).

[0174] In the twenty-second aspect of the present invention, a chimeric antigen receptor (CAR) is provided, the antigen binding domain of which comprises the VHH chain of the anti-nectin-4 single-domain antibody described in the second aspect of the present invention, the anti-nectin-4 single-domain antibody described in the third aspect of the present invention, the fusion protein described in the fourth aspect of the present invention, and the multivalent antibody described in the fifth aspect of the present invention.

[0175] The twenty-third aspect of the present invention provides an engineered immune cell, the cell surface of which expresses the chimeric antigen receptor as described in the twenty-second aspect of the present invention.

[0176] In another preferred embodiment, the immune cells include T cells, NK cells, and macrophages.

[0177] In another preferred embodiment, the immune cells are from humans or non-human mammals.

[0178] 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

[0179] FIG1 is the result of flow cytometry detection of the binding activity of Nectin-4 single domain antibody to BT474 cells.

[0180] FIG2 is the result of flow cytometry detection of the binding activity of nectin-4 single domain antibody to NCI-N87 cells.

[0181] FIG3 is an SDS-PAGE detection diagram of the bivalent humanized nectin-4 single domain antibody.

[0182] FIG4 is the result of flow cytometry detection of the binding activity of bivalent humanized nectin-4 single domain antibody to BT474 cells.

[0183] FIG5 is the result of flow cytometry detection of the binding activity of bivalent humanized nectin-4 single domain antibody to NCI-N87 cells.

[0184] FIG6 is a schematic diagram of the structure of Nectin-4 NDC.

[0185] FIG7 is the result of flow cytometry detection of cell binding activity of Nectin-4 NDC.

[0186] FIG8 shows the affinity detection result of Nectin-4 NDC by BLI method.

[0187] FIG9 shows the imaging distribution results of Nectin-4 NDC in N87 tumor-bearing mice.

[0188] FIG10 shows the results of weight changes in mice in the N87 tumor mouse model after administration of Nectin-4 NDC.

[0189] FIG11 shows the results of the changes in tumor volume in mice of the N87 tumor mouse model after administration of Nectin-4 NDC.

[0190] FIG12 shows the results of the change in tumor weight in mice of the N87 tumor mouse model after administration of Nectin-4 NDC. DETAILED DESCRIPTION

[0191] After extensive and in-depth research and extensive screening, the inventors unexpectedly discovered a class of anti-nectin-4 single-domain antibodies for the first time. Experimental results demonstrate that the single-domain antibodies of the present invention can specifically recognize nectin-4 and have excellent cell-binding activity. The single-domain antibodies of the present invention are particularly suitable for the development and application of antibody-drug conjugates. After humanization and modification, antibody-drug conjugates using the humanized single-domain antibodies of the present invention as targeting elements exhibit excellent in vivo pharmacokinetics, achieving excellent in vivo tumor targeting and anti-tumor efficacy. This completes the present invention.

[0192] the term

[0193] In order to better understand the present invention, the following terms are defined.

[0194] Unless otherwise stated, all singular terms also include the plural, active and past tenses of the terms.

[0195] Unless the context clearly dictates otherwise, the term "about" includes values ​​that are within a standard deviation of the stated value.

[0196] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be understood to have an inclusive sense, rather than an exclusive or exhaustive sense; that is, the sense of "including, but not limited to." Unless otherwise stated, "comprising" includes "consisting of."

[0197] A "subject" or "patient" according to the present invention is an animal, including a human patient, in need of anti-cancer therapy or treatment. In certain aspects, the present invention may also be applied in veterinary practice to any mammal or other animal in need of such nectin-4-targeted anti-cancer therapy. This may include, for example, non-human primates, canines, felines, porcines, horses, and any other animal for which nectin-4-targeted anti-cancer therapy is desired.

[0198] As used herein, the terms "single domain antibody of the present invention", "single domain antibody of the present invention", "anti-nectin-4 single domain antibody of the present invention", "nectin-4 single domain antibody of the present invention", "anti-nectin-4 single domain antibody", and "nectin-4 single domain antibody" have the same meaning and can be used interchangeably, all referring to single domain antibodies that specifically recognize and bind to nectin-4 (including human nectin-4).

[0199] 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.

