Anti-nectin-4 antibodies and uses thereof

Abstract

The present application provides anti-Nectin-4 antibodies, nucleic acid molecules encoding the antibodies, vectors comprising the nucleic acid molecules, host cells comprising the nucleic acid molecules or vectors, and conjugates comprising the anti-Nectin-4 antibodies and pharmaceutical compositions thereof. The present application also provides methods of making the antibodies and uses thereof.

Description

[0001] Related applications

[0002] This application claims priority to Chinese application CN202510538389.3, filed on April 27, 2025. The entire contents of the aforementioned application are expressly incorporated herein by reference. Technical Field

[0003] This application relates to the field of biomedicine, specifically to anti-Nectin-4 antibodies and their uses. Background Technology

[0004] Nectin-4, also known as PVRL4, is a member of the Nectin family within the immunoglobulin superfamily. It promotes tumor proliferation, metastasis, and angiogenesis by activating pathways such as PI3K / AKT and WNT-β-catenin, and induces lymphatic metastasis through the CXCR4 / CXCL12 axis. As a tumor-specific target with high expression, Nectin-4 has therapeutic potential in various solid tumors.

[0005] VHH antibodies, also known as nanobodies, are single-domain antibodies composed solely of the heavy chain variable region (VHH). Due to their unique structure, nanobodies offer numerous advantages over traditional antibodies, such as smaller molecular size, stronger tissue penetration, superior stability, multivalent / multifunctional design, ease of humanization, and wide applicability. Therefore, nanobodies hold significant value in disease treatment and diagnosis, and are gradually becoming an emerging force in next-generation therapeutic biopharmaceuticals and clinical diagnostic reagents.

[0006] There is a need in this field to develop VHH antibody drugs targeting the Nectin-4 protein. Summary of the Invention

[0007] In view of this, in a first aspect, this application provides an anti-Nectin-4 antibody or an antigen-binding fragment thereof, which comprises a VHH chain containing complementarity-determining regions CDR1, CDR2 and CDR3, wherein the sequences of the CDRs are defined according to Kabat.

[0008] In some embodiments, the heavy chain variable region of the anti-Nectin-4 antibody provided in this application comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any of SEQ ID NO: 4-9, 13-15, 17-18, 22-24, 28-30, 32, 36-38, 42, 44, 46, 48, 49, and 51.

[0009] In other embodiments, the heavy chain variable region of the anti-Nectin-4 antibody consists of any of the amino acid sequences shown in SEQ ID NO: 4-9, 13-15, 17-18, 22-24, 28-30, 32, 36-38, 42, 44, 46, 48, 49 and 51.

[0010] In some implementations, the anti-Nectin-4 antibody is a VHH antibody.

[0011] In other embodiments, the anti-Nectin-4 antibody is in monovalent or multivalent (e.g., bivalent, trivalent, or tetravalent).

[0012] In some embodiments, the anti-Nectin-4 antibody is a VHH-Fc antibody.

[0013] In some specific implementations, the Fc fragment is derived from human IgG.

[0014] In some other specific embodiments, the Fc fragment is derived from human IgG1.

[0015] In some other specific embodiments, the Fc fragment has an amino acid sequence as shown in SEQ ID NO: 53.

[0016] In some implementations, the VHH-Fc antibody is in a multivalent (e.g., bivalent) form.

[0017] Secondly, this application provides isolated nucleic acid molecules that encode the aforementioned anti-Nectin-4 antibody or its antigen-binding fragment.

[0018] In some implementations, this application also provides a vector, such as an expression vector, which contains the aforementioned nucleic acid molecules.

[0019] In some embodiments, this application further provides a host cell containing the aforementioned nucleic acid molecules or vectors.

[0020] Thirdly, this application provides an antibody conjugate comprising the above-mentioned anti-Nectin-4 antibody or its antigen-binding fragment and effector.

[0021] In some embodiments, the effector is selected from one or more of the following: radionuclides, antitumor agents, immunomodulators, bioresponse modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, imaging agents, chemiluminescent compounds, enzymes, and metal ions.

[0022] In some embodiments, the anti-Nectin-4 antibody or its antigen-binding fragment and the effector are linked by a linker.

[0023] In some embodiments, the linker includes cleavable linkers and non-cleavable linkers. In specific embodiments, those skilled in the art select the type of linker based on the type of effector.

[0024] Fourthly, this application provides a radionuclide-labeled antibody conjugate comprising the above-mentioned anti-Nectin-4 antibody or its antigen-binding fragment and a radionuclide-chelating agent.

[0025] In some embodiments, the radionuclide-labeled antibody conjugate further comprises a linker.

[0026] In some embodiments, the radionuclides in the radionuclide-labeled antibody conjugates provided in this application include alpha-ray nuclides, gamma-ray nuclides, beta-ray nuclides, or positron-emitting nuclides.

[0027] Fifthly, this application provides a pharmaceutical composition comprising the above-mentioned anti-Nectin-4 antibody or its antigen-binding fragment, the above-mentioned nucleic acid molecule, a vector or host cell, the above-mentioned antibody conjugate, or the above-mentioned radionuclide-labeled antibody conjugate.

[0028] Sixthly, this application provides the use of the above-mentioned anti-Nectin-4 antibody or its antigen-binding fragment, the above-mentioned nucleic acid molecule, the above-mentioned carrier, the above-mentioned host cell, the above-mentioned antibody conjugate, the above-mentioned radionuclide-labeled antibody conjugate, or the above-mentioned pharmaceutical composition in the preparation of a medicament for the prevention, diagnosis and / or treatment of diseases characterized by Nectin-4 expression (e.g., Nectin-4 positive tumors).

[0029] In addition, this application also provides a method for preventing, diagnosing and / or treating diseases characterized by Nectin-4 expression (e.g., Nectin-4 positive tumors) in an individual, comprising administering the above-mentioned antibody conjugate, the above-mentioned radionuclide-labeled antibody conjugate, or a pharmaceutical composition thereof to an individual in need.

[0030] In some implementations, the diseases characterized by Nectin-4 expression involved in the above uses or methods provided in this application are selected from: head and neck squamous cell carcinoma, bladder cancer, urothelial carcinoma, breast cancer, non-small cell lung cancer (NSCLC), gastric cancer, esophageal cancer, ovarian cancer, cervical cancer, melanoma, pancreatic cancer, liver cancer, and prostate cancer. Attached Figure Description

[0031] Figure 1The results show the cell binding activity assays for monovalent VHH-human IgG1-Fc candidate antibodies. A represents AHP53752, AHP53759, AHP53815, AHP53824, AHP53835, and AHP53691; B represents AHP53744, AHP54695, AHP54726, AHP54938, AHP54962, and AHP54966.

[0032] Figure 2 The results show the endocytic activity assay of the monovalent VHH-human IgG1-Fc candidate antibody.

[0033] Figure 3 The SDS-PAGE results for purified antibodies are shown below. Lane M indicates the marker; lanes 1-6 indicate AHP53744-G4SC, AHP54695-G4SC, AHP54938-G4SC, AHP54962-G4SC, AHP54966-G4SC, and AHP53835-G4SC under reducing conditions, respectively; lanes 7-12 indicate AHP53744-G4SC, AHP54695-G4SC, AHP54938-G4SC, AHP54962-G4SC, AHP54966-G4SC, and AHP53835-G4SC under non-reducing conditions, respectively; and lane 13 indicates human IgG.

[0034] Figure 4 The binding activity of the VHH-G4SC conformation antibody to human Nectin-4 protein was measured.

[0035] Figure 5 The binding activity of the VHH-G4SC conformation antibody to T47D cells was measured.

[0036] Figure 6 The binding activity of the VHH-G4SC conformation antibody to HT1376 cells was measured.

[0037] Figure 7 The binding activity of the bivalent VHH-hIgG1-Fc antibody to human Nectin-4 protein (EC) 50 value).

[0038] Figure 8 Cell binding activity of AHP54695-h7m1-G4SC-NOTA / H3RESCA in SW780 cells.

[0039] Figure 9 PET / CT images at different time points after administration of AHP54695-h7m1-G4SC-NOTA.

[0040] Figure 10PET / CT images at different time points after administration of AHP54966-h1m1-G4SC-NOTA.

[0041] Figure 11 for[ 68 PET-CT images at different time points after administration of Ga]AHP54695-h7m1-G4SC-H3RESCA.

[0042] Figure 12 for[ 18 PET-CT images at different time points after administration of F]AHP54695-h7m1-G4SC-H3RESCA.

[0043] Sequence Description

[0044] SEQ ID NO: 1-3 show the amino acid sequences of CDR1, CDR2 and CDR3 of the parental antibody clone AHP53835 against Nectin-4, respectively.

[0045] SEQ ID NO: 4 shows the amino acid sequence of the VHH chain of the parental antibody clone AHP53835 against Nectin-4.

[0046] SEQ ID NO: 5-9 show the amino acid sequences of the VHH chains of humanized antibody clones and variants AHP53835-h2, AHP53835-h5, AHP53835-h7, AHP53835-h8 and AHP53835-h7m1 based on the anti-Nectin-4 maternal antibody clone AHP53835, respectively. SEQ ID NO: 1-3 show the amino acid sequences of CDR1, CDR2 and CDR3 of these humanized antibodies, respectively.

[0047] SEQ ID NO: 10-12 show the amino acid sequences of CDR1, CDR2 and CDR3 of the parental antibody clone AHP54695 against Nectin-4, respectively.

[0048] SEQ ID NO: 13 shows the amino acid sequence of the VHH chain of the parental antibody clone AHP54695 against Nectin-4.

[0049] SEQ ID NO: 14, 15, 17 and 18 show the amino acid sequences of the VHH chains of humanized antibody clones and variants AHP54695-h1, AHP54695-h6, AHP54695-h7m1 and AHP54695-h7m1-2C based on the anti-Nectin-4 maternal antibody clone AHP54695, respectively. SEQ ID NO: 10-12 show the amino acid sequences of CDR1, CDR2 and CDR3 of AHP54695-h1 and AHP54695-h6, respectively. SEQ ID NO: 10, 11 and 16 show the amino acid sequences of CDR1, CDR2 and CDR3 of AHP54695-h7m1 and AHP54695-h7m1-2C, respectively.

[0050] SEQ ID NO: 19-21 show the amino acid sequences of CDR1, CDR2 and CDR3 of the parental antibody clone AHP54962 against Nectin-4, respectively.

[0051] SEQ ID NO: 22 shows the amino acid sequence of the VHH chain of the parental antibody clone AHP54962 against Nectin-4.

[0052] SEQ ID NO: 23 and 24 show the amino acid sequences of the VHH chains of humanized antibody clones AHP54962-h4 and AHP54962-h6 based on the parental antibody clone AHP54962 against Nectin-4, respectively, while SEQ ID NO: 19-21 show the amino acid sequences of CDR1, CDR2 and CDR3 of these humanized antibodies, respectively.

[0053] SEQ ID NO: 25-27 show the amino acid sequences of CDR1, CDR2 and CDR3 of the parental antibody clone AHP54966 against Nectin-4, respectively.

[0054] SEQ ID NO: 28 shows the amino acid sequence of the VHH chain of the parental antibody clone AHP54966 against Nectin-4.

[0055] SEQ ID NO: 29, 30 and 32 show the amino acid sequences of the VHH chain of the humanized antibody clone and variants AHP54966-h1, AHP54966-h4 and AHP54966-h1m1 based on the maternal antibody clone AHP54966 against Nectin-4, respectively. SEQ ID NO: 25-27 show the amino acid sequences of CDR1, CDR2 and CDR3 of AHP54966-h1 and AHP54966-h4, respectively. SEQ ID NO: 25, 26 and 31 show the amino acid sequences of CDR1, CDR2 and CDR3 of AHP54966-h1m1, respectively.

[0056] SEQ ID NO: 33-35 show the amino acid sequences of CDR1, CDR2 and CDR3 of the maternal antibody clone AHP54938 based on anti-Nectin-4, respectively.

[0057] SEQ ID NO: 36 shows the amino acid sequence of the VHH chain of the maternal antibody clone AHP54938 against Nectin-4.

[0058] SEQ ID NO: 37 and 38 show the amino acid sequences of the VHH chains of humanized antibody clones AHP54938-h1 and AHP54938-h7 based on the maternal antibody clone AHP54938 against Nectin-4, respectively, while SEQ ID NO: 33-35 show the amino acid sequences of CDR1, CDR2 and CDR3 of these humanized antibodies, respectively.

