A nanobody targeting nectin-4 and its application

Abstract

The present invention discloses a nanobody targeting Nectin-4 and its application. The nanobody comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is shown in SEQ ID NO: 2, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 3, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: 4. The nanobody targeting Nectin-4 provided by the present invention can bind to tumor cells with high affinity and has good endocytic activity. In in vivo imaging, the tumor targeting is strong, the tumor enrichment is fast and the retention time is long, which is conducive to the study of the diagnosis, prognosis and occurrence and development of tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a nano antibody targeting nectin-4 and applications thereof. Background Art

[0002] Nectin-4 (also known as poliovirus receptor-related-4, PVRL-4) is an immunoglobulin-like molecule. Nectin-4 works together with other junction proteins, such as nectin-1, nectin-2, and nectin-3, to participate in cell-cell adhesion. Compared with other nectins, nectin-4 is specifically enriched in human embryonic and placental tissues, and its expression decreases significantly in adulthood. In recent years, studies have found that nectin-4 is particularly overexpressed and acts as a tumor-related inducer in various malignant tumors, such as breast cancer, lung cancer, colorectal cancer, pancreatic cancer, and ovarian cancer. In some types of cancer, overexpression of nectin-4 is associated with various aspects of tumor progression, such as proliferation, angiogenesis, epithelial to mesenchymal transition, metastasis, DNA repair, tumor recurrence, and poor prognosis (Subhajit et al., 2021).

[0003] Currently, there are no reports on effective therapeutic drugs or detection products for tumors related to Nectin-4 expression that use Nectin-4 as a target or biomarker. Summary of the Invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides a nano antibody targeting nectin-4 and its application.

[0005] The present invention solves the above technical problems through the following technical solutions.

[0006] The first aspect of the present invention provides a nanobody targeting nectin-4, wherein the nanobody comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is shown in SEQ ID NO: 2, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 3, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: 4.

[0007] In some embodiments of the present invention, the framework region of the heavy chain variable region is an alpaca-derived framework region or a human-derived framework region.

[0008] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, or has a sequence identity of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% to SEQ ID NO: 1 and does not involve changes in the CDR sequence.

[0009] The second aspect of the present invention provides a nectin-4 binding molecule, which comprises the Nanobody as described in the first aspect of the present invention.

[0010] In some embodiments of the invention, the binding molecule is a multivalent Nanobody comprising a plurality of such Nanobodies; or the binding molecule is a fusion protein, such as a multispecific antibody or a heavy chain antibody.

[0011] In some embodiments of the invention, the binding molecule is a heavy chain antibody.

[0012] In some embodiments of the present invention, the Fc of the heavy chain antibody is IgG Fc, preferably derived from mouse or human.

[0013] In some embodiments of the present invention, the amino acid sequence of the Fc is as shown in SEQ ID NO: 7, or has a sequence identity of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% to SEQ ID NO: 7 and maintains the function of the Fc.

[0014] The third aspect of the present invention provides a chimeric antigen receptor, which comprises the Nanobody as described in the first aspect of the present invention or the binding molecule as described in the second aspect of the present invention.

[0015] The fourth aspect of the present invention provides a genetically modified cell, wherein the cell comprises the chimeric antigen receptor according to the third aspect of the present invention.

[0016] In some embodiments of the present invention, the genetically modified cell is a eukaryotic cell, preferably an isolated human cell.

[0017] In some embodiments of the present invention, the genetically modified cells are immune cells, such as T cells or NK cells.

[0018] A fifth aspect of the invention provides an isolated nucleic acid molecule encoding a Nanobody as described in the first aspect of the invention or a binding molecule as described in the second aspect of the invention.

[0019] The sixth aspect of the present invention provides a recombinant vector, wherein the recombinant expression vector comprises the nucleic acid molecule as described in the fifth aspect of the present invention.

[0020] In some embodiments of the present invention, the recombinant vector is a recombinant expression vector or a recombinant replication vector.

[0021] In some embodiments of the present invention, the recombinant vector is a plasmid, a cosmid, a phage or a viral vector, and the viral vector is preferably a retroviral vector, a lentiviral vector, an adenoviral vector or an adeno-associated viral vector.

[0022] The seventh aspect of the present invention provides a transformant, which comprises the nucleic acid molecule as described in the fifth aspect of the present invention or the recombinant vector as described in the sixth aspect of the present invention; or expresses the nanobody as described in the first aspect of the present invention or the binding molecule as described in the second aspect of the present invention.

[0023] In some embodiments of the present invention, the host cells used in constructing the transformant are selected from Escherichia coli cells, insect cells, yeast cells and mammalian cells.

[0024] In some embodiments of the present invention, the host cell is a 293 cell.

[0025] An eighth aspect of the present invention provides a method for preparing an antibody targeting nectin-4, the method comprising:

[0026] The transformant according to the seventh aspect of the present invention is cultured, and the antibody is obtained from the culture.

[0027] The ninth aspect of the present invention provides an antibody conjugate, which comprises the nanobody as described in the first aspect of the present invention or the binding molecule as described in the second aspect of the present invention, and an effector molecule; the effector molecule is a nuclide, a cytotoxic drug or a marker.

[0028] In some embodiments of the present invention, the cytotoxic drug is a toxin, such as one or more of a toxin (such as a small molecule toxin or an enzymatically active toxin of bacterial, fungal, plant or animal origin), a radioactive isotope, a toxic drug, a chemotherapeutic drug, an antibiotic, a nucleolytic enzyme and their derivatives.