[0200] As used herein, the terms "single-domain antibody," "VHH," "nanobody," and "single-domain antibody" (sdAb, or nanobody) have the same meaning and are used interchangeably. They refer to the construction of a single-domain antibody (VHH) consisting solely of a single heavy chain variable region by cloning the variable region of an antibody heavy chain. This is the smallest fully functional antigen-binding fragment. Typically, antibodies naturally lacking the light chain and heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting solely of a single heavy chain variable region.

[0201] 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 entire antibody variable region. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conservative 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-folded configuration, connected by three CDRs that form a connecting loop, and in some cases can form a partial β-folded 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.

[0202] 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-nectin-4 antibodies or fragments thereof.

[0203] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.

[0204] As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0205] 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.

[0206] 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.

[0207] 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 the nectin-4 protein, such as proteins or polypeptides having a heavy chain variable region. These may or may not contain an initial methionine.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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.

[0213] The antibodies of the present invention refer to polypeptides that have nectin-4 binding activity and include the above-mentioned CDR regions. The term also includes variant forms of polypeptides that have the same function as the antibodies of the present invention and include the above-mentioned CDR regions. These variant forms include (but are not limited to): deletion, insertion and / or substitution of one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids with similar or similar properties generally does not change the function of the protein. For another example, adding one or several amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0214] 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.

[0215] 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.

[0216] 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.

[0217] Table 1

[0218] 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.

[0219] 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.

[0220] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0221] 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%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0222] 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.

[0223] 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.

[0224] 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.

[0225] 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.

[0226] 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.

[0227] 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 with CaCl2, 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.

[0228] 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.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] 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 (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), etc.

[0233] Nectin-4

[0234] Nectin-4 (Nectin cell adhesion molecule 4) is a cell adhesion molecule of the nectin and nectin-like families. It is expressed at high levels in normal embryonic and fetal tissues, but at very low levels in healthy adult tissues. Nectin-4 is a type I membrane protein. The extracellular domain of the dimeric nectin-4 is composed of three Ig domains: IgV and IgC. The metalloproteinase TACE / ADAM-17 degrades the extracellular domain to produce soluble nectin-4. This secreted form has been found in breast cancer patients. Nectin-4 is overexpressed in various tumor cells and can promote tumor cell growth and proliferation. Solid tumors that highly express nectin-4 include bladder cancer, pancreatic cancer, triple-negative breast cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, ovarian cancer, and many other cancers. Studies have shown that nectin-4 promotes tumor cell proliferation, differentiation, migration, and invasion by activating the PI3K / AKT pathway, and is believed to play a key role in cancer development and metastasis.

[0235] Pharmaceutical composition

[0236] 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. Generally, 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 nature 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): intraperitoneal, intravenous, or topical administration.

[0237] 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, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. 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.

[0238] 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.

[0239] Anti-Nectin-4 single domain antibody

[0240] In the present invention, the anti-nectin-4 single domain antibody includes monomers, bivalents (divalent antibodies), trivalents (trivalent antibodies) and / or multivalents (multivalent antibodies).

[0241] In a preferred embodiment of the present invention, the anti-nectin-4 single domain antibody comprises one, two or more VHH chains having an amino acid sequence as shown in 8, 16, 24, 27, 29 or 32.

[0242] Typically, the anti-nectin-4 single domain antibody comprises two VHH chains having the amino acid sequence shown in SEQ ID NO: 27, 29 and / or 32.

[0243] Typically, the anti-nectin-4 single domain antibody has a VHH chain with an amino acid sequence as shown in SEQ ID NO: 27, 29 and / or 32.

[0244] In a preferred embodiment of the present invention, the two VHH chains having the amino acid sequences shown in SEQ ID NO: 27, 29 and / or 32 are connected via a linker.

[0245] In a preferred embodiment of the present invention, the two VHH chains having the amino acid sequence shown in SEQ ID NO: 1 are connected via a linker.

[0246] In a preferred embodiment of the present invention, the linker has a structure of (GGGGS)n, wherein n is a positive integer of 1-5.

[0247] In a preferred embodiment of the present invention, the sequence of the connecting peptide is

[0248] GGGGSGGGGSGGGGSGGGGS.

[0249] In a preferred embodiment of the present invention, the anti-nectin-4 single domain antibody has an amino acid sequence as shown in SEQ ID NO: 33, 34 or 35.

[0250] Labeled single domain antibodies

[0251] 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.