[0059] SEQ ID NO: 42, 44, 46, 48, 49, and 51 show the amino acid sequences of the VHH chains of affinity maturation antibodies AHF47750, AHF47752, AHF47753, AHF47765, AHF47772, and AHF47750E based on the humanized antibody AHP53835-h2, respectively. SEQ ID NO: 39-41 show the amino acid sequences of CDR1, CDR2, and CDR3 of AHF47750, respectively; SEQ ID NO: 43, 2, and 41 show the amino acid sequences of CDR1, CDR2, and CDR3 of AHF47752, respectively; and SEQ ID NO: 45, 40, and 41 show the amino acid sequences of CDR1, CDR2, and CDR3 of AHF47753, respectively. SEQ ID NO: 45, 2 and 47 show the amino acid sequences of CDR1, CDR2 and CDR3 of AHF47765, respectively; SEQ ID NO: 45, 2 and 41 show the amino acid sequences of CDR1, CDR2 and CDR3 of AHF47772, respectively; SEQ ID NO: 39, 50 and 41 show the amino acid sequences of CDR1, CDR2 and CDR3 of AHF47750E, respectively.

[0060] SEQ ID NO: 52 shows the amino acid sequence of human Nectin-4 protein, wherein amino acid sequence 32-349 is an extracellular epitope that binds to VHH antibody.

[0061] SEQ ID NO: 53 shows the amino acid sequence of a human IgG1 Fc fragment linked to an anti-Nectin-4 VHH antibody. Detailed Implementation

[0062] The inventors of this application have prepared novel antibodies against the Nectin-4 protein using antibody engineering technology. In various aspects of this application, novel antibodies against the Nectin-4 protein, nucleic acid molecules encoding said antibodies, vectors containing said nucleic acid molecules, cells containing said nucleic acid molecules or vectors, conjugates containing said antibodies, methods for preparing and purifying antibodies, and medical and biological uses of said antibodies are provided.

[0063] Unless otherwise specified, this application employs conventional molecular biology, microbiology, cell biology, biochemistry, and immunology techniques, and the terms used have the meanings commonly understood by those skilled in the art.

[0064] In this paper, the abbreviations of amino acid residues are the standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. The antibody variable region sequence adopts the Kabat coding scheme.

[0065] In this article, “anti-Nectin-4 antibody” refers to an antibody against Nectin-4 protein (e.g., human Nectin-4 protein), that is, an antibody that specifically binds to Nectin-4 protein (e.g., human Nectin-4 protein), including antibodies containing the VHH chain, such as VVH antibody and VHH-Fc antibody.

[0066] In this article, “VHH antibody”, also known as “nanobody (Nb)” or “sdAb”, is a variable region (VHH) of a heavy chain antibody that is naturally missing a light chain. Its antigen binding site depends only on three complementarity-determining regions (CDR1, CDR2, and CDR3).

[0067] In this paper, “VHH-Fc antibody” refers to an engineered fusion protein of VHH and the Fc fragment of immunoglobulin G (IgG), which has both the targeting function of small molecule antibodies and the effector function mediated by Fc. The Fc fragment is derived from the constant region of IgG (e.g., human IgG1) and contains CH2 and CH3 domains.

[0068] In a first aspect, this application provides an anti-Nectin-4 antibody or an antigen-binding fragment thereof.

[0069] In some embodiments, the anti-Nectin-4 antibody provided in this application can be prepared by the following methods: immunizing alpacas with human Nectin-4 protein to construct a phage display library; screening target phage clones by liquid-phase cell panning; identifying positive clones using FACS; and expressing and purifying the anti-Nectin-4 antibody.

[0070] In some embodiments, the positive clone antibody AHP53835 (parent) obtained through screening contains complementarity-determining regions CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 1-3, respectively.

[0071] In a specific implementation scheme, the maternal antibody AHP53835 has the amino acid sequence shown in SEQ ID NO: 4.

[0072] In a specific implementation scheme, the positive clone antibody AHP53835 obtained through screening is humanized to obtain humanized antibody AHP53835-h2, whose complementarity-determining regions CDR1, CDR2, and CDR3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively. In a specific implementation scheme, humanized antibody AHP53835-h2 has the amino acid sequence shown in SEQ ID NO: 5.

[0073] In a specific implementation scheme, the positive clone antibody AHP53835 obtained through screening is humanized to obtain the humanized antibody AHP53835-h5, whose complementarity-determining regions CDR1, CDR2, and CDR3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively. In a specific implementation scheme, the humanized antibody AHP53835-h5 has the amino acid sequence shown in SEQ ID NO: 6.

[0074] In a specific implementation scheme, the positive clone antibody AHP53835 obtained through screening is humanized to obtain the humanized antibody AHP53835-h7, whose complementarity-determining regions CDR1, CDR2, and CDR3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively. In a specific implementation scheme, the humanized antibody AHP53835-h7 has the amino acid sequence shown in SEQ ID NO: 7.

[0075] In a specific implementation scheme, the positive clone antibody AHP53835 obtained through screening is humanized to obtain the humanized antibody AHP53835-h8, whose complementarity-determining regions CDR1, CDR2, and CDR3 amino acid sequences are shown in SEQ ID NO: 1-3, respectively. In a specific implementation scheme, the humanized antibody AHP53835-h8 has the amino acid sequence shown in SEQ ID NO: 8.

[0076] In a specific embodiment, the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the humanized antibody AHP53835-h7 variant AHP53835-h7m1 are shown in SEQ ID NO: 1-3, respectively. In another specific embodiment, variant AHP53835-h7m1 has the amino acid sequence shown in SEQ ID NO: 9.

[0077] In some embodiments, the positive clone antibody AHP54695 (parent) obtained through screening contains complementarity-determining regions CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 10-12, respectively.

[0078] In a specific implementation scheme, the maternal antibody AHP54695 has the amino acid sequence shown in SEQ ID NO: 13.

[0079] In a specific implementation scheme, the positive clone antibody AHP54695 obtained through screening is humanized to obtain humanized antibody AHP54695-h1, whose complementarity-determining regions CDR1, CDR2, and CDR3 amino acid sequences are shown in SEQ ID NO: 10-12, respectively. In a specific implementation scheme, humanized antibody AHP54695-h1 has the amino acid sequence shown in SEQ ID NO: 14.

[0080] In a specific implementation scheme, the positive clone antibody AHP54695 obtained through screening is humanized to obtain humanized antibody AHP54695-h6, whose complementarity-determining regions CDR1, CDR2, and CDR3 amino acid sequences are shown in SEQ ID NO: 10-12, respectively. In a specific implementation scheme, humanized antibody AHP54695-h6 has the amino acid sequence shown in SEQ ID NO: 15.

[0081] In a specific implementation scheme, the positive clone antibody AHP54695 obtained through screening is humanized to obtain the humanized antibody AHP54695-h7m1, whose complementarity-determining regions CDR1, CDR2, and CDR3 amino acid sequences are shown in SEQ ID NO: 10, 11, and 16, respectively. In a specific implementation scheme, the humanized antibody AHP54695-h7m1 has the amino acid sequence shown in SEQ ID NO: 17.

[0082] In a specific embodiment, the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the humanized antibody AHP54695-h7m1-2C are shown in SEQ ID NO: 10, 11, and 16, respectively. In another specific embodiment, the humanized antibody AHP54695-h7m1-2C has the amino acid sequence shown in SEQ ID NO: 18.

[0083] In some embodiments, the positive clone antibody AHP54962 (parent) obtained through screening contains complementarity-determining regions CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 19-21, respectively.

[0084] In a specific implementation scheme, the maternal antibody AHP54962 has the amino acid sequence shown in SEQ ID NO: 22.

[0085] In a specific implementation scheme, the positive clone antibody AHP54962 obtained through screening is humanized to obtain the humanized antibody AHP54962-h4, whose complementarity-determining regions CDR1, CDR2, and CDR3 amino acid sequences are shown in SEQ ID NO: 19-21, respectively. In a specific implementation scheme, the humanized antibody AHP54962-h4 has the amino acid sequence shown in SEQ ID NO: 23.

[0086] In a specific implementation scheme, the positive clone antibody AHP54962 obtained through screening is humanized to obtain the humanized antibody AHP54962-h6, whose complementarity-determining regions CDR1, CDR2, and CDR3 amino acid sequences are shown in SEQ ID NO: 19-21, respectively. In a specific implementation scheme, the humanized antibody AHP54962-h6 has the amino acid sequence shown in SEQ ID NO: 24.

[0087] In some embodiments, the positive clone antibody AHP54966 obtained through screening contains complementarity-determining regions CDR1, CDR2, and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 25-27, respectively.

[0088] In a specific implementation scheme, the maternal antibody AHP54966 has the amino acid sequence shown in SEQ ID NO: 28.

[0089] In a specific implementation scheme, the positive clone antibody AHP54966 obtained through screening is humanized to obtain humanized antibody AHP54966-h1, whose complementarity-determining regions CDR1, CDR2, and CDR3 amino acid sequences are shown in SEQ ID NO: 25-27, respectively. In a specific implementation scheme, humanized antibody AHP54962-h1 has the amino acid sequence shown in SEQ ID NO: 29.

[0090] In a specific implementation scheme, the positive clone antibody AHP54966 obtained through screening is humanized to obtain the humanized antibody AHP54966-h4, whose complementarity-determining regions CDR1, CDR2, and CDR3 amino acid sequences are shown in SEQ ID NO: 25-27, respectively. In a specific implementation scheme, the humanized antibody AHP54966-h4 has the amino acid sequence shown in SEQ ID NO: 30.

[0091] In a specific implementation scheme, the positive clone antibody AHP54966 obtained through screening is humanized to obtain the humanized antibody AHP54966-h1m1, whose complementarity-determining regions CDR1, CDR2, and CDR3 amino acid sequences are shown in SEQ ID NO:25, 26, and 31, respectively. In a specific implementation scheme, the humanized antibody AHP54966-h1m1 has the amino acid sequence shown in SEQ ID NO:32.

[0092] In some embodiments, the positive clone antibody AHP54938 (parent) obtained through screening contains complementarity-determining regions CDR1, CDR2 and CDR3, the amino acid sequences of which are shown in SEQ ID NO: 33-35, respectively.

[0093] In a specific implementation scheme, the maternal antibody AHP54938 has the amino acid sequence shown in SEQ ID NO: 36.

[0094] In a specific implementation scheme, the positive clone antibody AHP54938 obtained through screening is humanized to obtain humanized antibody AHP54938-h1, whose complementarity-determining regions CDR1, CDR2, and CDR3 amino acid sequences are shown in SEQ ID NO: 33-35, respectively. In a specific implementation scheme, humanized antibody AHP54938-h1 has the amino acid sequence shown in SEQ ID NO: 37.

[0095] In a specific implementation scheme, the positive clone antibody AHP54938 obtained through screening is humanized to obtain humanized antibody AHP54938-h7, whose complementarity-determining regions CDR1, CDR2, and CDR3 amino acid sequences are shown in SEQ ID NO: 33-35, respectively. In a specific implementation scheme, humanized antibody AHP54938-h7 has the amino acid sequence shown in SEQ ID NO: 38.

[0096] In some implementations, affinity-matured antibodies are obtained based on the humanized antibody AHP53835-h2.

[0097] In a specific embodiment, the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the affinity maturation antibody AHF47750 are shown in SEQ ID NO: 39-41, respectively. In a specific embodiment, the affinity maturation antibody AHF47750 has the amino acid sequence shown in SEQ ID NO: 42.

[0098] In a specific embodiment, the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the affinity maturation antibody AHF47752 are shown in SEQ ID NO: 43, 2, and 41, respectively. In a specific embodiment, the affinity maturation antibody AHF47752 has the amino acid sequence shown in SEQ ID NO: 44.

[0099] In a specific embodiment, the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the affinity maturation antibody AHF47753 are shown in SEQ ID NO: 45, 40, and 41, respectively. In a specific embodiment, the affinity maturation antibody AHF47753 has the amino acid sequence shown in SEQ ID NO: 46.

[0100] In a specific embodiment, the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the affinity maturation antibody AHF47765 are shown in SEQ ID NO: 45, 2, and 47, respectively. In a specific embodiment, the affinity maturation antibody AHF47765 has the amino acid sequence shown in SEQ ID NO: 48.

[0101] In a specific embodiment, the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the affinity maturation antibody AHF47772 are shown in SEQ ID NO: 45, 2, and 41, respectively. In a specific embodiment, the affinity maturation antibody AHF47772 has the amino acid sequence shown in SEQ ID NO: 49.