[0029] In some embodiments of the present invention, the label is any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means, selected from enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase), radionuclides (e.g., 3 H. 125 I. 35 S. 14 C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate, fluorescein, tetramethylrhodamine isothiocyanate, phycoerythrin, Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., IR808, Alexa 750)), acridinium ester compounds, magnetic beads, calorimetric labels (e.g., colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads), and biotin for binding to avidin (e.g., streptavidin) modified with the above labels.

[0030] In some embodiments of the invention, the C-terminus of the Nanobody is connected to a His-Cys tag, and the amino acid sequence of the Nanobody with a His-Cys tag connected to the C-terminus is, for example, shown in SEQ ID NO: 8.

[0031] In some embodiments of the present invention, the antibody conjugate is an antibody probe, which is obtained by connecting the nanobody with a His-Cys tag at the C-terminus to IR808.

[0032] The tenth aspect of the present invention provides a pharmaceutical composition, which comprises the Nanobody as described in the first aspect of the invention, the binding molecule as described in the second aspect of the invention, the chimeric antigen receptor as described in the third aspect of the invention, the cell as described in the fourth aspect of the invention, the transformant as described in the seventh aspect of the invention or the antibody conjugate as described in the ninth aspect of the invention, and a pharmaceutically acceptable excipient.

[0033] The eleventh aspect of the present invention provides a detection reagent for Nectin-4, which comprises the Nanobody as described in the first aspect of the present invention, the binding molecule as described in the second aspect of the present invention, or the antibody conjugate as described in the ninth aspect of the present invention.

[0034] In some embodiments of the present invention, the nectin-4 detection reagent is used for in vivo imaging technology, Western blot, enzyme-linked immunosorbent assay and / or flow cytometry.

[0035] The twelfth aspect of the present invention provides a kit for detecting nectin-4 and / or diagnosing cancers related to nectin-4 expression, the kit comprising the nanobody as described in the first aspect of the present invention, the binding molecule as described in the second aspect of the present invention, the antibody conjugate as described in the ninth aspect of the present invention, or the detection reagent as described in the eleventh aspect of the present invention.

[0036] In some embodiments of the present invention, the cancer is selected from one or more of breast cancer, lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, and ovarian cancer.

[0037] In some embodiments of the present invention, the kit further comprises other reagents for detecting nectin-4 using in vivo imaging technology, Western blot, enzyme-linked immunosorbent assay and / or flow cytometry, such as fluorescently labeled secondary antibodies.

[0038] The thirteenth aspect of the present invention provides the use of the nanoantibody as described in the first aspect of the present invention, the binding molecule as described in the second aspect of the present invention, the chimeric antigen receptor as described in the third aspect of the present invention, the cell as described in the fourth aspect of the present invention, the nucleic acid molecule as described in the fifth aspect of the present invention, the recombinant vector as described in the sixth aspect of the present invention, the transformant as described in the seventh aspect of the present invention, the antibody conjugate as described in the ninth aspect of the present invention, the pharmaceutical composition as described in the tenth aspect of the present invention, and the detection reagent as described in the eleventh aspect of the present invention in the preparation of a product for diagnosing cancer, or a drug for preventing and / or treating cancer; the cancer is a cancer related to Nectin-4 expression.

[0039] In some embodiments of the present invention, the cancer is selected from one or more of breast cancer, lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, and ovarian cancer.

[0040] The fourteenth aspect of the present invention provides a method for detecting nectin-4 for non-diagnostic purposes, the method comprising contacting a sample to be tested with the nanoantibody as described in the first aspect of the present invention, the binding molecule as described in the second aspect of the present invention, the antibody conjugate as described in the ninth aspect of the present invention, the detection reagent as described in the eleventh aspect of the present invention, or the kit as described in the twelfth aspect of the present invention to detect the expression level of nectin-4 in the sample to be tested.

[0041] In some embodiments of the present invention, the test sample is a cell or an animal.

[0042] In some embodiments of the present invention, the method uses in vivo imaging technology, Western blot, enzyme-linked immunosorbent assay and / or flow cytometry to detect nectin-4.

[0043] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0044] The reagents and raw materials used in the present invention are commercially available.

[0045] The positive progress effect of the present invention is:

[0046] The nanoantibody targeting nectin-4 provided by the present invention can bind to tumor cells with high affinity and has good endocytic activity. In in vivo imaging, it has strong tumor targeting, rapid tumor enrichment and long retention time, which is beneficial to the study of tumor diagnosis, prognosis and occurrence and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 In vivo fluorescence imaging of the anti-nectin-4 antibody conjugate 39-IR808. DETAILED DESCRIPTION

[0048] In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the procedures in molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA used herein are conventional procedures widely used in the corresponding fields. In addition, for a better understanding of the present invention, the following definitions and explanations of relevant terms are provided:

[0049] "Nectin-4" or "PVRL-4" are used interchangeably and include variants, isoforms, species homologs, and analogs of human nectin-4 that share at least one common epitope with nectin-4.

[0050] In the present invention, the letters in the amino acid sequence represent the single-letter abbreviations of amino acids known in the art, such as those described in J. Biol. Chem, 243, p3558 (1968): alanine: Ala-A, arginine: Arg-R, aspartic acid: Asp-D, cysteine: Cys-C, glutamine: Gln-Q, glutamic acid: Glu-E, histidine: His-H, glycine: Gly-G, asparagine: Asn-N, tyrosine: Tyr-Y, proline: Pro-P, serine: Ser-S, methionine: Met-M, lysine: Lys-K, valine: Val-V, isoleucine: Ile-I, phenylalanine: Phe-F, leucine: Leu-L, tryptophan: Trp-W, threonine: Thr-T.