[0252] Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, a single domain antibody against nectin-4 is labeled with colloidal gold to obtain a colloidal gold-labeled single domain antibody.

[0253] The novel Nectin-4 single-domain antibody of the present invention has good specificity and high titer.

[0254] Antibody Drug Conjugates

[0255] The present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention, also referred to as "Nanobody Drug Conjugate / NDC" in the present invention.

[0256] The term "antibody-drug conjugate" refers to a monoclonal antibody or antibody fragment linked to a biologically active toxic drug via a linker. The antibodies or antibody fragments described herein can be coupled to effector molecules by any means. For example, the antibody or antibody fragment can be attached to the toxic drug by chemical or recombinant means. Chemical methods for preparing fusions or conjugates are known in the art. The method used to couple the antibody or antibody fragment and the drug must be able to connect the antibody and the toxic drug without interfering with the ability of the antibody or antibody fragment to bind to the target molecule.

[0257] The drug can be any cytotoxic, cell growth inhibiting or immunosuppressive drug, such as. In an embodiment, a linker connects the antibody and the drug, and the drug has a functional group that can form a bond with the linker. For example, the drug can have an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, or a ketone group that can form a bond with the linker. In the case where the drug is directly connected to the linker, the drug has a reactive group that reacts before being connected to the antibody. Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc.

[0258] Cytotoxic drugs are substances that inhibit or prevent cell function and / or cause cell death or destruction. In principle, cytotoxic drugs can kill tumor cells at sufficiently high concentrations. However, due to their lack of specificity, they can also cause apoptosis of normal cells while killing tumor cells, leading to serious side effects. Cytotoxic drugs include toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, radioactive isotopes (such as At 211 , I 131 , I 125 、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 and radioactive isotopes of Lu), chemotherapeutic drugs, antibiotics, and nucleolytic enzymes.

[0259] The antibody of the present invention and the cytotoxic drug can be coupled via a coupling agent. Examples of the coupling agent can 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 can 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).

[0260] 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.

[0261] Antibodies can be coupled to drugs to form antibody-drug conjugates (ADCs). Typically, ADCs contain a linker (or linker) located between the drug and the antibody. The term "linker unit" or "linker fragment" or "linker unit" refers to a chemical structure fragment or bond that is connected to an antibody or its antigen-binding fragment at one end and to a drug at the other end, and can also be connected to other linkers before being connected to the drug. The linker can be a degradable or non-degradable linker. Degradable linkers are typically easily degraded in the intracellular environment, for example, the linker degrades at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including linkers containing peptide groups that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers, such as linkers containing glucuronides that can be degraded by glucuronidases. Peptide linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine or valine-alanine; or tripeptides such as glycine-phenylalanine-glycine; or tetrapeptides such as glycine-glycine-phenylalanine-glycine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at a pH of less than 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.

[0262] 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.

[0263] In the present invention, drug-linker compounds can be used to form ADCs in a single step. In other embodiments, bifunctional linker compounds can be used to form ADCs 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, the functional group on the linker reacts with the drug to form an ADC.

[0264] 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.

[0265] The present invention also provides a method for preparing an ADC, which may further comprise: combining an antibody with a drug-linker compound (or a drug-linker compound (LD)) under conditions sufficient to form an antibody conjugate (ADC).

[0266] In certain embodiments, the methods of the present invention comprise conjugating an antibody to a 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.

[0267] Drug loading, also known as the drug-to-antibody ratio (DAR), is the average number of drugs conjugated to each antibody in the ADC. It can be, for example, in the range of about 1 to about 10 drugs conjugated to each antibody, and in certain embodiments, in the range of about 1 to about 8 drugs conjugated to each antibody, preferably in the range of 2-8, 2-7, 2-6, 2-5, 2-4, 3-4, 3-5, 5-6, 5-7, 5-8, and 6-8. Exemplary, the drug loading can be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The ADC formula herein includes a collection of antibody drug conjugates within the aforementioned range. In embodiments herein, the drug loading can be expressed as n, which is a decimal or integer. Drug loading can be determined by conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assay, and HPLC.

[0268] In one embodiment herein, the cytotoxic drug is coupled to the antibody via a linker unit.

[0269] The loading capacity of the ligand drug conjugate can be controlled by the following non-limiting methods, including:

[0270] (1) Control the molar ratio of drug linker fragment and monoclonal antibody,

[0271] (2) Control reaction time and temperature,

[0272] (3) Select different reaction reagents.