[0102] In a specific embodiment, the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the affinity maturation antibody AHF47750E are shown in SEQ ID NO: 39, 50, and 41, respectively. In a specific embodiment, the affinity maturation antibody AHF47750E has the amino acid sequence shown in SEQ ID NO: 51.

[0103] The term "identity" is defined as the percentage of identical residues in an amino acid or nucleotide sequence variant after sequence alignment and vacancy introduction, reaching the maximum percentage of identity if desired. The methods and computer programs used for alignment are well known in the art.

[0104] In some embodiments, the amino acid sequence of the antibody provided in this application can be modified in various ways known in the art, such as introducing site-directed amino acid deletions, insertions, or substitutions (1-25 residues) in the CDR region or frame region (FR) of VHH, or fusing the VHH antibody with functional elements, such as Fc tags, His tags, or flexible linker peptides (e.g., G4SC, GSC). G4SC is an abbreviation for GGGGSC.

[0105] In some embodiments, the amino acid sequence of the VHH antibody provided in this application may be linked to a human IgG1-Fc fragment as shown in SEQ ID NO: 53 to form a VHH-Fc antibody.

[0106] In this application, when measuring cell binding activity, the "half-maximum effective concentration (EC50)" is used. 50 "Value" refers to the antibody concentration at which 50% binding occurs to a cell surface target antigen (e.g., Nectin-4 protein). 50 The lower the value, the stronger the antibody binding ability and the greater the affinity. "Bmax" represents the maximum capacity that the antibody can bind to the target antigen on the cell surface, reflecting the total receptor density or number of binding sites of the target antigen on the cell surface. The higher the Bmax, the higher the density of the target antigen on the cell surface. When measuring antibody affinity, "binding constant Ka" represents the rate at which the antibody binds to the antigen. The larger the Ka, the higher the affinity. "Dissociation constant Kd" represents the dissociation rate of the antibody-antigen complex into free antibody and antigen. The smaller the Kd, the higher the affinity. "Equilibrium dissociation constant KD" is the ratio of the binding rate constant (kon) to the dissociation rate constant (koff). It is a global indicator of the antibody-antigen binding strength in equilibrium. The smaller the KD, the higher the affinity.

[0107] In a second aspect, this application provides a nucleic acid molecule encoding an anti-Nectin-4 antibody or antigen-binding fragment provided in this application, a vector containing the nucleic acid molecule, such as an expression vector, and a host cell containing the nucleic acid molecule or the vector.

[0108] In some embodiments, the nucleic acid molecule is operatively linked to a regulatory sequence that can be recognized by a host cell transformed with the vector. The choice of vector depends on the choice of host cell, such as a mammalian cell. In some embodiments, the host cell is a eukaryotic cell, such as yeast cells, mammalian cells, or other cells suitable for preparing antibodies or antigen-binding fragments thereof. In some embodiments, the host cell is a prokaryotic cell, such as *Escherichia coli*.

[0109] In a third aspect, this application provides an antibody conjugate comprising the anti-Nectin-4 antibody or antigen-binding fragment provided in this application.

[0110] In this paper, "antibody conjugates" are a class of composite molecules that conjugate a target carrier (e.g., an antibody) with an effector payload through a specific linking technology, thereby precisely delivering the effector payload through the targeted recognition of the antibody.

[0111] In some implementations, the anti-Nectin-4 antibody or antigen-binding fragment provided in this application can be conjugated with a variety of different effectors to form antibody conjugates.

[0112] In some implementations, effectors that can be conjugated with the nanobodies provided in this application include, but are not limited to: radionuclides, antitumor agents, immunomodulators, bioresponse modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, imaging agents, chemiluminescent compounds, enzymes, metal ions, etc.

[0113] In other embodiments, the anti-Nectin-4 antibody or antigen-binding fragment provided in this application is linked to a radionuclide to form a radionuclide conjugate (RDC). The antibody is used to target and deliver the radionuclide, and the ionizing radiation of the radionuclide is used to directly kill cancer cells or to visualize tumors, thus having both diagnostic and therapeutic functions.

[0114] In a fourth aspect, this application provides an antibody conjugate comprising the anti-Nectin-4 antibody or antigen-binding fragment of this application and a radionuclide, also known as a radionuclide-labeled antibody conjugate or radionuclide conjugate (RDC), which acquires diagnostic and / or therapeutic functions by linking different types of radionuclides to the anti-Nectin-4 antibody.

[0115] In some embodiments, the radionuclide-labeled antibody conjugates provided in this application comprise the anti-Nectin-4 antibody of this application and a radionuclide-chelating agent. In some embodiments, the radionuclide-labeled antibody conjugates provided in this application further comprise a linker.

[0116] Any radionuclide known in the art that can be used for treatment or diagnosis can be conjugated to the nanobody of this application. For example, diagnostic radionuclides that are mainly gamma-ray or positron-emitting radionuclides, and therapeutic radionuclides that are mainly beta-ray or alpha-ray radionuclides.

[0117] In some implementations, the applicable radionuclides include, but are not limited to: 11 C 13 N、 15 O、¹ 8 F, 32 P, 44 Sc、 47 Sc、 51 Mn, 52m Mn, 52Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 68 Ge 72 As、 75 Br、 76 Br、 82m Rb、 83 Sr、 86 Y、 89 Sr、 89 Zr、 90 Y、 99m Tc, 105 Rh、 110 In、 111 In、 117 mSn, 120 I, 123 I, 124 I, 125 I、¹³¹I、 134 Ce、 148 Gd, 149 Tb, 149 Pm, 152 Tb, 153 Sm、 154-158 Gd, 160 Gd, 161 Tb, 165 Ho、 166 Ho、 167 Tm、 175 Yb、¹ 77 Lu、 186 Re、 188 Re、 197 Au、 198 Au、 199 Au、 201 Ti、 209 Bi、 211 At、 212 Pb, 212 Bi、 213 Bi、 213 Po、²²³Ra、 223 Fr、²² 5 Ac、 227 Th and 229 Th.

[0118] Any chelating agent known in the art for labeling radionuclides can be used in this application, wherein the chelating agent stably binds the radionuclide to the nanobody, preventing premature dissociation of the nuclide during in vivo circulation and avoiding damage to non-target tissues.

[0119] In some embodiments, suitable chelating agents include, but are not limited to: 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), N,N'-bis(2-hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED-CC), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) or triazacyclononane phosphate (TRAP), EDTA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG3, MAG2, HYNIC, AAZTA, BAT, BAT-TM, Crown, Cyclen, DO2A, CB-DO2A, DO3A, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA, MACROPA, HEHA, DOTA-GA, p-BnDOTA, p-Bn-SCN-DOTA, NH2-DOTA, NH2-DOTA-GA, p-NC S-Bn-DOTA-GA, p-NH2-Bn-OxODO3A, p-SCN-Bn-oxo-DO3A, NODA-GA, NH2-NODA-GA, p-NCS-Bn-NODA-GA, p-NH2Bn-NOTA, p-SCN-Bn-NOTA, NCS-MP-NOD A, NH2-MPAA-NODA, PCTA, p-NH2-Bn-PCTA, pSCN-Bn-PCTA, p-SCN-Bn-HEHA, H2-MACROPA-NCS, H1-MACROPA, H2-MACROPA-NH2, H4OCTAPA, tetra-(S,S,S,S)-Me-DOTA, SHBED, (HBED-CC)TFP, DMSA, DMPS, DHLA, tetra-(S,S,S,S)-Et-DOTA, tetra-(S,S,S,S)-iBu-DOTA, and maleimide-nBu-DOTA and their derivatives.

[0120] In some implementations, suitable chelating agents include, but are not limited to: H3RESCA, (+-) H3RESCA-TFP, and (+-) H3RESCA-Mal.

[0121] In some embodiments, the chelating agent is selected from H3RESCA, (+-)H3RESCA-TFP, and (+-)H3RESCA-Mal.

[0122] In some embodiments, the radionuclide-labeled antibody conjugate is: radionuclide-VHH-Mal-NOTA, radionuclide-VHH-G4SC-Mal-NOTA, radionuclide-VHH-GSC-Mal-NOTA, radionuclide-VHH-Mal-H3RESCA, radionuclide-VHH-GSC-Mal-H3RESCA, or radionuclide-VHH-G4SC-Mal-H3RESCA. In some embodiments, for example, a nucleic acid may be... 68 Ga、 18 F, 177 Lu、 225 Ac, etc.

[0123] In this article, nuclide refers to radioactive nuclide; VHH-Mal-NOTA, VHH-linker-Mal-NOTA, VHH-Mal-H3RESCA, VHH-linker-Mal-H3RESCA, etc. are precursors for radioactive nuclide labeling. The nuclide is labeled on the chelating agent NOA or H3RESCA and chelates with the chelating agent multidentate.

[0124] In this document, "VHH-chelating agent" is an abbreviation for "VHH-Mal-chelating agent," "VHH-linker-chelating agent," or "VHH-linker-Mal-chelating agent," and these terms are interchangeable. For example, "VHH-NOTA" is an abbreviation for "VHH-Mal-NOTA," "VHH-G4SC-NOTA," or "VHH-G4SC-Mal-NOTA," and "VHH-H3RESCA" is an abbreviation for "VHH-Mal-H3RESCA," "VHH-G4SC-H3RESCA," or "VHH-G4SC-Mal-H3RESCA." For the abbreviations "VHH-NOTA" and "VHH-Mal-NOTA," the specific linker used can be determined according to the embodiment. Variants obtained by introducing one or more pairs of cysteine ​​residues into the VHH sequence can be directly coupled to a Mal-chelating agent, for example [ 68 Ga]AHP54695-h7m1-2C-NOTA([ 68 [Ga]AHP54695-h7m1-2C-Mal-NOTA (abbreviation), without the VHH sequence introducing cysteine, can generally be coupled to a Mal-chelating agent via a linker (e.g., G4SC, GSC), for example, it could be [ 68 Ga]AHP54695-h7m1-G4SC-H3RESCA ([ 68 (The abbreviation of Ga]AHP54695-h7m1-G4SC-Mal-H3RESCA).

[0125] In some embodiments, the radionuclide-labeled antibody conjugate is selected from: [ 68 Ga]AHP54695-h7m1-G4SC-H3RESCA, [ 18 F]AHP54695-h7m1-G4SC-H3RESCA, [ 68 Ga]AHP53835-h7-G4SC-NOTA, [ 68 Ga]AHP53835-h7m1-NOTA、[ 68 Ga]AHP54938-h7-G4SC-NOTA, [ 68 Ga]AHP54962-h4-GSC-NOTA, [ 68 Ga]AHP54966-h1m1-G4SC-NOTA,[ 68 Ga]AHP54695-h7m1-G4SC-NOTA,[ 68 Ga]AHP54695-h7m1-2C-NOTA.

[0126] In a fifth aspect, this application provides pharmaceutical compositions comprising the different forms of VHH antibodies described above. For example, pharmaceutical compositions comprising any one of the aforementioned VHH antibodies, pharmaceutical compositions comprising nucleic acid molecules encoding any one of the aforementioned VHH antibodies, pharmaceutical compositions comprising a carrier or host cell of the aforementioned nucleic acid molecules, and pharmaceutical compositions comprising the aforementioned antibody conjugates (such as radionuclide-labeled antibody conjugates).

[0127] In some embodiments, the pharmaceutical composition further comprises pharmaceutical excipients.

[0128] In a sixth aspect, this application provides the use of the aforementioned different forms of antibodies, such as for the prevention, diagnosis and / or treatment of diseases characterized by Nectin-4 expression, or for use in the preparation of medicaments for the prevention, diagnosis and / or treatment of diseases characterized by Nectin-4 expression, such as Nectin-4 positive cancers / tumors.

[0129] Accordingly, this application provides methods for the prevention, diagnosis and / or treatment of diseases characterized by Nectin-4 expression in individuals, comprising administering the above-described different forms of antibody conjugates or pharmaceutical compositions thereof to individuals in need.

[0130] In some implementations, diseases characterized by Nectin-4 expression are defined as Nectin-4 positive tumors, including but not limited to: head and neck squamous cell carcinoma, bladder cancer, urothelial carcinoma, breast cancer, non-small cell lung cancer (NSCLC), gastric cancer, esophageal cancer, ovarian cancer, cervical cancer, melanoma, pancreatic cancer, liver cancer, and prostate cancer, etc.