[0051] In the present invention, "single domain antibody", "heavy chain variable region domain of heavy chain antibody", "VHH", "VHH domain" and "nanoantibody" are used interchangeably, all referring to nanoantibodies that specifically recognize and bind to Nectin-4. Nanoantibodies are the variable region of heavy chain antibodies. Typically, nanoantibodies contain three CDRs and four FRs. Nanoantibodies are the smallest functional antigen-binding fragments. "VHH domain" includes but is not limited to natural antibodies produced by camelids, and may also be antibodies produced by camelids that are then humanized, or obtained through phage display technology screening. The total number of amino acid residues in the VHH domain will generally be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in the present invention. The VHH domain (alone or as part of a larger polypeptide) provides many significant advantages over the use of conventional VH and VL domains, scFv or conventional antibody fragments (such as Fab- or F(ab')2-fragments):

[0052] Only a single domain is required to bind the antigen with high affinity and selectivity, thus eliminating the need for two separate domains and ensuring that the two domains are in the appropriate spatial conformation and configuration (e.g., scFv generally requires the use of a specially designed linker);

[0053] - VHH domains can be expressed from a single gene and do not require post-translational folding or modification;

[0054] - VHH domains can be easily engineered into multivalent and multispecific formats;

[0055] - VHH domains are highly soluble and have no tendency to aggregate;

[0056] - VHH domains are highly stable to heat, pH, proteases, and other denaturing agents or conditions, and therefore can be prepared, stored, or transported without the use of refrigeration equipment, thereby achieving cost, time, and environmental savings;

[0057] - VHH domains are easy and relatively cheap to prepare, even on the scale required for production;

[0058] - The VHH domain is relatively small compared to conventional tetrapeptide-structured antibodies (approximately 15 kDa or 1 / 10 the size of conventional IgG), and therefore exhibits higher tissue penetration and can be administered at higher doses than conventional tetrapeptide-structured antibodies;

[0059] - VHH domains can display so-called cavity-binding properties (especially due to their extended CDR3 loop compared to conventional VH domains), thus being able to reach targets and epitopes that are inaccessible to conventional tetrapeptide-structured antibodies.

[0060] Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240 (2000) 185-195; Li et al., J Biol Chem., 287 (2012) 13713-13721; Deffar et al., African Journal of Biotechnology Vol. 8 (12), pp. 2645-2652, 17 June, 2009 and WO94 / 04678.

[0061] Typically, the antibodies of the present invention targeting Nectin-4 will be expressed as preferably 10 -7 to 10 -10 mol / L (M), more preferably 10 -8 to 10 -10 mol / L, even more preferably 10 -9 to 10 -10 or lower dissociation constant (KD), and / or with a dissociation constant of at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, more preferably at least 10 -10 The association constant (KA) of M binds to the antigen to be bound (ie, nectin-4). Any value greater than 10 -4 A KD value of M is generally considered to indicate nonspecific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be determined in any suitable manner known in the art, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competitive assays) as described herein.

[0062] When "competes" is used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, it means competition between antigen binding proteins, which is determined by the following assay: the antigen binding protein to be tested (e.g., antibody or immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., nectin-4 antigen or fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, such as: solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid phase direct label assays, solid phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1992). Press); solid phase direct labeling RIA using an I-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology 176:546-552); and directly labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, the assay involves the use of purified antigen that binds to an unlabeled test antigen-binding protein and a labeled reference antigen-binding protein (the antigen being on a solid surface or on the surface of cells). Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Antigen binding proteins identified by competitive assays (competing antigen binding proteins) include those that bind to the same epitope as a reference antigen binding protein, and those that bind to an epitope sufficiently proximal to the epitope bound by the reference antigen binding protein that the two epitopes sterically interfere with each other's binding. Further details regarding methods for determining competitive binding are provided in the Examples. Typically, when the competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) specific binding of the reference antigen binding protein to the common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more.In certain instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0063] Conventional techniques known to those skilled in the art can be used to competitively screen antibodies for binding to the same epitope. For example, competition and cross-competition studies can be conducted to obtain antibodies that compete with or cross-compete for antigen binding. High-throughput methods for obtaining antibodies that bind to the same epitope based on their cross-competition are described in International Patent Publication WO 03 / 48731. Therefore, conventional techniques known to those skilled in the art can be used to obtain antibodies that compete with the antibody molecules of the present invention for binding to the same epitope on nectin-4.

[0064] "Cross-reactivity" refers to, for example, the nectin-4 binding protein of the present invention and nectin-4 from a different species. For example, a single domain antibody or derivative protein of the present invention that binds to human nectin-4 may also bind to nectin-4 from another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (e.g., SPR and ELISA), or binding or functional interaction with cells that physiologically express nectin-4. Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.

[0065] "Antigen" refers to a molecule used to immunize an immunocompetent vertebrate to produce antibodies that recognize the antigen, or to screen an expression library (e.g., particularly a phage, yeast, or ribosome display library). In the present invention, antigen is defined more broadly to include target molecules specifically recognized by antibodies, as well as portions or mimetics of molecules used in immunization procedures for antibody production or library screening for antibody selection. For example, with respect to the antibodies of the present invention that bind to human nectin-4, monomers and multimers (e.g., dimers, trimers, etc.) of human nectin-4, as well as truncated and other variants of human nectin-4, are all referred to as antigens.

[0066] In the present invention, the amino acid sequences of the complementarity determining regions (CDRs) listed are shown according to the definition of the Kabat numbering convention. However, it is well known to those skilled in the art that antibody CDRs can be defined by a variety of methods in the art, such as Chothia (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)) based on the three-dimensional structure of the antibody and the topology of the CDR loops, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability, AbM (University of Bath), Contact (University College London), the international ImMunoGeneTicsdatabase (IMGT, World Wide Web imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions as defined by any of the above-mentioned known schemes described in the present invention.