[0273] Detection method

[0274] The present invention also relates to a method for detecting Nectin-4 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 Nectin-4 protein in the dissolved sample.

[0275] 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.

[0276] Reagent test kit

[0277] 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.

[0278] The present invention also provides a detection kit for detecting nectin-4 levels. The kit includes an antibody that recognizes the nectin-4 protein, a lysis medium for dissolving the sample, and common reagents and buffers required for detection, such as various buffers, a detection marker, and a detection substrate. The detection kit can be an in vitro diagnostic device.

[0279] application

[0280] As described above, the single-domain antibodies of the present invention have broad biological and clinical applications, encompassing a variety of fields, including the diagnosis and treatment of nectin-4-related diseases, basic medical research, and biological research. A preferred application is in clinical diagnosis and targeted therapy for nectin-4.

[0281] The main advantages of the present invention include:

[0282] (1) The single-domain antibody of the present invention can efficiently bind to the Nectin-4 protein on the cell surface.

[0283] (2) The single-domain antibody of the present invention is very suitable for the development of antibody-drug conjugates and multispecific antibody drugs targeting nectin-4.

[0284] (3) The antibody-drug conjugate prepared using the single-domain antibody of the present invention has excellent in vivo tumor targeting function and long half-life.

[0285] (4) The antibody-drug conjugate prepared using the single-domain antibody of the present invention has both high therapeutic effect and excellent safety against tumor models.

[0286] The following specific examples further illustrate the present invention. 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, were generally performed under conventional conditions, such as those described in (Sambrook and Russell et al., Molecular Cloning: A Laboratory Manual (3rd Edition) (2001) CSHL Press), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0287] Example 1: Establishment and screening of immune phage display library

[0288] The human nectin-4 extracellular domain gene was cloned into the pFUSE vector, mixed with polyethyleneimine (PEI), and transfected into HEK 293F cells to express the nectin-4-Fc fusion protein. The fusion protein was purified from the cell culture supernatant using protein A affinity chromatography. A Bactrian camel was injected weekly with a mixture of the nectin-4-Fc fusion protein and Freund's adjuvant. After seven immunizations, 100 mL of camel blood was collected to separate peripheral blood mononuclear cells (PBMCs), from which total RNA was extracted. This RNA was reverse-transcribed into cDNA and subjected to two rounds of PCR to generate heavy chain antibody VHH variable domain fragments. The phage vector pMECS containing the VHH fragments was transformed into Escherichia coli to generate a phage display library. The library was screened using phage display technology and identified by PE-ELISA. Finally, all positive clones were sequenced and expressed in E. coli for production and characterization of nectin-4 single-domain antibodies.

[0289] Example 2: Cellular Activity Identification of Candidate Nectin-4 Single Domain Antibodies

[0290] NCI-N87 and BT474 cells were cultured at 2 × 10 5Cells were seeded at a density of 100 μL in a 96-well plate and washed twice with PBS. The purified nectin-4 single-domain antibody was diluted with PBS containing 2% FBS. Subsequently, 100 μL of nectin-4 single-domain antibody solution was added to the 96-well plate and incubated at 4°C for 30 minutes. After two PBS washes, diluted APC anti-HA antibody was added and incubated at 4°C for 30 minutes. After two PBS washes, the cells were resuspended in PBS, mixed thoroughly, and analyzed by flow cytometry. The results are shown in Figures 1 and 2. The candidate single-domain antibodies Nb6, Nb10, and Nb26 all showed binding activity against NCI-N87 and BT474 cells.

[0291] Example 3: Modification of candidate Nectin-4 single domain antibodies

[0292] The above three single-domain antibodies were humanized, and the humanized transformation was carried out on the framework sequence of each nanobody, while keeping the variable region unchanged. The humanization method refers to the method of Example 4 in patent CN110144009A. The humanized antibody sequences are shown in Table 2 and Table 3 as SEQ ID NO: 27, SEQ ID NO: 29, and SEQ ID NO: 32, respectively. On this basis, a bivalent single-domain antibody was constructed to improve the antibody activity. The bivalent nectin-4 single-domain antibody is composed of two homologous humanized nectin-4 single-domain antibodies connected by a linker. The sequences after connection are shown in SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively. The recombinant antibody sequence was humanized, amplified, and ligated into the pPICZaA vector, and the vector was transformed into Pichia pastoris X-33 receptor cells, and the expression of the multivalent nectin-4 single-domain antibody was induced with methanol. After purification by protein A affinity chromatography, the purity of the multivalent nectin-4 single-domain antibody was assessed using SDS-PAGE. The purification results are shown in Figure 3, indicating that the humanized bivalent nectin-4 single-domain antibody was successfully constructed.