[0131] The anti-Nectin-4 antibody provided in this application has a stable structure, high affinity, strong specificity, strong tissue penetration, and good tolerability, making it widely applicable in research, diagnosis, and treatment. For example, radionuclide conjugates containing the anti-Nectin-4 antibody of this application combine the advantages of both VHH antibodies and radionuclides, showing broad application prospects in tumor imaging and the diagnosis and / or treatment of related tumors (e.g., integrated diagnosis and treatment).

[0132] It should be understood that the above detailed description is only intended to provide a clearer understanding of the contents of this application to those skilled in the art, and is not intended to limit in any way. Those skilled in the art can make various modifications and variations to the described embodiments.

[0133] Example

[0134] The following embodiments are for illustrative purposes only and are not intended to limit the scope of this application. Any modifications or substitutions made to the methods, steps, or conditions of this application without departing from the spirit and substance of this application shall fall within the scope of this application.

[0135] Unless otherwise specified, all reagents used in the examples are commercially available and all technical means used in the examples are conventional means well known to those skilled in the art.

[0136] Example 1: Generation and screening of Nectin-4 antibodies

[0137] 1. Preparation of Nectin-4 antigen

[0138] His-tagged human Nectin-4 was used as the immunogenic antigen, biotin-tagged human Nectin-4 was used as the panning antigen, and His-tagged human Nectin-4 was used as the screening antigen. All of these were purchased from AcroBiosystems.

[0139] The sequence of human Nectin-4 protein (Accession # Q96NY8-1) is as follows, where the underlined part is the extracellular region sequence, and the amino acid sequence starts and ends at Gly32-Ser349.

[0140] The amino acid sequence of human Nectin-4 protein (SEQ ID NO: 52): MPLSLGAEMWGPEAWLLLLLLLASFTGRCPA GELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWAR VDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVL VPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTC VVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTL GFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSAS VVVVGVIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIRRLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRAEEEEDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV

[0141] 2. Alpaca Immunization

[0142] Two healthy alpacas were immunized. On day 0, the initial immunization was performed by subcutaneous injection of 0.5 mg human Nectin-4 antigen mixed with 1 ml of Freund's complete adjuvant (CFA). On days 14, 28, and 42, 0.25 mg human Nectin-4 antigen was mixed with 1 ml of Freund's incomplete adjuvant (IFA) and injected subcutaneously. Blood samples of 5 ml, 50 ml, and 50 ml were collected on days 21, 35, and 49, respectively. Serum titers were measured, and alpaca peripheral lymphocytes meeting the library construction criteria were used to construct a nanobody phage display library.

[0143] 3. Phage library construction

[0144] Peripheral blood (50 mL) was collected from two alpacas after their third and fourth immunizations. Total RNA was extracted from isolated PBMCs (#132-3&4©-3) according to the TRIzol Reagent instructions. Total cDNA was then obtained via reverse transcription. The cDNA was used as the template for the first round of PCR. After two rounds of PCR amplification, the second-round PCR product fragment was ligated into the phage library vector AH-V21S. The ligation product was then electroporated into competent *E. coli* cells. 100 μL of transformation buffer was titrated, and the library size was calculated based on the number of clones. Ninety-six clones were randomly selected for sequencing to assess library quality. Sequencing results, based on library insertion rate and library diversity analysis, indicated a library volume of 1.09 × 10⁻⁶. 9 The library has 100% diversity and insertion rate.

[0145] 4. Nanobody Screening

[0146] Human Nectin-4 protein, labeled with three rounds of biotin, was panned using liquid chromatography in a phage display library. M-280 magnetic bead suspension was washed three times with PBS, resuspended in 3% MPBS, and blocked for 1 hour. 1×10⁻⁶ samples were then collected.12 The phage library solution in pfu / pool was replenished to 1 ml with 3% MPBS and incubated slowly by rotation at room temperature for 1 h. Then, 100 nM (or 50 nM, or 25 nM) of biotin-labeled human Nectin-4 was added and mixed well. The mixture was then incubated by rotation at room temperature for 1 h. The phage library was then placed in the wells of a magnetic rack to adsorb magnetic beads, and the blocking solution was removed. The antigen-bound phage library was transferred to the blocked magnetic beads and resuspended, mixed well, and incubated by rotation at room temperature for 20-30 min. The phage library was washed 8-10 times with PBST. Wash 3-5 times with PBS. Add 800 μl of 0.1 M TEA elution buffer to the tube and elute by gently shaking for 8-10 minutes. Then add 0.4 ml of 1 M Tris-HCl (pH 7.4) to neutralize for 10 minutes. Take 1 ml of the eluted and neutralized phage product and add it to 10 ml of TG1 bacterial culture in the logarithmic growth phase. Mix well and incubate at 37°C for 45 minutes for infection.

[0147] 5. Monoclonal supernatant ELISA screening

[0148] Single-clone bacterial cultures were cultured in 96-well plates. IPTG induction medium was added to each well, and the plates were incubated overnight at 30°C on a shaker. Simultaneously, 100 μl / well of 1 μg / ml human Nectin-4 protein was added to each well of a 96-well microplate and incubated overnight at 4°C. After coating, the plates were blocked with 3% MPBS at 37°C for 1 hour. The plates were washed with PBST, and 50 μl of overnight bacterial supernatant and 50 μl of 0.05% PBST were added to each well. The plates were incubated at room temperature for 2 hours. After washing, the plates were incubated with the secondary antibody MonoRabR. TM Rabbit Anti-Camelid VHHCocktail [HRP] was incubated at room temperature for 45 minutes. The plate was washed 6 times with 0.05% PBST. 100 μl of TMB chromogenic buffer was added to each well, and the plate was incubated at room temperature for 10 minutes. 50 μl of 2M H₂SO₄ stop solution was added to each well. The plate was read using a microplate reader, and positive clones were selected based on the OD value at 450 nm.

[0149] 6. Single-clonal supernatant FACS screening

[0150] The binding activity of the supernatant from ELISA-selected positive monoclonal cells to CHO-K1 / human Nectin-4 cells (CHO-K1 cells as a control) was detected by flow cytometry. Cells were washed with PBS and aliquoted into 96-well plates at 100 μl / well × 10⁶ cells / well. 5Add 100 μl of cells / well and 50 μl of supernatant / well sample, and incubate at 4°C for 40 min. Wash cells twice with pre-chilled PBS. Add 100 μl / well of 1 μg / ml secondary antibody GenScript MonoRab. TM Rabbit anti-Camelid VHH Cocktail [iFluor 647] was incubated at 4°C. Cells were washed twice and resuspended in PBS, and analyzed using flow cytometry (FACS).

[0151] Based on the results of ELISA and FACS tests, a total of 28 positive clones were selected as candidate antibodies.

[0152] 7. Expression and purification of monovalent VHH-Fc candidate antibodies

[0153] The selected candidate antibody sequences were inserted into the eukaryotic expression vector pcDNA3.4, with one strand's Fc linked to a VHH, and the other strand's Fc not linked to a VHH, constructing a monovalent VHH-Fc structure. The Fc was derived from human IgG1. Antibody expression was induced by co-transfection of ExpiCHO-S cells with both plasmids. Protein A Magnetic Beads were added to the purified supernatant and incubated at room temperature in a shaking incubator for 2 hours. Protein A Magnetic Beads were equilibrated with 10 column volumes of binding buffer. Elution buffer was added to the Protein A Magnetic Beads, and the eluent was collected. Neutralization buffer (1 / 10 of the collected eluent volume) was added simultaneously to adjust the pH to neutral. After elution and neutralization, the collected eluted sample was transferred to a dialysis bag and subjected to 1... PBS was incubated at room temperature for 2 hours for dialysis, then the buffer was changed, and dialysis was continued at 2-8°C for 16 hours. The concentration and purity of the antibody were determined by OD280 and SDS-PAGE.

[0154] 8. ELISA detection of monovalent VHH-Fc candidate antibodies

[0155] The binding levels of purified antibodies to Nectin-4 protein of different species and domains were detected by ELISA. 1 μg / ml of human / macaque / mouse / rat Nectin-4 protein and human Nectin-4 (32-153) / (154-243) / (244-349) protein were added to each well of a 96-well ELISA plate and incubated overnight at 4°C. After washing three times, 3% MPBS was added to each well, and the plate was blocked at 37°C for 1 hour. The antibody to be tested was diluted to a concentration of 100 nM in the first well, followed by 3-fold serial dilutions, for a total of 11 dilutions. The diluted antibody was then added to the blocked ELISA plate at 100 µl / well and incubated at 37°C for 1 hour. After washing three times, mouse anti-human IgG Fc-HRP secondary antibody was added, and the plate was incubated at 37°C for 30 minutes. The plate was washed six times, then TMB chromogenic buffer was added and incubated at room temperature for 10 minutes. Finally, 50 μl of stop buffer was added to each well. The OD values ​​at 450 nm were read using a microplate reader, and the results are shown in Table 1.

[0156] As can be seen, after preparing monovalent VHH-Fc antibodies, the binding specificity of 28 candidate antibodies was detected using MDA-MB-231 cells (human breast cancer cells, nectin-4 negative). The results showed that 12 antibodies, AHP53752, AHP53759, AHP53815, AHP53824, AHP53835, AHP53691, AHP53744, AHP54695, AHP54726, AHP54938, AHP54962, and AHP54966, did not bind nonspecifically.

[0157] Table 1. ELISA results of the binding specificity of candidate antibodies.

[0158] 9. FACS detection of monovalent VHH-Fc candidate antibodies

[0159] The binding activity of 12 purified antibodies with no nonspecific binding activity to HT1376 cells was detected by FACS. Cells were washed with PBS and aliquoted into 96-well plates (100 μl / well, 1 × 10⁶ cells / well). 5 Cells / well, antibody diluted to an initial concentration of 300 nM, serially diluted 11 times in 3-fold increments, with a blank control included. Add 100 μl / well to a 96-well plate and incubate at 4°C for 40 minutes. Wash cells twice with pre-chilled PBS. Add 100 μl / well of 1 μg / ml secondary antibody GenScript MonoRab. TMRabbit anti-Camelid VHH Cocktail [iFluor 647] was incubated at 4°C for 40 minutes. After washing twice, the cells were resuspended in PBS and analyzed by flow cytometry. The results are shown in Table 2. Figure 1 .

[0160] Table 2. FACS assay results of the binding activity of candidate antibodies

[0161] The results showed that, except for the AHP53759 candidate antibody which had weak cell-binding activity, the binding activities of the other candidate antibodies were similar.

[0162] 10. SPR detection of monovalent VHH-Fc candidate antibodies

[0163] The affinity between the antibody and the antigen was determined by surface plasmon resonance (SPR). The antigen, as a ligand, was immobilized on the sensor chip CM5, and the antibody, as an analyte, flowed through the surface of the sensor chip. The dissociation rate (kd) and binding rate constant (ka) were obtained using evaluation software. The equilibrium dissociation constant (KD) was calculated based on the ratio of kd to ka. Exemplary affinity data are shown in Table 3.

[0164] Table 3 SPR test results of candidate antibodies

[0165] The results showed that, using SRP to determine antibody affinity, AHP53835, AHP54695, AHP54938, and AHP54962 exhibited good affinity. Based on SRP data and combined with FACS data, although AHP54966 showed lower affinity for soluble proteins as determined by SRP, it demonstrated good binding activity to cell surface antigens.

[0166] 11. Detection of endocytosis of monovalent VHH-Fc candidate antibodies

[0167] Antibody internalization assays were used to evaluate the in vitro biological activity of antibodies on different target cells. HT-1376 target cells were digested and collected, and resuspended in complete culture medium. The target cell density was adjusted, and the cell suspension (50 μl / well) was transferred to a 96-well plate. The plate was incubated overnight in a cell culture incubator (37℃ / 5% CO2). Antibody working solution and labeling reagent working solution (4×) were prepared using experimental buffer. The antibody working solution and labeling reagent working solution were mixed at a 1:3 molar ratio and a 1:1 volume ratio, and incubated in a cell culture incubator (37℃ / 5% CO2) for 15 minutes to ensure complete coupling. The coupling mixture working solution (50 μl / well) was transferred to the corresponding wells of the 96-well plate. The plate was incubated in a cell culture incubator (37℃ / 5% CO2) for a specified time (48 h), and images were taken at designated time points using an Incucyte instrument that matched the detection wavelength. Raw experimental data and results were analyzed and exported using the Incucyte® Live-Cell Analysis System, and analyzed using Microsoft Office Excel 2021 and GraphPad Prism 6 software. Example results are shown below. Figure 2 This indicates that all the antibodies tested have endocytic activity, and five clones with relatively good endocytic activity were selected as the antibodies of this application.