[0067] Therefore, when referring to antibodies defined by specific CDR sequences defined herein, the scope of such antibodies also encompasses antibodies whose variable region sequences comprise such specific CDR sequences, but whose claimed CDR boundaries differ from the specific CDR boundaries defined herein due to the application of a different scheme (e.g., a different assignment system rule or combination). Although the scope of protection claimed in the present invention is based on the sequences defined according to the Kabat numbering convention, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of the present invention.

[0068] In the present invention, "Nectin-4 binding molecules" are proteins that have the function of recognizing and binding to Nectin-4, including but not limited to antibodies, antigen-binding fragments of antibodies, heavy chain antibodies, nanobodies, minibodies, multispecific antibodies, fusion proteins, affibodies, target binding regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines and chemokines.

[0069] In the present invention, a binding molecule comprising two or more Nanobodies is a multivalent Nanobody; a binding molecule comprising two or more Nanobodies with different specificities is a multispecific Nanobody. A multivalent Nanobody or a multispecific Nanobody is connected to multiple Nanobodies via a linker. The linker is typically composed of 1-15 amino acids selected from G and S, for example (G4S)3.

[0070] In the present invention, "multispecific antibody" refers to a type of antibody that can simultaneously recognize and bind to at least two different antigenic epitopes, such as a bispecific antibody.

[0071] In the present invention, "fusion protein" refers to a protein formed by linking two or more different protein or polypeptide sequences together through genetic engineering. Among them, the structure of the Fc fusion protein (such as a heavy chain antibody) consists of two parts, namely the Fc segment of the immunoglobulin and the nanobody described in the present invention. The two have relatively independent structural domains and functions, and can affect their own physicochemical properties and biological activities from different aspects. In some embodiments, the Fc region is an Fc region that increases effector function, for example, increasing antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC) with increased effector function. In some embodiments, the Fc region contained in the aforementioned Fc fusion protein can enable the binding protein to form a dimeric molecule, while prolonging the in vivo half-life of the binding protein.

[0072] Exemplary IgG1 Fc regions include those with the following substitutions: 239D; 239E; 239K, 241A; 262A; 264D; 264L; 264A; 264S; 265A; 265S; 265V; 296A; 296A; 301A; 332E; 239D / 332E; 239D / 330S / 332E; 239D / 330L / 332E; 298A / 333A / 334A; 247I / 339D; 247I / 339Q; 280H / 290S; 280H / 290S / 298D; 80H / 290S / 298V;243L / 292P / 300L;243L / 292P / 300L / 396L;243L / 292P / 300L / 305I / 396L;236A / 239D / 332E;326A / 333A;326W / 333S;290E / 298G / 299A;290N / 298G / 299A;290E / 298G / 299A / 326E;or 290N / 298G / 299A / 326E;or any combination of any of the above positions. Said mutations are defined according to the EU numbering system.

[0073] Exemplary IgG1 Fc regions include those with the following substitutions: S239D; S239E; S239K, F241A; V262A; V264D; V264L; V264A; V264S; D265A; D265S; D265V; F296A; Y296A; R301A; I332E; S239D / I332E; S239D / A330S / I332E; S239D / A330L / I332E; S298A / D333A / K334A; P247I / A339D; P247I / A339Q; D280H / K290S; D280H / K290S / S298D; D or K290N / S298G / T299A / K326E, or any combination of the foregoing positions.

[0074] For the purposes of this invention, "heavy chain antibodies" are antibodies derived from camelids or elasmobranchs. Compared to the aforementioned full-length antibodies, heavy chain antibodies lack the light chain and heavy chain constant region 1 (CH1), consisting of only two heavy chains consisting of a variable region (VHH) and other constant regions. The variable regions are connected to the constant regions by a hinge-like structure. Each heavy chain of a camelid heavy chain antibody contains one variable region (VHH) and two constant regions (CH2 and CH3), while each heavy chain of an elasmobranch heavy chain antibody contains one variable region and five constant regions (CH1-CH5). Antigen-binding fragments of heavy chain antibodies include VHH and single-chain heavy chain antibodies. By fusion with the constant region of human IgG Fc, heavy chain antibodies can possess the CH2 and CH3 of human IgG Fc.

[0075] In some embodiments, the aforementioned Nanobody is connected to the Fc region via a linker. The linker can be a non-functional amino acid sequence of 1-20 or more amino acids in length and without secondary or higher structure.

[0076] In some embodiments, the aforementioned anti-nectin-4 Nanobody or heavy chain antibody comprises a Cys at the C-terminus or N-terminus.

[0077] In some embodiments, the KD value of the aforementioned anti-nectin-4 nanobody or heavy chain antibody binding to nectin-4 may be ≤1×10 -7 M.

[0078] In some embodiments, the aforementioned anti-nectin-4 nanobody or heavy chain antibody binds to tumor cells, such as the detection method in Example 3.

[0079] In some embodiments, the aforementioned anti-nectin-4 nanobody or heavy chain antibody has endocytosis activity, such as the detection method in Example 4.

[0080] In some embodiments, the aforementioned anti-Nectin-4 Nanobody or heavy chain antibody can inhibit tumor growth by at least about 10%, for example, at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%.

[0081] In the present invention, heavy chain antibodies and antibodies are intended to distinguish different combinations of antibodies.