[0293] Table 2

[0294] Example 4: Activity identification of humanized bivalent antibodies

[0295] NCI-N87 and BT474 cells were cultured at 2 × 10 5Cells were seeded at a density of 100 μg / mL in a 96-well plate and washed twice with PBS. Purified humanized bivalent single-domain antibodies were diluted in PBS containing 2% FBS and added to the wells for incubation with the cells at 4°C for 30 minutes. After washing the cells twice with PBS, diluted goat anti-VHH polyclonal antibody was added and incubated at 4°C for 30 minutes. After washing twice with PBS, diluted donkey anti-goat IgG H&L antibody was added to each well and incubated at 4°C for 30 minutes. The cells were then resuspended in PBS, mixed thoroughly, and analyzed by flow cytometry.

[0296] The results are shown in Figures 4 and 5 . All three humanized bivalent nectin-4 single-domain antibodies showed excellent binding activity to NCI-N87 and BT474 cells, and unexpectedly, huNb26 / Nb26 showed the best binding activity in both NCI-N87 and BT474 cells.

[0297] Example 5: Construction of candidate Nectin-4 NDC and cell binding activity detection

[0298] The trivalent nectin-4 single-domain antibody is composed of a bivalent nectin-4 single-domain antibody and an HSA single-domain antibody connected by a linker. The sequences after connection are shown in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively.

[0299] The reaction was incubated overnight at 4°C in PBS containing TECP, followed by purification to remove excess TCEP. The reaction was then treated with an equimolar ratio of Mc-Vc-PBC-MMAE (VcMMAE) at room temperature for two hours. The reaction was terminated by the addition of an excess of acetylcysteine. The resulting NDC structure is shown in Figure 6.

[0300] NCI-N87 cells and 293T-human-Nectin-4 cells were seeded in 96-well plates at a density of 1E5 cells per well. After washing the cells, the trivalent nectin-4 single-domain antibody huNb26 / Nb26-Nbh (SEQ ID NO: 38) and nectin-4 NDC were diluted in PBS containing 2% FBS and added to the 96-well plates for 30 minutes. After washing the cells, a goat anti-VHH polyclonal antibody was added to each well and incubated for 30 minutes. Subsequently, diluted donkey anti-goat IgG H&L antibody was added to each well and incubated at 4°C for 30 minutes. Finally, the cells were resuspended for flow cytometry analysis.

[0301] The results are shown in Figure 7. After being linked to the HSA single-domain antibody, the trivalent nectin-4 single-domain antibody still has good cell binding activity, and the Nectin-4 NDC constructed on this basis retains cell binding activity that is basically equivalent to that of the trivalent nectin-4 single-domain antibody.

[0302] Example 6: Affinity of candidate Nectin-4 NDCs

[0303] The affinity of nectin-4 NDC for human nectin-4 antigen was assessed using the Fortebio Octet system. After equilibration of the streptavidin A biosensor with PBST buffer for 10 minutes, the biosensor streptavidin A was exposed to diluted biotinylated nectin-4 protein, mixed, and then immersed in PBST buffer. The biosensor was then bound to serially diluted nectin-4 NDC samples. The association rate (Kon), dissociation rate (Koff), and dissociation constant (Kd) were analyzed using ForteBio Data Analysis 9.0 software.

[0304] The results are shown in FIG8 , which show that the Nectin-4 NDC of the present invention exhibits a strong binding affinity to the human Nectin-4 antigen.

[0305] Example 7: Distribution of Candidate Nectin-4 NDC in Mouse N87 Tumor Model

[0306] To evaluate the distribution of nectin-4 NDC in animals, imaging was performed immediately before and after intravenous injection of Cy7-labeled nectin-4 NDC in mice bearing NCI-N87 human gastric cancer xenografts (n=3). Imaging was performed 0.5, 3, 6, 24, and 48 hours later. Images of three mice were acquired simultaneously using the IVIS Lumina system (Caliper Life Science), and data were analyzed using Living Image software.