[0168] The sequences of the five antibody variable regions are shown below. The variable regions are encoded using the Kabat scheme, and CDRs are shown in bold and underlined (the same applies below): AHP53835 (SEQ ID NO: 4): QVQLVESGGGLVQPGGSLRLSCAASRSDFS LNVMG WFRQAPGNQRELVA SITRGGSVRYVDSVKG RFTISRDGAENTMSLQMNSLKPEDTAVYYCFA ELSPWSAPA WGQGTQVIVSS AHP54695 (SEQ ID NO: 13): QVKLEESGGGLVQPGGSLRLSCAASRSVLS PNVMG WYRQAPGKQREQVA TITPGGSTNYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYICTA DFWPSDSAPYDS WGQGTQVTVSS AHP54962 (SEQ ID NO: 22): DVQLVESGGGLVQPGGSLRLSCTASGSIFS VNLMGWHRQVPGKQRELVA SISRGGSTKYGDSVKG RFTLSRDNAKNTVYLQMNSLKPEDTAVYYCLA DSVGEEGFYDY WGQGTQVTVSS AHP54966 (SEQ ID NO: 28): QVQLVESGGGLVQPGGSLRLSCAASGSIFS IYAMG WYRQAPGKQRELVA SISSGGSTNYADSVKG RFTISRDNAKNTVYLQMNSLKPEDTAVYYCNA DLAYGFDPKYDY WGQGTQVTVSS AHP54938 (SEQ ID NO: 36):AVQLVESGGGLVQPGGSLRLSCAASGSFLS INVMG WYRQVPGKQRELVA TITRGGSLNYAEDVRG RFTVSRDNAKNTVYLQMNSLKLEDTAVYYCTA DEWATPRGWSDY WGQGTQVTVSS

[0169] Example 2: Humanization of anti-Nectin-4 antibody

[0170] 1. Humanization of anti-Nectin-4 antibody

[0171] Five VHH antibodies obtained from screening were humanized using the CDR-Grafting method. The parental anti-Nectin-4 antibody was compared with fully human germline genes in the IMGT or NCBI / IGBAST databases using the IMGT or NCBI website. The human germline gene IGHV 3-23 (IGHV3-23), which is highly homologous to the Nectin-4 nanobody, was selected. 01; IGHV3-23 04) As a humanization template, CDR was transplanted, and four key residues (37, 44, 45, 47) in the FR2 of the nanobody were retained. The key core residues in the framework region close to the CDR region and the Vernier position residues that interact with CDR were analyzed and reversed to obtain humanized antibodies.

[0172] The variable region of the antibody sequence uses the Kabat coding scheme. The following is an example of the humanized antibody molecule sequence of AHP53835: AHP53835-h2 (SEQ ID NO: 5): EVQLVESGGGLVQPGGSLRLSCAASRSDFS LNVMGWFRQAPGKQRELVS SITRGGSVRYVDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCFA ELSPWSAPA WGQGTLVTVSS AHP53835-h5 (SEQ ID NO: 6): EVQLVESGGGLVQPGGSLRLSCAASRSDFS LNVMG WFRQAPGKQRELVA SITRGGSVRYVDSVKG RFTISRDNAENTLYLQMNSLRAEDTAVYYCFA ELSPWSAPA WGQGTLVTVSS AHP53835-h7 (SEQ ID NO: 7): EVQLVESGGGLVQPGGSLRLSCAASRSDFS LNVMG WFRQAPGKQRELVA SITRGGSVRYVDSVKG RFTISRDGAENTLYLQMNSLRAEDTAVYYCFA ELSPWSAPA WGQGTLVTVSS AHP53835-h8 (SEQ ID NO: 8): EVQLVESGGGLVQPGGSLRLSCAASRSDFS LNVMG WFRQAPGNQRELVA SITRGGSVRYVDSVKG RFTISRDGAENTLYLQMNSLRAEDTAVYYCFA ELSPWSAPA WGQGTLVTVSS Based on the AHP53835-h7 sequence, four cysteine ​​residues were added inside VHH, resulting in the following variant molecular sequence: AHP53835-h7m1(SEQ ID NO: 9): EVCLVESGGGLVQPGGSLRLSCAACRSDFS LNVMG WFRQAPGKQRELVA SITRGGSVRYVDSVKG RFTISRDGAENTLYLQMNSLRCEDTAVYYCFA ELSPWSAPA WGQGTLVTVSC The following is an example of a humanized antibody molecule sequence for the AHP54695 molecule: AHP54695-h1 (SEQ ID NO: 14): EVQLVESGGGLVQPGGSLRLSCAASGFTVS PNVMG WYRQAPGKGREQVSTITPGGSTNYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCTA DFWPSDSAPYDS WGQGTLVTVSS AHP54695-h6 (SEQ ID NO: 15): EVQLVESGGGLVQPGGSLRLSCAASRSVLS PNVMG WYRQAPGKQREQVA TITPGGSTNYADSVKG RFTISRDNAKNTVYLQMNSLRAEDTAVYICTA DFWPSDSAPYDS WGQGTLVTVSS AHP54695-h7m1(SEQ ID NO: 17): EVQLVESGGGLVQPGGSLRLSCAASGFTVS PNVMG WYRQAPGKGREQVS TITPGGSTNYADSVKG RFTISRDNAKNTVYLQMNSLRAEDTAVYYCTA DFWPSDTAPYDS WGQGTQVTVSS Based on the AHP54695-h7m1 sequence, two cysteine ​​residues were added inside VHH, resulting in the following variant molecule sequence: AHP54695-h7m1-2C (SEQ ID NO: 18): EVQLVESGGGLVQPGGSLRLSCAASGFTVS PNVMG WYRQAPGKGREQVS TITPGGSTNYADSVKG RFTISRDNAKNTVYLQMNSLRCEDTAVYYCTA DFWPSDTAPYD SWGQGTQVTVSC The following is an example of a humanized antibody molecule sequence for the AHP54692 molecule: AHP54962-h4 (SEQ ID NO: 23): EVQLVESGGGLVQPGGSLRLSCAASGSIFS VNLMG WHRQAPGKQRELVA SISRGGSTKYGDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCLA DSVGEEGFYDY WGQGTLVTVSS AHP54962-h6 (SEQ ID NO: 24): EVQLVESGGGLVQPGGSLRLSCAASGSIFS VNLMG WHRQAPGKQRELVASISRGGSTKYGDSVKG RFTLSRDNAKNTVYLQMNSLRAEDTAVYYCLA DSVGEEGFYDY WGQGTLVTVSS The following is an example of a humanized antibody molecule sequence for the AHP54966 molecule: AHP54966-h1 (SEQ ID NO: 29): EVQLVESGGGLVQPGGSLRLSCAASGSIFS IYAMG WYRQAPGKGRELVS SISSGGSTNYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCNA DLAYGFDPKYDY WGQGTTVTVSS AHP54966-h4 (SEQ ID NO: 30): EVQLVESGGGLVQPGGSLRLSCAASGSIFS IYAMG WYRQAPGKQRELVA SISSGGSTNYADSVKG RFTISRDNAKNTVYLQMNSLRAEDTAVYYCNA DLAYGFDPKYDY WGQGTTVTVSS AHP54966-h1m1(SEQ ID NO: 32): EVQLVESGGGLVQPGGSLRLSCAASGSIFS IYAMG WYRQAPGKGRELVS SISSGGSTNYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCNA DLAYGFDAKYDY WGQGTQVTVSS The following is an example of a humanized antibody molecule sequence for the AHP54938 molecule: AHP54938-h1 (SEQ ID NO: 37): EVQLVESGGGLVQPGGSLRLSCAASGSTVS INVMG WYRQAPGKGRELVS TITRGGSLNYAEDVRG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCTA DEWATPRGWSDY WGQGTTVTVSS

[0173] AHP54938-h7 (SEQ ID NO: 38): EVQLVESGGGLVQPGGSLRLSCAASGSTVS INVMG WYRQAPGKGRELVS TITRGGSLNYAEDVRGRFTVSRDNSKNTVYLQMNSLRAEDTAVYYCTA DEWATPRGWSDY WGQGTQVTVSS

[0174] 2. Affinity assay of humanized anti-Nectin-4 antibody

[0175] The affinity of humanized AHP53835 molecules for the Nectin-4 protein with its His Tag antigen (Acro, NE4-H52H3) was detected using surface plasmon resonance (SPR) technology with a Biacore T200 (Cytiva) instrument. A Series S Sensor Chip Protein A (Cytiva) was used, with HBS-EP+ buffer pH 7.4 (Genscript) as the mobile phase. Each antibody was prepared as a ligand using HBS-EP+ buffer pH 7.4 (Genscript) and captured by Protein A on the chip channel. The antibody was captured onto the Protein A sensor chip at a flow rate of 10 μl / min for 12 seconds. Human Nectin-4 Protein and His Tag antigen (Acro, NE4-H52H3, 2823a-21BAF1-1CX) were used as analytes and prepared with HBS-EP+ buffer. The analytes were serially diluted twofold and flowed through the experimental and reference channels at a flow rate of 30 µL / min for 2 minutes for binding and 2 minutes for dissociation. Regeneration buffer (10 mM Glycine-HCl, pH 1.5) was added and run at a flow rate of 30 µL / min for 30 seconds. The data were processed using Biacore T200 evaluation software version 4.0. The binding rate Ka, dissociation rate Kd, and dissociation constant (KD) were calculated.

[0176] For the AHP53835 molecule, six humanized antibodies (AHP53835-h1 to AHP53835-h6) were synthesized in the first round of humanization design. After expression and purification, and based on SPR affinity assays, antibody purity, number of reversion mutations, and expression levels, two antibodies (AHP53835-h5 and AHP53835-h6) showed similar binding affinity to the chimeric antibody. A second round of humanization design was then conducted based on AHP53835-h5. The results showed that the antibody affinity increased after the second round of humanization, with AHP53835-h7 and AHP53835-h8 approaching the affinity of the AHP53835 alpaca maternal antibody. Exemplary affinity data are shown in Table 4.

[0177] Table 4. Affinity assay results of humanized AHP53835 antibody molecules.

[0178] In addition, the intermolecular affinity (antibody-antigen binding) of humanized molecules AHP54695, AHP54962, AHP54966, and AHP54938 was determined using ForteBio Octet biolayer interferometry (BLI). Humanized antibodies were immobilized using an AHC2 sensor at a concentration of 5 μg / ml for 60 seconds. The buffer was PBST (PBS + 0.02% Tween 20 + 0.1% BSA), diluted with human-Nectin-4-His (Acro, NE4-H52H3) to 25, 12.5, 6.25, 3.13, 1.56, and 0 nM. Affinity assays were performed: equilibration for 120 seconds, binding for 180 seconds, dissociation for 180 seconds, and detection at 30°C. Using Octet data analysis software, select a fitting model (commonly the 1:1 Langmuir binding model), and calculate the binding rate constant (kon), dissociation rate constant (koff), and equilibrium dissociation constant (KD=koff / kon).

[0179] In the first round of humanization design, six humanized antibodies (AHP54695-h1 to AHP54695-h6) were synthesized. Then, a second round of humanization molecules was designed based on AHP54695-h1 and AHP54695-h6. The results showed that AHP54695-h7m1 removed a potential PTM risk point in CDR3 while retaining the affinity of the alpaca maternal antibody. Exemplary affinity data are shown in Table 5.

[0180] Table 5. Affinity assay results of humanized AHP54695 antibody molecules.

[0181] In the first round of humanization design, six humanized antibodies (AHP54962-h1 to AHP54962-h6) were synthesized. Then, a second round of humanization molecules was designed based on AHP54962-h6. Based on the expression data and affinity assay data of the humanized antibodies, AHP54962-h4 was selected as a candidate humanized molecule. Exemplary affinity data are shown in Table 6.

[0182] Table 6. Affinity assay results of humanized AHP54962 antibody molecules

[0183] In the first round of humanization design, four humanized antibodies (AHP54966-h1 to AHP54966-h4) were synthesized. Then, based on AHP54966-h1 and AHP54966-h4, a second-round humanized molecule, AHP54966-h1m1, was designed. Results showed that the affinity of AHP54966-h1m1 largely retained that of the alpaca maternal antibody, while simultaneously eliminating a potential PTM risk point in CRD3. Exemplary affinity data are shown in Table 7.

[0184] Table 7. Affinity assay results of humanized AHP54966 antibody molecules.