[0082] Without materially affecting the activity of the antibody, those skilled in the art may modify one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the sequences of the present invention to obtain variants of the antibody or its functional fragment sequence. These variants include (but are not limited to): deletion, insertion, and / or substitution of one or more (generally 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, as well as addition of one or more (generally within 20, preferably within 10, and more preferably within 5) amino acids to the C-terminus and / or N-terminus. In the art, conservative substitutions with amino acids having similar or similar properties generally do not alter protein function. For example, amino acids with similar properties may be substituted in the FR and / or CDR regions of the variable region. Amino acid residues suitable for conservative substitutions are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. For another example, adding one or more amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein and is considered to be within the scope of protection of the present invention.

[0083] Variants of the various antibodies of the present invention include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the DNA encoding the various antibodies of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antisera against the various antibodies of the present invention.

[0084] In some embodiments, the sequence of a variant of the present invention can be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to its source sequence. Sequence identity as described herein can be measured using sequence analysis software, such as the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. The present invention also includes molecules having antibody heavy chain variable regions with CDRs, provided that their CDRs have greater than 90% (preferably greater than 95%, and most preferably greater than 98%) homology to the CDRs identified herein.

[0085] The antibodies of the present invention can be prepared using conventional methods in the art, such as hybridoma technology, which is well known in the art. Nanobodies and heavy chain antibodies of the present invention can be prepared using conventional methods in the art, such as phage display technology, which is well known in the art. Alternatively, the various antibodies of the present invention can be expressed in other cell lines. Sequences encoding the various antibodies of the present invention can be used to transform suitable mammalian host cells. Transformation can be carried out using any known method, including, for example, packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, and direct microinjection of DNA into the nucleus. Mammalian cell lines that can be used as hosts for expression are well known in the art and include, but are not limited to, various immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), etc. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with substantial GPC3 binding properties.

[0086] In the present invention, "nucleic acid molecule" refers to a nucleotide chain of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.

[0087] In the present invention, the term "recombinant vector" means a construct capable of delivering and, in some embodiments, expressing one or more target genes or sequences in a host cell. The term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell when the construct comprises a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and the vector is in contact with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell. The vectors of the present invention are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of the present invention may comprise any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthesized or partially obtained from natural sources, and may contain natural, non-natural, or altered nucleotides. The recombinant expression vector may comprise naturally occurring or non-naturally occurring nucleotides, or both types of connections. In exemplary aspects, the altered nucleotides or non-naturally occurring nucleotides are connected without hindering transcription or replication of the vector.

[0088] The recombinant expression vector of the present invention can be any suitable recombinant expression vector that can be used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably express the genes or sequences in the host cell. Suitable vectors include those designed for expansion and amplification or for expression or both, examples of vectors include but are not limited to viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0089] As used herein, the term "host cell" refers to any type of cell that can contain a nucleic acid or vector described herein. In exemplary aspects, the host cell is a eukaryotic cell, such as a plant, animal, fungus, or algae; or it can be a prokaryotic cell, such as a bacterium or protozoa. Easily transformed bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary) cells, NS0 cells, and 293 cells.

[0090] In the present invention, a chimeric antigen receptor (CAR) is an engineered transmembrane protein that combines the specificity of an antigen-specific antibody with the function of a T cell receptor. Generally, a CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In exemplary aspects, the extracellular domain of the CAR comprises an antigen recognition region, which may be a scFv of an antigen-specific antibody.

[0091] In the present invention, the term "antibody conjugate" includes the aforementioned nanobodies or binding molecules and effector molecules. The effector molecule is a molecule that can exhibit the desired target activity. For example, the effector molecule is selected from radioisotopes, antitumor agents, immunomodulators, biological response modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.

[0092] In some embodiments, the effector molecule is a label. As an example, the label can be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. The label is any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means, and is selected from enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase), radionuclides (e.g., 3 H. 125 I. 35 S. 14 C or 32 P), fluorescent dyes (e.g., fluorescein isothiocyanate, fluorescein, tetramethylrhodamine isothiocyanate, phycoerythrin, Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., IR808, Alexa750)), acridinium ester compounds, magnetic beads, calorimetric labels (e.g., colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin modified with the above labels (e.g., streptavidin). The labels encompassed by the present invention can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or a scintillation counter, and fluorescent labels can be detected using a photodetector to detect emitted light. Enzyme labels are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simple visualization of the colored label.

[0093] In some embodiments, the labels described above can be attached to the antibodies of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0094] In some embodiments, the labels described above can be linked to the antibodies of the present invention via chelating agents, such as NOTA, DOTA, and the like.

[0095] In some embodiments, the effector molecule is a cytotoxic drug.

[0096] In the present invention, the term "Antibody-Drug Conjugate" or "Antibody Conjugate" generally refers to an antibody linked to a biologically active cytotoxic drug, nuclide, or label via a stable linker. The "cytotoxic drug" may include toxins, such as small molecule toxins or enzymatic toxins of bacterial, fungal, plant, or animal origin, toxic drugs, chemotherapeutic drugs, antibiotics, or nucleolytic enzymes, or their derivatives.

[0097] In the present invention, pharmaceutical compositions may include suitable pharmaceutically acceptable carriers, such as pharmaceutical excipients, such as those known in the art, including buffers. "Pharmaceutically acceptable carriers" include any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delaying agents. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid carriers, particularly for injectable solutions. Pharmaceutical compositions comprising the present invention can be prepared by mixing an antibody of the present invention of the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of a lyophilized formulation or aqueous solution. In some embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of the aforementioned Nanobodies or aforementioned binding molecules per unit dose, or the amount of the aforementioned Nanobodies or aforementioned binding molecules per unit dose of the pharmaceutical composition may be 0.1-2000 mg, and in some embodiments, 1-1000 mg.