[0307] The results are shown in Figure 9. Within 3 hours after intravenous injection, Nectin-4 NDC was gradually concentrated in the tumor site in the mouse N87 tumor model, indicating that Nectin-4 NDC has excellent in vivo targeting effect and a long half-life, and can stay in the body for up to 48 hours.

[0308] Example 8: Efficacy of Candidate Nectin-4 NDC in Mouse N87 Tumor Model

[0309] The anti-cancer effect of NDC in vivo was evaluated using a cell line xenograft model. NCI-N87 cells were mixed with Matrix and injected subcutaneously into 6-week-old female BALB / c nude mice. When the tumor volume reached 100 mm 3Mice were randomly divided into 5 groups (n=8 per group). The positive drug group was given 10 mg / kg 5-FU (5-fluorouracil), and the negative control group was given PBS. The experimental groups were given Nectin-4NDC at doses of 2.5 mg / kg, 5 mg / kg, and 10 mg / kg, respectively. All groups were dosed every other day (Q2D) for a total of 10 doses. The tumor size of individual mice was recorded and measured regularly. Finally, all mice were euthanized, and the tumor tissues were removed and weighed.

[0310] The results are shown in Figures 10 to 12. Compared with the positive drug group (10 mg / kg 5-FU), after administration of different doses of Nectin-4 NDC, the tumor volume and weight of mice in the 5 mg / kg dose group decreased significantly, and the tumor in the 10 mg / kg dose group almost completely disappeared, indicating that Nectin-4 NDC exhibited significantly better anti-tumor activity than the positive drug group in the mouse N87 tumor model. In addition, there was no significant decrease in mouse body weight in the Nectin-4 NDC experimental group compared with the control group (Figure 10), indicating that Nectin-4 NDC had no side effects on mouse body weight, that is, it had a better safety profile than the positive drug group.

[0311] The antibody sequences obtained by the present invention are shown in Table 3 below:

[0312] Table 3 Antibody sequences of the present invention Note: Underline indicates linker sequence.

[0313] 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. A complementary determining region (CDR) of an anti-nectin-4 single domain antibody VHH chain, characterized in that: The complementarity determining region (CDR) region of the VHH chain is selected from the following group: (1) CDR1 shown in SEQ ID NO. 17, CDR2 shown in SEQ ID NO. 18, and CDR3 shown in SEQ ID NO. 19; or (2) CDR1 shown in SEQ ID NO. 9, CDR2 shown in SEQ ID NO. 10, and CDR3 shown in SEQ ID NO. 11; or, (3) CDR1 shown in SEQ ID NO. 1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO.

3.

2. A VHH chain of an anti-nectin-4 single-domain antibody, characterized in that: The VHH chain comprises a framework region FR and a complementarity determining region CDR according to claim 1 .

3. The VHH chain according to claim 2, characterized in that The framework region FR includes: (a) FR1 shown in SEQ ID NO: 20, FR2 shown in SEQ ID NO: 21, FR3 shown in SEQ ID NO: 22, and FR4 shown in SEQ ID NO: 23; (b) FR1 shown in SEQ ID NO: 12, FR2 shown in SEQ ID NO: 13, FR3 shown in SEQ ID NO: 14, and FR4 shown in SEQ ID NO: 15; (c) FR1 shown in SEQ ID NO: 4, FR2 shown in SEQ ID NO: 5, FR3 shown in SEQ ID NO: 6, and FR4 shown in SEQ ID NO: 7; (d) FR1 set forth in SEQ ID NO:25, FR2 set forth in SEQ ID NO:30, FR3 set forth in SEQ ID NO:22, and FR4 set forth in SEQ ID NO:31; (e) FR1 shown in SEQ ID NO: 28, FR2 shown in SEQ ID NO: 13, FR3 shown in SEQ ID NO: 14, and FR4 shown in SEQ ID NO: 26; or (f) FR1 shown in SEQ ID NO: 25, FR2 shown in SEQ ID NO: 5, FR3 shown in SEQ ID NO: 6, and FR4 shown in SEQ ID NO:

26.

4. The VHH chain according to claim 1, wherein The VHH chain sequence of the anti-nectin-4 single domain antibody is shown in SEQ ID NO: 8, 16, 24, 27, 29 or 32.