[0185] Six humanized antibodies (AHP54938-h1 to AHP54938-h6) were synthesized through the design of AHP54938 antibody. Then, a second-round humanized molecule, AHP54938-h7, was designed based on AHP54938-h1. The results showed that the AHP54938-h7 humanized antibody retained the affinity of the alpaca maternal antibody. Exemplary affinity data are shown in Table 8.

[0186] Table 8. Affinity assay results of humanized AHP54938 antibody molecules.

[0187] 3. Antibody affinity maturation

[0188] Antibody affinity maturation studies were conducted based on a precise saturation mutagenesis and screening strategy, aiming to improve the targeting and binding ability of the parent antibody to human Nectin4 protein. Taking AHP53835-h2 as an example, 35 key residue sites were selected within its complementarity-determining region (CDR), and a mutant library containing 18 amino acids (excluding cysteine) was designed for each site. DNA oligonucleotide libraries were constructed using high-throughput synthesis technology, and next-generation sequencing (NGS) was used for quality monitoring to ensure that the coverage of all mutation sites was no less than 90%.

[0189] For each saturated mutant library, 48 clones were randomly selected for expression in *E. coli*. Specifically, 44-48 clones from each library were inoculated into 96-well deep-plates for induced expression. The binding activity of crude antibody protein to human Nectin4 protein in the culture supernatant was then quantitatively analyzed using ELISA. Positive clones with enhanced affinity were screened, and the mutation sites were verified by sequencing.

[0190] The obtained dominant mutations were further randomly combined using multiplex PCR to construct a combined mutant library. Two rounds of ELISA screening were performed on the lead clones in the library, and affinity ranking was performed based on binding signal intensity. High-potential clones were finally selected for DNA sequence analysis. After preparing and purifying antibodies using a mammalian expression system, the kinetic parameters (KD value) of the antibody were matured using surface plasmon resonance (SPR) affinity.

[0191] The results showed that the Top 5 clones with enhanced affinity were selected from the combinatorial library for antibody purification and synthesis.

[0192] The variable region sequence after affinity maturation is as follows (according to Kabat's definition, where the CDR of VHH is shown in bold and underline): AHF47750 (SEQ ID NO: 42): EVQLVESGGGLVQPGGSLRLSCAASRSVFS FNIWG WFRQAPGKQRELVS SISRGGSVRYVDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCFA EESIWSAPA WGQGTLVTVSS AHF47752 (SEQ ID NO: 44): EVQLVESGGGLVQPGGSLRLSCAASRSVFS FNVWG WFRQAPGKQRELVS SITRGGSVRYVDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCFA EESIWSAPA WGQGTLVTVSS AHF47753 (SEQ ID NO: 46): EVQLVESGGGLVQPGGSLRLSCAASRSDFS LNIWG WFRQAPGKQRELVS SISRGGSVRYVDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCFA EESIWSAPA WGQGTLVTVSS AHF47765 (SEQ ID NO: 48): EVQLVESGGGLVQPGGSLRLSCAASRSVFS LNIWG WFRQAPGKQRELVS SITRGGSVRYVDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCFA ELSIWSAPA WGQGTLVTVSS AHF47772 (SEQ ID NO: 49): EVQLVESGGGLVQPGGSLRLSCAASRSVFS LNIWG WFRQAPGKQRELVS SITRGGSVRYVDSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCFA EESIWSAPA WGQGTLVTVSS AHF47750E (SEQ ID NO: 51): EVQLVESGGGLVQPGGSLRLSCAASRSVFS FNIWG WFRQAPGKQRELVS SISRGGSVRYVDEVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCFA EESIWSAPA WGQGTLVTVSS SPR multi-concentration assays of the Top5 clones and human Nectin4 antigen protein showed that the affinity of these five antibodies was more than 10-fold higher than that of the parent strain AHP53835-h2. Specific results are shown in Table 9. Since AHF47750, constructed in VHH form, is not expressed, to optimize antibody expression, AHF47750E was obtained by amino acid mutation based on AHF47750. BLI assays showed that the affinity of AHF47750E was slightly lower than that of AHF47750.

[0193] Table 9. Antibody affinity determined after maturation.

[0194] Example 3: Expression, purification, and detection of VHH-G4SC conformation antibody

[0195] 1. Expression and purification of VHH-G4SC conformation antibody

[0196] Selected antibody sequences were inserted into the eukaryotic expression vector pcDNA3.4 and transfected into ExpiCHO-S cells for antibody expression. The supernatant was added to MonoRab Anti-Camelid VHH Affinity Resin and incubated at room temperature for 2 hours on a rotary incubator. The Resin affinity column was equilibrated with binding buffer to 10 column volumes. The Resin affinity column was eluted with Elution Buffer and collected according to the A280 of the UV detector. After collection, neutralization buffer was added to adjust the sample pH to approximately 5.0. The eluted and neutralized antibody was transferred to a dialysis bag and dialyzed at 25°C with dialysis buffer for 2–3 hours. The dialysis buffer was then replaced, and the sample was dialyzed at 4°C for 16–18 hours. The sample was transferred from the dialysis bag to centrifuge tubes and filtered through a 0.22 μm filter in a biosafety cabinet. The sample volume was recorded. The concentration was determined and recorded using Nanodrop One.

[0197] The six VHH antibodies obtained through screening were recombinantly expressed with the linker G4SC to obtain VHH-G4SC conformation antibodies. The SDS-PAGE results of the purified antibodies are shown below. Figure 3 All antibodies were expressed normally, and the molecular weight of the purified antibodies was correct; the SDS purity was >90%.

[0198] 2. ELISA detection of VHH-G4SC conformation antibody

[0199] The binding activity of purified antibody to human Nectin-4 protein was detected using an ELISA assay. 1 μg / ml of human Nectin-4 protein was added to each well of a 96-well microplate and incubated overnight at 4°C. After washing three times, 300 μl of 3% MPBS was added to each well, and the plate was blocked at 37°C for 1 hour. The initial concentration of the antibody to be tested was 100 nM, and it was serially diluted 3-fold for a total of 11 dilutions, with a blank control included. 100 μl of the diluted antibody was added to each well of the blocked microplate and incubated at 37°C for 60 minutes. After washing three times, 100 μl of the secondary antibody MonoRab™ Rabbit Anti-Camelid VHH Cocktail [HRP] was added, and the plate was incubated at 37°C for 30 minutes. After washing six times, 100 μl of TMB chromogenic buffer was added to each well, and after incubation at room temperature for 10 minutes, 50 μl of stop buffer was added to each well. The OD450 nm value was read using a microplate reader.

[0200] See results Figure 4 See Table 10. PC represents the positive control antibody (Enfortumab), and NC represents the negative control human IgG isotype antibody. This indicates that all six recombinant antibodies (VHH antibodies) have binding activity to human Nectin-4 protein.

[0201] Table 10 ELISA results of VHH-G4SC conformation antibody and human Nectin-4 protein

[0202] 3. FACS detection of VHH-G4SC conformation antibody

[0203] The binding activity of purified antibodies to T47D and HT1376 cells was detected by flow cytometry. Digested cells were washed with PBS and aliquoted into 96-well plates at 100 μl / well, 1×10⁻⁶ cells / well. 5 Cells / well, starting antibody concentration of 300 nM, serially diluted 3-fold for a total of 11 dilutions, with a blank control included. Add 100 μl / well to each 96-well plate and incubate at 4°C for 40 minutes. Wash cells twice with pre-chilled PBS. Add 100 μl of 1 μg / ml GenScript MonoRab secondary antibody to each well. TM Rabbit anti-Camelid VHH Cocktail [iFluor 647] was incubated at 4°C for 40 minutes. After washing twice, the cells were resuspended in PBS, and the fluorescence intensity was detected by flow cytometry.

[0204] The results are shown below. Figure 5 and Table 11 and Figure 6 The results are shown in Table 12, where PC is the positive control antibody Enfortumab and the human IgG isotype antibody is the negative control. This indicates that all six recombinant antibodies have binding activity with T47D and HT1376 cells.

[0205] Table 11. Binding activity of VHH-G4SC conformation antibody to T47 cells (EC) 50 value

[0206] Table 12. Binding activity of VHH-G4SC conformation antibody to HT1376 cells (EC) 50 value

[0207] Example 4: Expression, purification, and detection of bivalent VHH-Fc conformation antibodies

[0208] 1. Expression and purification of bivalent VHH-Fc conformation antibodies

[0209] The antibody sequence was inserted into the eukaryotic expression vector pcDNA3.4 and transfected into ExpiCHO-S cells for antibody expression. Protein A Magnetic Beads were added to the sample to be purified and incubated in a room temperature shaking incubator for 2 hours. The Protein A Magnetic Beads were equilibrated with 10 column volumes of binding buffer. Elution buffer was added to the Protein A Magnetic Beads, and the eluent was collected. Neutralization buffer (1 / 10 of the collected eluent volume) was added simultaneously to adjust the pH to neutral. After elution and neutralization, the collected sample was transferred to a dialysis bag and purified using 1... The antibody was dialyzed against PBS at room temperature for 2 hours, then the medium was changed and dialyzed again at 2-8°C for 16 hours. The concentration and purity of the antibody were determined by OD280 and SDS-PAGE.

[0210] Five VHH antibodies obtained through screening were recombinantly expressed with human IgG1-Fc to obtain bivalent VHH-Fc antibodies. Antibody expression information is shown in Table 13. Results showed that all antibodies were expressed normally, the molecular weight of the purified antibodies was correct, and the SDS purity was >95%.

[0211] Table 13 Information on bivalent VHH-Fc conformation antibodies

[0212] Human IgG1 Fc sequence (SEQ ID NO: 53): DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0213] 2. ELISA detection results of bivalent VHH-Fc antibodies

[0214] The binding level of bivalent VHH-Fc antibody to human Nectin-4 protein was detected by ELISA. 1 μg / ml of human Nectin-4 protein was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. After washing three times, 300 μl of 3% MPBS was added to each well, and the plate was blocked at 37°C for 1 hour. The antibody was diluted to a concentration of 100 nM in the first well, followed by 11 serial dilutions (3-fold), with a blank control included. The diluted antibody was then added to the blocked ELISA plate at 100 μl / well and incubated at 37°C for 1 hour. After washing three times, 100 μl of mouse anti-human IgG Fc-HRP secondary antibody was added, and the plate was incubated at 37°C for 30 minutes, followed by six washes. 100 μl of TMB chromogenic buffer was added to each well, and the plate was incubated at room temperature for 10 minutes, followed by 50 μl of stop solution per well. The OD value at 450 nm was read using an ELISA reader.

[0215] The test results are shown in Table 14 and Figure 7 PC is the positive control antibody (Enfortumab), and NC is the negative control human IgG isotype antibody. It is evident that all five recombinant antibodies exhibit binding activity to human Nectin-4 protein.

[0216] Table 14 Results of ELISA detection of bivalent VHH-Fc anti-antibody

[0217] Example 5: Application of anti-Nectin-4 antibody

[0218] 1. VHH antibody conjugated with maleimide-NOTA / H3RESCA

[0219] VHH antibodies (such as VHH-G4SC, VHH-GSC, and VHH-Fc) were added to TCEP at a molar ratio of VHH:TCEP = 1:10, and incubated at 37°C for 60 min to release free cysteine. PBS was used as the buffer in the experiment. After the reaction was complete, Mal-NOTA or / Mal-H3RESCA was added at a molar ratio of VHH:Mal-NOTA / Mal-H3RESCA = 1:10, and incubated at room temperature for 2 hours. The experiment was performed in PBS buffer. The reacted samples were purified using a Superdex 75pg molecular sieve, with PBS as the mobile phase at a flow rate of 1 ml / min. The average conjugation ratio (CAR) of the chelating agent to the antibody was calculated using mass spectrometry. The results showed that the average CAR values ​​of the conjugated VHH-NOTA and VHH-H3RESCA (abbreviations for VHH-Mal-NOTA and VHH-Mal-H3RESCA, respectively) were close to 1. Exemplary results are shown in Table 15.

[0220] Table 15 CAR values ​​of VHH-NOTA / H3RESCA by mass spectrometry analysis

[0221] The cell-binding activity and affinity assay protocol of the antibodies conjugated with NOA or H3RESCA were evaluated according to Example 3. The results showed that AHP54695-h7m1-G4SC-NOTA and AHP54695-h7m1-G4SC-H3RESCA exhibited good cell-binding activity and affinity. For specific details on the cell-binding activity of the VHH-chelating agent in SW780 cells (Wuhan Pronosei Life Sciences Co., Ltd., catalog number CL-0449B), please refer to Table 16. Figure 8 .