[0098] In some embodiments, an article of manufacture or product is provided, comprising the aforementioned anti-nectin-4 antibody or antibody conjugate. Optionally, the article of manufacture comprises a container and a label. Containers such as bottles, syringes, and test tubes contain a composition effective for treating a condition. The label on or associated with the container indicates that the composition is used to treat the selected condition. The composition contains the aforementioned anti-nectin-4 antibody or antibody conjugate.

[0099] The pharmaceutical composition of the present invention can also comprise more than one active ingredient, the active ingredient being required for the specific indication being treated, preferably having those active ingredients of complementary activity that do not adversely affect each other. For example, it is desirable to also provide other active ingredients, such as other antibodies, antiviral active agents, small molecule drugs or immunomodulators, etc. The active ingredients are suitably combined in an amount effective for the intended use. Sustained release formulations can be prepared, and suitable examples thereof include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody of the present invention, the matrix being a shaped article, such as a film or microcapsule form.

[0100] "Tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. "Cancer," "cancerous," "proliferative disorder," and "tumor" are not mutually exclusive when used herein. In some embodiments, the cancer is breast cancer or bladder cancer.

[0101] In the present invention, application scenarios of "non-diagnostic purposes" include but are not limited to: for example, in vitro detection of the presence or absence of antigens (proteins containing the extracellular region of nectin-4) in the laboratory; or as a positive antibody to screen other antibodies targeting nectin-4; or competing with other antibodies targeting nectin-4 to detect whether there is competition between antibodies, that is, whether the antigen epitopes are the same or similar, etc.

[0102] As used herein, the term "effective amount" refers to an amount of a drug or pharmaceutical agent that elicits the biological or pharmaceutical response of a tissue, system, animal, or human that is being sought, for example, by a researcher or clinician. Furthermore, the term "effective amount" refers to an amount that results in improved treatment, cure, prevention, or alleviation of a disease, condition, or side effect, or reduces the rate of progression of a disease or condition, compared to a corresponding subject that has not received that amount. The term also includes within its scope amounts that are effective to enhance normal physiological function.

[0103] The "subject" and "patient" described in the present invention refer to mammals, especially primates, and especially humans.

[0104] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0105] Example 1. Screening and Preparation of Anti-Nectin-4 Antibodies

[0106] In this example, alpacas were immunized using His-tagged human nectin-4 (Acro, NE4-H52H3) as the immunizing antigen. Peripheral blood was collected, and PBMCs were isolated. RNA from the PBMCs was extracted and reverse transcribed to obtain total cDNA. A yeast library was constructed for antibody screening.

[0107] After two rounds of sorting, multiple unique VHH sequences binding to the human nectin-4 antigen were obtained through monoclonal identification, sequencing, and sequence analysis. The sequence of NB656-M1-39 is shown below.

[0108] >NB656-M1-39 variable region

[0109] EVQLVESGGGLVQPGGSLRLSCAASGSISDINVMGWFRQAPSQQRELVATITPGGSTNYADSVKGRFTIARDNAKNTVYLQMNSLKPEDTAVYFCLAEELGSLTPTEHWGQGTQVTVSS (SEQ ID NO: 1)

[0110] The CDR sequences are shown in Table 1 below: (Kabat numbering convention)

[0111] Table 1 CDR sequences in the variable region of NB656-M1-39

[0112]

[0113] The above sequences were ligated to the human IgG1 Fc fragment (including the hinge region) to construct VHH-Fc antibodies. Plasmids were constructed and transiently transfected into 293 cells. The cells were cultured for antibody expression and purified using a protein A column. The column was washed with 1× PBS buffer and eluted with 0.1 M glycine buffer (pH 2.5). The column was then dialyzed into 1× PBS buffer (pH 7.4). The sequence of the human IgG1 Fc fragment (including the hinge region) is as follows:

[0114] > IgG1 Fc

[0115] EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7)

[0116] >BS025-PC HC

[0117] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLSLQMNSLRDEDTAVYYCARAYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5)

[0118] > BS025-PC LC

[0119] DIQMTQSPSSVSASVGDRVTITCRASQGISGWLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO: 6)

[0120] Example 2. Affinity Identification of Anti-Nectin-4 Antibody and Antigen Nectin-4

[0121] The binding ability of anti-Nectin-4 antibody to Nectin-4 antigen protein was detected by ELISA and BLI methods.

[0122] 1. ELISA

[0123] Experimental Methods: Antigens were coated overnight at 4°C in 100 μL / well of 50 mM NaHCO₃, pH 9.6 buffer containing human nectin-4 his (Acro, NE4-H52H3), cyno nectin-4 his (Acro, NE4-C52H4), and mouse nectin-4 his (Acro, NE4-M52H3) at a final concentration of 2 μg / mL. The plates were washed three times with PBST and blocked with 5% milk at 37°C. After washing once with PBST, the proteins were serially diluted 1:5 from 100 nM in 5% milk. 100 μL of these diluted antibodies were added to the microtiter plate wells and incubated at 37°C for 1 hour. The isotype control antibody was purchased from Syd Labs, catalog number PA007165.m2a. Wash with PBST 5 times, add the secondary antibody (Anti-Human IgG Fc, HRP, 1:10K) diluted in blocking solution. Wash the ELISA plate incubated with secondary antibody 5 times with PBST, add 100μl TMB single component colorimetric solution to each well and incubate at 37℃ for 7min. Add 1M HCl (50μl / well) to stop the reaction, and OD 450 Reading, calculation of EC 50 .

[0124] Experimental results: The results are shown in Table 2, which show that the anti-nectin-4 antibodies have strong binding affinity to the nectin-4 antigen, and are stronger than the control antibody BS025-PC.