5. An anti-nectin-4 single domain antibody, characterized in that: It is a single-domain antibody directed against the nectin-4 epitope and has the VHH chain according to claim 2.

6. The single domain antibody according to claim 5, wherein The anti-nectin-4 single-domain antibody has an amino acid sequence as shown in SEQ ID NO: 33, 34 or 35.

7. An anti-nectin-4 single domain antibody Fc fusion protein, characterized in that: The structure of the fusion protein from N-terminus to C-terminus is as shown in Formula Ia or Ib: ALB (Ia); BLA (Ib); in, A is the anti-nectin-4 single domain antibody according to claim 5; B is the Fc fragment of IgG; and L is no or flexible joint.

8. A multivalent antibody, characterized in that The multivalent antibody comprises the CDR region of the anti-nectin-4 single-domain antibody VHH chain of claim 1, the VHH chain of the anti-nectin-4 single-domain antibody of claim 2, the anti-nectin-4 single-domain antibody of claim 5, and / or the fusion protein of claim 7.

9. A polynucleotide, characterized in that The polynucleotide encodes a protein selected from the following group: the CDR region of the anti-nectin-4 single-domain antibody VHH chain of claim 1, the VHH chain of the anti-nectin-4 single-domain antibody of claim 2, the anti-nectin-4 single-domain antibody of claim 5, the fusion protein of claim 7, and / or the multivalent antibody of claim 8.

10. An expression vector, characterized in that The expression vector contains the polynucleotide according to claim 9.

11. A host cell, characterized in that The host cell contains the expression vector according to claim 10, or the polynucleotide according to claim 9 is integrated into its genome.

12. A method for producing an anti-nectin-4 single domain antibody or an Fc fusion protein thereof, comprising the steps of: (a) culturing the host cell according to claim 11 under conditions suitable for producing the single-domain antibody or Fc fusion protein thereof, thereby obtaining a culture containing the anti-nectin-4 single-domain antibody or Fc fusion protein thereof; (b) isolating or recovering the anti-nectin-4 single domain antibody or Fc fusion protein thereof from the culture; and (c) Optionally, purifying and / or modifying the anti-nectin-4 single domain antibody or Fc fusion protein thereof obtained in step (b).

13. An immunoconjugate comprising: (a) the VHH chain of the anti-nectin-4 single domain antibody according to claim 2, the anti-nectin-4 single domain antibody according to claim 5, the fusion protein according to claim 7, or the multivalent antibody according to claim 8; and (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, a gold nanoparticle / nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

14. An antibody-drug conjugate or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the antibody drug conjugate is shown in Formula I: Ab-(JU)n (I) Where, Ab is an anti-nectin-4 antibody; the antibody comprises the CDR region of the anti-nectin-4 single-domain antibody VHH chain of claim 1, the VHH chain of the anti-nectin-4 single-domain antibody of claim 2, the anti-nectin-4 single-domain antibody of claim 5, and / or the fusion protein of claim 7; U is the drug; J is a chemical bond or linker; n is 0 or a positive integer; "-" represents a chemical bond, a linker, or a connector.

15. Use of the VHH chain of the anti-nectin-4 single-domain antibody according to claim 2, the anti-nectin-4 single-domain antibody according to claim 5, the fusion protein according to claim 7, or the multivalent antibody according to claim 8, characterized in that: Used for preparing drugs for preventing or treating diseases or disorders related to nectin-4 signal transduction.

16. A pharmaceutical composition, characterized in that The pharmaceutical composition contains: (i) the complementarity determining region (CDR) of the anti-nectin-4 single domain antibody VHH chain of claim 1, the VHH chain of the anti-nectin-4 single domain antibody of claim 2, the anti-nectin-4 single domain antibody of claim 5, or the fusion protein of claim 7, or the multivalent antibody of claim 8, or the antibody-drug conjugate of claim 14; and (ii) a pharmaceutically acceptable carrier.

17. A method for detecting Nectin-4 protein in a sample, the method comprising the steps of: (1) contacting a sample with the VHH chain according to claim 2, the single domain antibody according to claim 5, the fusion protein according to claim 7, the multivalent antibody according to claim 8, the immunoconjugate according to claim 13, and / or the antibody-drug conjugate according to claim 14; (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of nectin-4 protein in the sample.

Images