[0222] BLI assay for the affinity between VHH-chelating agent and antigen: Fc-Nectin4 (purchased from Arco) was cured using an AHC2 sensor at a concentration of 10 μg / mL for 90 s. After equilibration in buffer for 60 s, unbound antigen was washed away. Then, 200 nM serially diluted antibody was bound for 60 s, followed by antigen dissociation in buffer for 180 s. Octet data analysis software was used to select a fitting model and calculate the binding rate constant (kon), dissociation rate constant (koff), and equilibrium dissociation constant (KD = koff / kon). The results of the VHH-chelating agent affinity assay are shown in Table 17.

[0223] Table 16 FACS detection results of VHH-chelator in SW780 cells

[0224] Table 17. Affinity of VHH-chelating agent to antigen detected by BLI method.

[0225] 2. Low pH stability analysis of VHH-NOTA and VHH-H3RESCA

[0226] The low-pH stability tests of the RDC precursors of VHH-NOTA and VHH-H3RESCA were conducted in an acetate-sodium acetate system at three pH conditions (pH 3.5; 4.5; 5.5), with the temperature controlled at 37℃ and the incubation time at 30 minutes. The control group consisted of samples of the same concentration in a PBS system at pH 7.4. After the stability test samples were incubated, the purity of the main peak of the antibody was analyzed by SEC-HPLC, and the appearance of the antibody solution under different pH conditions (e.g., opalescence or precipitation) was observed visually.

[0227] The results showed that, compared with the sample at pH 7.4, the purity of the main peak of VHH-NOTA / VHH-H3RESCA SEC stored at different low pH values ​​did not decrease significantly, and no opalescence or precipitation was observed in the appearance. Exemplary results are shown in Table 18.

[0228] Table 18 Results of SEC-HPLC analysis for low pH stability

[0229] 3. Preliminary accelerated stability experiments of VHH-NOTA / H3RESCA or VHH-Fc

[0230] Preliminary accelerated stability analysis of the purified VHH-NOTA / H3RESCA RDC precursor or VHH-Fc was performed using a high-temperature accelerated method to evaluate whether VHH-NOTA / H3RESCA or VHH-Fc would precipitate, aggregate, or degrade. Samples were placed at 40°C, and samples were taken at 3 days, 1 week, 2 weeks, and 4 weeks for supernatant content and SEC-HPLC purity analysis. Data were compared with those of samples not placed at 40°C.

[0231] The results showed that the supernatant content of the samples placed at high temperatures did not change significantly, nor did the purity of the SEC main peak. Exemplary results are shown in Tables 19 and 20.

[0232] Table 19 Accelerated stability test results of VHH-NOTA / H3RESCA

[0233] Table 20 Accelerated stability test results of VHH-Fc

[0234] 4. Preliminary analysis of the freeze-thaw stability of VHH-NOTA

[0235] VHH-NOTA was aliquoted into multiple tubes of equal volume (e.g., 100 μL / tube). The control group consisted of fresh, un-frozen samples for direct testing (0 freeze-thaw cycles). The freeze-thaw experimental group consisted of samples that underwent 5 freeze-thaw cycles. Sample tubes were placed at the target low temperature (-80°C) for ≥2 hours and then thawed at room temperature until completely thawed. A portion of the sample was used for testing, while the remaining sample underwent further freeze-thaw cycles. This freeze-thaw cycle was repeated until 5 cycles were completed. The purity of the main peak was determined using SEC-HPLC to analyze the formation of aggregates or fragments.

[0236] The results showed that the purity of the SEC main peak did not change significantly after five freeze-thaw cycles, indicating good freeze-thaw stability. Exemplary results are shown in Table 21.

[0237] Table 21 Results of VHH-NOTA freeze-thaw stability test

[0238] 5. VHH-NOTA / H3RESCA precursor 68 Ga / 18 F marking and quality control

[0239] 5.1 VHH-NOTA precursor 68 Ga labeling and quality control

[0240] Achieving this through chelation coupling technology 68 The specific steps for Ga radioactive labeling are as follows: (1) Preparation of NOTA-VHH coupling solution: Take VHH-NOTA precursor (5 nmol), add 0.5 mL of 0.35 mol / L sodium acetate buffer (containing 1 mg / mL gentian acid) to redissolve, mix well and transfer all to reaction flask.

[0241] (2) Preparation of radionuclides: Rinse with 0.1 mol / L sterile hydrochloric acid solution 68 Ge / 68 Ga generator, collection 68 Pour the GaCl3 eluent into a vial.

[0242] (3) Construction of chelation reaction system (labeling reaction): Take 1 mL 68 Add GaCl3 eluent to the reaction flask; place the reaction flask in a constant temperature metal bath and react at 37°C for 15-30 minutes.

[0243] (4) Purification and displacement: The reaction solution was purified by a PD-10 desalting column. Column equilibration: 15 mL of sodium chloride injection containing 15 mg / mL sodium ascorbate and 1 mg / mL gentic acid; loading volume: 1.5 mL; elution buffer: sodium chloride injection containing 15 mg / mL sodium ascorbate and 1 mg / mL gentic acid, and 1.5-3.0 mL of elution peak were collected; the solution was filtered using a 0.22 μm PVDF sterile filter.

[0244] (5) Quality Control Analysis: Radioactivity purity was determined using a high-performance liquid chromatograph with a radioactivity detector; the radiochemical purity was ≥90%. Free 68 Ga ions and 68 Ga colloids were all detected using a radiometric scanner; free Ga colloids were examined. 68 Ga ions and 68 Ga colloid ≤5%.

[0245] The results showed that the nuclide coupling efficiency was high after coupling. An example of a VHH-NOTA precursor is shown.68 The Ga labeling detection results are shown in Table 22.

[0246] Table 22 VHH-NOTA precursors 68 Ga marker detection results

[0247] 5.2 VHH-H3RESCA precursor 68 Ga labeling and quality control

[0248] Achieving this through chelation coupling technology 68 The specific steps for Ga radioactive labeling are as follows: (1) Preparation of H3RESCA-VHH coupling solution: Take VHH-H3RESCA precursor (5 nmol), add 1.5 mL of 0.35mol / L sodium acetate buffer (containing 1 mg / mL gentian acid and appropriate amount of Tween 80) to reconstitute, mix well and transfer all to reaction flask.

[0249] (2) Preparation of radionuclides: Rinse with 0.1 mol / L sterile hydrochloric acid solution 68 Ge / 68 Ga generator, collection 68 Pour the GaCl3 eluent into a vial.

[0250] (3) Construction of the chelation reaction system (labeling reaction): Take 3 mL 68 Add GaCl3 eluent to the reaction flask; place the reaction flask in a constant temperature metal bath and react at 37°C for 30 minutes.

[0251] (4) Sterilization filtration: Filtration is carried out using a 0.22 μm PVDF sterile filter.

[0252] (5) Quality Control Analysis: Radioactivity purity was determined using a high-performance liquid chromatograph with a radioactivity detector; the radiochemical purity was ≥90%. Free 68 Ga ions and 68 Ga colloids were all detected using a radiometric scanner; free Ga colloids were examined. 68 Ga ions and 68 Ga colloid ≤5%.

[0253] The results showed that the nuclide coupling efficiency was high after coupling. VHH-H3RESCA precursor 68 The Ga labeling detection results are shown in Table 23.

[0254] Table 23 VHH-H3RESCA precursors 68 Ga marker detection results

[0255] 5.3 VHH-H3RESCA precursor 18 F marking and quality control

[0256] Achieving this through chelation coupling technology 18 The specific steps for F radioactive labeling are as follows: (1) Preparation of aluminum chloride solution: Take 10 mg of aluminum chloride or aluminum chloride hydrate and add it to 50 mL of 0.1 mol / L sodium acetate buffer. After it is completely dissolved, take 0.2 mL of the solution and add it to the reaction flask.

[0257] (2) Preparation of radionuclides: fluorine-containing [ 18 F] ions H2 18 After O was enriched by QMA column, fluoride was removed using sodium chloride injection. 18 F] ions were eluted into the reaction flask and reacted at 37°C for 5 minutes.

[0258] (3) Preparation of H3RESCA-VHH coupling solution: Take VHH-H3RESCA precursor (500 nmol), add 0.5 mL of 0.1 mol / L sodium acetate buffer (containing 1-5 mg / mL gentian acid and appropriate amount of Tween 80) to redissolve, mix well and transfer all to reaction flask.

[0259] (4) Construction of chelation reaction system (labeling reaction): Place the reaction flask in a constant temperature metal bath or synthesizer and react at 37°C for 15-30 minutes.

[0260] (5) Purification and replacement: The reaction solution was purified by an antibody purification column. Column equilibration: 15 mL sodium chloride injection containing 15 mg / mL sodium ascorbate and 1 mg / mL gentianic acid; loading volume: 1.5 mL; elution buffer: sodium chloride injection containing 15 mg / mL sodium ascorbate and 1 mg / mL gentianic acid, and 1.5-3.0 mL of elution peak was collected; the solution was filtered using a 0.22 μm PVDF sterile filter.

[0261] (6) Quality control analysis: Radioactivity purity was determined using a high-performance liquid chromatograph with a radioactivity detector; the radiochemical purity was ≥90%. Free 18 F ions were detected using a radiometric scanner; free ions... 18 F ions ≤ 5%.

[0262] The results showed that the nuclide coupling efficiency was high after coupling. VHH-H3RESCA precursor 18 The results of F-label detection are shown in Table 24.

[0263] Table 24 VHH-H3RESCA precursors 18 F-mark detection results

[0264] 6. 68 Ga / 18 In vivo imaging of F-NOTA / H3RESCA-VHH

[0265] 6.1 68 In vivo imaging of Ga-NOTA-VHH

[0266] Using the SW780 mouse model for... 68 In vivo distribution study of Ga-NOTA-VHH. Female nude mice aged 6–8 weeks (Beijing Vital River Laboratory Animal Technology Co., Ltd., SPF, 14–16 g) were selected and xenografted with SW780 cells into the upper right back of the mice. The cell seeding density was approximately 7–8 × 10⁶ cells / mL. 6 Cells / mouse. Usable tumors (150-200 mm) were obtained after 3 weeks. 3 ).

[0267] Radiolabeled VHH antibody was administered to mice via tail vein injection at approximately 100 μCi. Imaging studies were conducted at 30 min, 1 h, 2 h, and 4 h post-administration to investigate drug targeting and analyze radioactive uptake in tumors, heart, muscle, and kidneys at multiple time points. Simultaneously, corresponding regions of the kidneys, tumors, and muscles were delineated on PET / CT fusion images to obtain the %ID / g of the respective organs. In the blocking experiment, mice were pre-administered the therapeutic drug VHH antibody. After drug distribution was complete, mice were injected via tail vein injection, and imaging studies were performed at 30 min, 1 h, 2 h, and 4 h post-administration.

[0268] Example PET-CT imaging results can be found in Figure 9 and Figure 10 The results indicate that VHH antibodies were taken up in the tumor sites of SW780 tumor-bearing mice. The drug was mainly excreted in the urine via the kidneys and bladder. Among them, AHP54695-h7m1-G4SC-NOTA, AHP54966-h1m1-G4SC-NOTA and AHP54962-h4-GSC-NOTA showed higher tumor uptake, and AHP54695-h7m1-G4SC-NOTA had the best tumor / kidney ratio.

[0269] In addition, Tables 25 to 31 show PET-CT animal imaging data of the exemplary VHH-G4SC-NOTA.

[0270] Table 25 Animal imaging data of AHP53835-h7-G4SC-NOTA

[0271] (%ID / g and its ratios, average ± SD, n = 3)

[0272] Table 26 Animal Imaging Data for AHP53835-h7m1-NOTA

[0273] (%ID / g and its ratios, average ± SD, n = 3)

[0274] Table 27 Animal Imaging Data for AHP54938-h7-G4SC-NOTA

[0275] (%ID / g and its ratios, average ± SD, n = 3)

[0276] Table 28 Animal Imaging Data for AHP54962-h4-GSC-NOTA

[0277] (%ID / g and its ratios, average ± SD, n = 3)

[0278] Table 29 Animal Imaging Data for AHP54695-h7m1-G4SC-NOTA

[0279] (%ID / g and its ratios, average ± SD, n = 3)

[0280] Table 30 Animal Imaging Data of AHP54966-h1m1-G4SC-NOTA

[0281] (%ID / g and its ratios, average ± SD, n = 3)

[0282] Table 31 Animal Imaging Data for AHP54695-h7m1-2C-NOTA

[0283] (%ID / g and its ratios, average ± SD, n = 3)

[0284] 6.2 68 In vivo imaging of Ga-H3RESCA-VHH

[0285] Using the SW780 mouse model for... 68In vivo distribution study of Ga-H3RESCA-VHH. Female nude mice aged 6–8 weeks (Beijing Vital River Laboratory Animal Technology Co., Ltd., SPF, 14–16 g) were selected and xenografted with SW780 cells into the upper right back of the mice. The cell seeding density was approximately 7–8 × 10⁻⁸ g. 6 Cells / mouse. Usable tumors (150-200 mm) were obtained after 3 weeks. 3 ).