[0125] Table 2 ELISA detection of anti-nectin-4 antibody binding to antigen

[0126]

[0127] 2. BLI

[0128] Experimental method: First, immerse the biosensor in PBST buffer for 15 minutes to equilibrate, then immerse it in a solution containing a known concentration of antibody (2μg / ml). Then, immerse the sensor with the immobilized antigen in the buffer for baseline equilibrium. Then, immerse the biosensor with the immobilized antigen at a known concentration (200nM, 100nM, 50nM, or 25nM) in a sample solution containing the test antibody. Finally, immerse the sensor bound to the test antibody in the buffer for dissociation. The Octet instrument monitors the biofilm thickness of the biosensor in real time during the experiment to obtain the kinetic constant of the test sample. The specific steps are as follows:

[0129] (1) Turn on the power of the Octet K2 instrument (Sartorius) and the operating software to initialize the instrument. This process takes about 1 minute.

[0130] (2) Add 200 μL PBST to the well plate to pre-wet the sensor (the sensor is ProA sensor - Sartorius) for 15 minutes;

[0131] (3) Programming: antibody solidification for 60 s, antigen binding for 180 s, and dissociation for 240 s;

[0132] (4) Prepare the sample according to the set procedure and add the sample to the detection plate;

[0133] (5) Place the sensor and sample plate into the instrument together and perform the test in sequence;

[0134] (6) After the test is completed, recycle the sensor, clean the sample plate, and turn off the instrument power.

[0135] (7) Use the analysis software DataAnalysis12 to perform fitting, export the images and the fitted affinity constant values.

[0136] Experimental results: The results are shown in Table 3, which show that the anti-nectin-4 antibodies have strong binding affinity with the nectin-4 antigen.

[0137] Table 3 Detection of binding ability of anti-nectin-4 antibodies

[0138]

[0139] Example 3. In vitro cell binding assay of anti-nectin-4 antibodies

[0140] Experimental purpose: To detect the binding ability of anti-nectin-4 antibodies using human breast cancer cells MCF-7 (Punosai, CL-0149).

[0141] Experimental method: Wash the cells twice with 1× PBS buffer and resuspend the cells with 1× PBS buffer to a cell concentration of 3×10 5 cells / μL, aliquot the cells into a PCR plate, 50 μL per well; dilute the antibody to be detected with 1× PBS buffer to a concentration of 200 nM and add it to the detection wells, then make 7 serial dilutions according to 5-fold dilution, take 50 μl / well and add it to the plate, incubate at 4°C for 1 hour; after the incubation is completed, wash the cells 3 times with 1× PBS buffer, add 100 μL of fluorescent secondary antibody (647 Anti-HumanIgG Fc) to each well, and incubate in a refrigerator at 4°C for 1 hour; after the incubation is completed, wash the cells once with PBS, transfer the cells to a 96-well cell plate, and analyze them on a flow cytometer.

[0142] Experimental results: The results are shown in Table 4, which show that the anti-nectin-4 antibodies have strong binding affinity to the nectin-4 antigen on human breast cancer MCF-7 cells, and are stronger than the control antibody BS025-PC.

[0143] Table 4 EC binding of anti-nectin-4 antibodies to cell surface antigen nectin-4 50

[0144]

[0145] Example 4. Endocytic activity of anti-nectin-4 antibodies

[0146] Experimental purpose: To detect the endocytic activity of anti-nectin-4 antibody using human breast cancer cells MCF-7.

[0147] Experimental method: Wash the cells twice with 1× PBS buffer and resuspend the cells with 1× PBS buffer to a cell concentration of 3×10 5 cells / μL, and cells were aliquoted into PCR plates, 50 μL per well; the test antibody was diluted to 4 μg / mL with 1× PBS buffer, and 50 μL / well was added to the plate to make the final antibody concentration of 2 μg / mL, with 5 wells for each concentration; the antibody and cells were incubated in a 4°C refrigerator for 1 hour; after the incubation, the cells were washed once with 1× PBS buffer, and the cells were resuspended in 200 μL 1× PBS buffer. The antibodies and cells were transferred to a 96-well cell plate according to a time gradient of 0 h, 0.5 h, 1 h, 2 h, and 4 h, and incubated at 37°C; after the incubation, the cells were washed once with 1× PBS buffer, and 100 μL of fluorescent secondary antibody (647 Anti-Human IgGFc) was added to each well; incubated in a 4°C refrigerator for 1 hour; after the incubation, the cells were washed once with 1× PBS buffer, and the cells were resuspended in 200 μL 1× PBS buffer, transferred to a 96-well cell plate, and analyzed on a flow cytometer.

[0148] Experimental results: As shown in Table 5, the anti-nectin-4 antibody has endocytic activity.

[0149] Table 5 Endocytic activity of anti-nectin-4 antibodies in MCF-7 cells

[0150]

[0151] Example 5. Anti-Nectin-4 Antibody

[0152] The variable region of the antibody was linked to a His-Cys tag to construct a VHH-His-Cys antibody. The plasmid was constructed and transformed into Escherichia coli BL21(DE3) strain. Single colonies were selected and activated overnight in LB medium at 37°C. The cells were transferred to 300 mL of LB medium at a ratio of 1:100. When the OD value reached 0.6, IPTG was added to a final concentration of 1 mmol / L and expression was induced at 30°C for 6 hours. The resulting culture was centrifuged at 6,000 rpm for 5 minutes to collect the cells. The cells were resuspended in 1× PBS buffer, polymyxin was added to a final concentration of 0.5 mM, and the cells were disrupted at 37°C for 2 hours. The supernatant was then centrifuged at 8,000 rpm for 30 minutes. The supernatant was filtered and bound to Ni Sepharose (GE) for 2 hours. The supernatant was washed with Tris buffer containing 40 mM imidazole, and the target protein was eluted with Tris buffer containing 250 mM imidazole. The protein was concentrated by ultrafiltration using an ultrafiltration tube, and the Tris buffer was replaced with a 0.1M NaHCO3 solution (pH 8.3). The protein purity was determined to be >90% by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The sequence of NB656-M1-39-His-Cys is as follows:

[0153] >NB656-M1-39-His-Cys

[0154] EVQLVESGGGLVQPGGSLRLSCAASGSISDINVMGWFRQAPSQQRELVATITPGGSTNYADSVKGRFTIARDNAKNTVYLQMNSLKPEDTAVYFCLAEELGSLTPTEHWGQGTQVTVSSHHHHHHC (SEQ ID NO: 8)

[0155] Example 6. Anti-Nectin-4 Antibody Conjugate

[0156] The anti-nectin-4 antibody conjugate is prepared by coupling the anti-nectin-4 antibody to the anthocyanin dye derivative IR808 (also known as MHI-808). The specific preparation steps are as follows: 1 mg of the anti-nectin-4 antibody NB656-M1-39-His-Cys was diluted to 1 mg / ml in 20 mM Tris-HCl (pH 8.0) buffer. 2.8 μL of 100 mM TCEP was added to prevent protein aggregation. At a molar ratio of MHI-808:anti-nectin-4 antibody of 2:1, approximately 32 μL of MHI-808 (5 mg / ml) was added per 1 mg of protein. The reaction was carried out at 4°C for 30 minutes. The sample was then dialyzed against 20 mM Tris-HCl, pH 8.0 (5 kD dialysis bag). The buffer was changed every 3-4 hours for a total of five changes.

[0157] The anti-nectin-4 antibody conjugate 39-IR808 was obtained according to the above method.

[0158] Example 7. Targeting of anti-nectin-4 antibody conjugates

[0159] Objective: To investigate the tumor targeting and sustained tumor accumulation of anti-nectin-4 antibodies using bladder cancer cell line T24 (Punosai, CL-0277).

[0160] Experimental method: First, resuspend T24 cells (5×10 6 cells) were injected subcutaneously into BALB / cNj-Foxn1 nu The right hind limb of Gpt mice (Jicui Yaokang, strain number: D000521, female) was used to establish a T24 xenograft model. When the tumor volume reached approximately 200-400 mm 3 At the same time, 10 T24 xenograft mice were intravenously injected with the corresponding anti-nectin-4 antibody conjugate (concentration of 1 mg / mL, 100 μL each), and the mice were imaged in vivo with an in vivo imaging system (IVIS) at different time points (1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h and 96h).

[0161] Experimental results: The results showed that the anti-nectin-4 antibody conjugate 39-IR808 has the advantages of strong tumor targeting, rapid tumor accumulation and long retention time (see Figure 1 ).

Claims

1. A nanobody targeting nectin-4, characterized in that: The Nanobody comprises a heavy chain variable region, which comprises CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is shown in SEQ ID NO: 2, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 3, and the amino acid sequence of the CDR3 is shown in SEQ ID NO:

4.

2. The Nanobody according to claim 1, wherein The framework region of the heavy chain variable region is an alpaca-derived framework region or a human-derived framework region.

3. The Nanobody according to claim 2, wherein The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

1.

4. A nectin-4 binding molecule, characterized in that: The binding molecule comprises the Nanobody according to any one of claims 1 to 3; wherein the binding molecule is a heavy chain antibody.

5. The binding molecule according to claim 4, wherein The amino acid sequence of the Fc of the heavy chain antibody is shown in SEQ ID NO:

7.

6. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule encodes the Nanobody according to any one of claims 1 to 3 or the binding molecule according to claim 4 or 5.

7. An antibody conjugate, characterized in that The antibody conjugate comprises the Nanobody according to any one of claims 1 to 3 or the binding molecule according to claim 4 or 5, and an effector molecule; the effector molecule is a label.

8. The antibody conjugate according to claim 7, wherein The effector molecule is a nuclide.

9. The antibody conjugate according to claim 7 or 8, wherein The C-terminus of the nanobody is connected to a His-Cys tag; and / or the antibody conjugate is an antibody probe, and the label is IR808.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the Nanobody according to any one of claims 1 to 3, the binding molecule according to claim 4 or 5, or the antibody conjugate according to any one of claims 7 to 9, and a pharmaceutically acceptable excipient.

11. A kit for detecting nectin-4 and / or diagnosing cancers associated with nectin-4 expression, characterized in that: The kit comprises the Nanobody according to any one of claims 1 to 3, the binding molecule according to claim 4 or 5, or the antibody conjugate according to any one of claims 7 to 9; the cancer is selected from one or more of breast cancer, lung cancer, colorectal cancer, pancreatic cancer, bladder cancer and ovarian cancer.

12. Use of the Nanobody according to any one of claims 1 to 3, the binding molecule according to claim 4 or 5, the nucleic acid molecule according to claim 6, the antibody conjugate according to any one of claims 7 to 9, or the pharmaceutical composition according to claim 10 in the preparation of a product for diagnosing cancer; the cancer is a cancer associated with Nectin-4 expression; the cancer is selected from one or more of breast cancer, lung cancer, colorectal cancer, pancreatic cancer, bladder cancer and ovarian cancer.

13. A method for detecting nectin-4 for non-diagnostic purposes, characterized in that: The method comprises contacting the nanobody according to any one of claims 1 to 3, the binding molecule according to claim 4 or 5, the antibody conjugate according to any one of claims 7 to 9, or the kit according to claim 11 with a test sample to detect the expression level of nectin-4 in the test sample.

Images