[0286] Radiolabeled VHH antibody was administered to mice via tail vein injection at approximately 100 μCi. Imaging studies were conducted at 30 min, 1 h, 2 h, and 4 h post-administration to investigate drug targeting and analyze radioactive uptake in tumors, heart, muscle, and kidneys at multiple time points. Simultaneously, corresponding regions of the kidneys, tumors, and muscles were delineated on PET / CT fusion images to obtain the %ID / g of the respective organs. In the blocking experiment, mice were pre-administered VHH antibody, and after drug distribution was complete, they were injected via tail vein. Imaging studies were performed at 30 min, 1 h, 2 h, and 4 h post-administration.

[0287] [ 68 PET-CT imaging results at different time points after administration of Ga]AHP54695-h7m1-G4SC-H3RESCA are as follows: Figure 11 And as shown in Table 32. Through [ 68 In PET-CT images of SW780 tumor-bearing mice, Ga]AHP54695-h7m1-G4SC-H3RESCA showed good imaging efficacy against Nectin-4-positive tumors, with significant uptake at the tumor tissue site and low renal uptake, resulting in an excellent tumor / kidney ratio. Uptake in non-target organs was rapidly cleared over time. The drug is primarily excreted in the urine via the kidneys and bladder.

[0288] Table 32 [ 68 Ga]AHP54695-h7m1-G4SC-H3RESCA Animal Imaging Data

[0289] (%ID / g and its ratios, average ± SD, n = 3)

[0290] 6.3 18 In vivo imaging of F-H3RESCA-VHH

[0291] Using the SW780 mouse model for... 18In vivo distribution study of F-H3RESCA-VHH. Female nude mice aged 6–8 weeks (Beijing Vital River Laboratory Animal Technology Co., Ltd., SPF, 14–16 g) were selected and xenografted with SW780 cells into the upper right back of the mice. The cell seeding density was approximately 7–8 × 10⁶ cells / cm². 6 Cells / mouse. Usable tumors (150-200 mm) were obtained after 3 weeks. 3 ).

[0292] Radiolabeled VHH antibody was administered to mice via tail vein injection at approximately 100 μCi. Imaging studies were conducted 30 min and 2 h post-administration to investigate drug targeting, analyzing radioactive uptake in tumors, heart, muscle, and kidneys at multiple time points. Simultaneously, corresponding regions of the kidneys, tumors, and muscles were delineated on PET / CT fusion images to obtain the %ID / g of the respective organs. In the blockade experiment, VHH antibody was administered pre-administered, and after drug distribution was complete, mice were injected via tail vein. Imaging studies were performed 30 min and 2 h post-administration.

[0293] [ 18 PET-CT imaging results at different time points after administration of F]AHP54695-h7m1-G4SC-H3RESCA are as follows: Figure 12 And as shown in Table 33. Through [ 18 In PET-CT images of SW780 tumor-bearing mice, [F]AHP54695-h7m1-G4SC-H3RESCA showed good imaging efficacy against Nectin-4-positive tumors, with significant uptake at the tumor tissue site and low renal uptake, resulting in an excellent tumor / kidney ratio. Uptake in non-target organs was rapidly cleared over time. The drug is primarily excreted in the urine via the kidneys and bladder.

[0294] Table 33 [ 18 F]AHP54695-h7m1-G4SC-H3RESCA Animal Imaging Data

[0295] (%ID / g and its ratios, average ± SD, n = 3)

[0296] All patents, patent application publications, and non-patent documents mentioned and / or listed in this application are incorporated herein by reference in their entirety. Exemplary embodiments of the inventions described above have been described; however, those skilled in the art can modify or improve the exemplary embodiments described herein without departing from the spirit and scope of this application, and such variations or equivalents also fall within the scope of this application.

Claims

1. An anti-Nectin-4 antibody or its antigen-binding fragment, comprising a VHH chain, wherein the VHH chain comprises CDR1, CDR2, and CDR3, wherein: The amino acid sequence of CDR1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 3; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 10, the amino acid sequence of CDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 12; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 10, the amino acid sequence of CDR2 is shown in SEQ ID NO: 11, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 16; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 19, the amino acid sequence of CDR2 is shown in SEQ ID NO: 20, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 21; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 25, the amino acid sequence of CDR2 is shown in SEQ ID NO: 26, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 27; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 25, the amino acid sequence of CDR2 is shown in SEQ ID NO: 26, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 31; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 33, the amino acid sequence of CDR2 is shown in SEQ ID NO: 34, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 35; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 39, the amino acid sequence of CDR2 is shown in SEQ ID NO: 40, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 41; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 43, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 41; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 45, the amino acid sequence of CDR2 is shown in SEQ ID NO: 40, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 41; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 45, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 47; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 45, the amino acid sequence of CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 41; or The amino acid sequence of CDR1 is shown in SEQ ID NO: 39, the amino acid sequence of CDR2 is shown in SEQ ID NO: 50, and the amino acid sequence of CDR3 is shown in SEQ ID NO:

41. The above CDR sequence is defined according to Kabat.

2. The anti-Nectin-4 antibody or its antigen fragment as described in claim 1, wherein: The VHH chain comprises an amino acid sequence having at least 85% identity with any of the amino acid sequences shown in SEQ ID NO: 4-9, 13-15, 17-18, 22-24, 28-30, 32, 36-38, 42, 44, 46, 48, 49, and 51, or consists of any of the amino acid sequences shown in SEQ ID NO: 4-9, 13-15, 17-18, 22-24, 28-30, 32, 36-38, 42, 44, 46, 48, 49, and 51.

3. The anti-Nectin-4 antibody or its antigen fragment as described in claim 1, wherein the anti-Nectin-4 antibody is a VHH antibody or a VHH-Fc antibody; Optionally, the Fc fragment is derived from IgG; Optionally, the Fc fragment is derived from human IgG1; and / or Optionally, the anti-Nectin-4 antibody may be in monovalent or multivalent form.

4. An isolated nucleic acid molecule encoding the anti-Nectin-4 antibody or an antigen fragment thereof as described in any one of claims 1 to 3.

5. A vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the nucleic acid molecule of claim 4 or the vector of claim 5.

7. An antibody conjugate comprising the anti-Nectin-4 antibody as described in any one of claims 1 to 3, an antigen fragment thereof, a linker, and an effector; Optionally, the effector is selected from: radionuclides, antitumor agents, immunomodulators, bioreaction modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, imaging agents, chemiluminescent compounds, enzymes, and metal ions.

8. A radionuclide-labeled antibody conjugate comprising the anti-Nectin-4 antibody or its antigen-binding fragment as described in any one of claims 1 to 3 and a radionuclide-chelating agent; Optionally, the radionuclide-labeled antibody conjugate further comprises a linker; Optionally, the radioactive nuclides include alpha-ray nuclides, gamma-ray nuclides, beta-ray nuclides, and positron-emitting nuclides; Optionally, the radionuclide is selected from: 11 C 13 N、 15 O、¹ 8 F, 32 P, 44 Sc、 47 Sc、 51 Mn, 52m Mn, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 68 Ge 72 As、 75 Br、 76 Br、 82m Rb、 83 Sr、 86 Y、 89 Sr、 89 Zr、 90 Y、 99m Tc, 105 Rh、 110 In、 111 In、 117 mSn, 120 I, 123 I, 124 I, 125 I、¹³¹I、 134 Ce、 148 Gd, 149 Tb, 149 Pm, 152 Tb, 153 Sm、 154-158 Gd, 160 Gd, 161 Tb, 165 Ho、 166 Ho、 167 Tm、 175 Yb、¹ 77 Lu、 186 Re、 188 Re、 197 Au、 198 Au、 199 Au、 201 Ti、 209 Bi、 211 At、 212 Pb, 212 Bi、 213 Bi、 213 Po、²²³Ra、 223 Fr、²² 5 Ac, 227 Th and 229 Th; Optionally, the chelating agent is selected from 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), N,N'-bis(2-hydroxybenzyl)-ethylenediamine-N,N'-diacetic acid (HBED-CC), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), or triazacyclononane phosphate (TRAP), EDTA, NETA, CB-TE2A, Cyclen, Cyclam, Bispidine, TACN, ATSM, SarAr, AmBaSar, MAG3, MAG2, HYNIC, AAZTA, BAT, BAT-TM, Crown, Cyclen, DO2A, CB-DO2A, DO3A, DADT, EC, NS3, H2dedpa, HBED, DFO, PEPA, MACROPA, HEHA, DOTA-GA, p-BnDOTA, p-Bn-SCN-DOTA, NH2-DOTA, NH2-DOTA-GA, p-N CS-Bn-DOTA-GA, p-NH2-Bn-OxODO3A, p-SCN-Bn-oxo-DO3A, NODA-GA, NH2-NODA-GA, p-NCS-Bn-NODA-GA, p-NH2Bn-NOTA, p-SCN-Bn-NOTA, NCS-MP-N ODA, NH2-MPAA-NODA, PCTA, p-NH2-Bn-PCTA, pSCN-Bn-PCTA, p-SCN-Bn-HEHA, H2-MACROPA-NCS, H1-MACROPA, H2-MACROPA-NH2, H4OCTAPA, tetra-(S,S,S,S)-Me-DOTA, SHBED, (HBED-CC)TFP, DMSA, DMPS, DHLA, tetra-(S,S,S,S)-Et-DOTA, tetra-(S,S,S,S)-iBu-DOTA, and maleimide-nBu-DOTA and their derivatives; Optionally, the chelating agent is selected from H3RESCA, (+-)H3RESCA-TFP, and (+-)H3RESCA-Mal; Optionally, the radionuclide-labeled antibody conjugate is: radionuclide-VHH-Mal-NOTA, radionuclide-VHH-G4SC-Mal-NOTA, radionuclide-VHH-GSC-Mal-NOTA, radionuclide-VHH-Mal-H3RESCA, radionuclide-VHH-GSC-Mal-H3RESCA, or radionuclide-VHH-G4SC-Mal-H3RESCA; Optionally, the radionuclide-labeled antibody conjugate is selected from: [ 68 Ga]AHP54695-h7m1-G4SC- H3RESCA、[ 18 F]AHP54695-h7m1-G4SC-H3RESCA、 [ 68 Ga]AHP53835-h7-G4SC-NOTA、[ 68 Ga]AHP53835-h7m1-NOTA、 [ 68 Ga]AHP54938-h7-G4SC-NOTA,[ 68 Ga]AHP54962-h4-GSC-NOTA, [ 68 Ga]AHP54966-h1m1-G4SC-NOTE [ 68 Ga]AHP54695-h7m1-G4SC-NOTE, [ 68 Ga]AHP54695-h7m1-2C-NOTA。 9. A pharmaceutical composition comprising the anti-Nectin-4 antibody or its antigen-binding fragment as described in any one of claims 1 to 3, the nucleic acid molecule as described in claim 4, the vector as described in claim 5, the host cell as described in claim 6, the antibody conjugate as described in claim 7, or the radionuclide-labeled antibody conjugate as described in claim 8.

10. Use of any one of the anti-Nectin-4 antibody or antigen-binding fragment thereof, the nucleic acid molecule of claim 4, the vector of claim 5, the host cell of claim 6, the antibody conjugate of claim 7, the radionuclide-labeled antibody conjugate of claim 8, or the pharmaceutical composition of claim 9 in the preparation of a medicament for the prevention, diagnosis and / or treatment of diseases characterized by Nectin-4 expression; Optionally, the diseases characterized by Nectin-4 expression are selected from: head and neck squamous cell carcinoma, bladder cancer, urothelial carcinoma, breast cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, ovarian cancer, cervical cancer, melanoma, pancreatic cancer, liver cancer, and prostate cancer.

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