Nectin-4 targeted nano antibody and application thereof in preparation of products for treating bladder cancer

Abstract

The invention discloses a Nectin-4 targeted nano antibody and an application of the Nectin-4 targeted nano antibody in preparation of a product for treating bladder cancer. The nano antibody comprises a heavy chain variable region, and the heavy chain variable region comprises CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is as shown in SEQ ID NO: 4; the amino acid sequence of the CDR2 is as shown in SEQ ID NO: 17; the amino acid sequence of the CDR3 is as shown in SEQ ID NO: 8, 9 or 10. The Nectin-4 targeted nano antibody provided by the invention has strong binding force with a Nectin-4 antigen, and has good endocytosis activity, and the prepared related antibody conjugate has the advantages of strong tumor targeting, fast tumor enrichment and long residence time, is beneficial to research on diagnosis, prognosis and development of tumors, and has broad application prospects. And an effective choice is provided for treating tumors (such as bladder cancer).

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a nectin-4 targeted nano antibody and an application thereof in preparing a product for treating bladder cancer. Background Art

[0002] Nectin-4 is a type I transmembrane glycoprotein and a member of the Nectin family in the immunoglobulin superfamily. In normal tissues, the expression level of Nectin-4 is low, but in malignant tumors such as bladder cancer, its expression is significantly upregulated. Especially in bladder cancer, the high expression of Nectin-4 is closely related to the tumor's invasiveness, metastatic ability and poor prognosis. Therefore, Nectin-4 has become an important target for the treatment and diagnosis of bladder cancer.

[0003] In terms of treatment, the overexpression of Nectin-4 makes it an ideal target for antibody-drug conjugates (ADCs). For example, Enfortumab Vedotin is an ADC drug targeting Nectin-4 and has been approved by the FDA for the treatment of locally advanced or metastatic urothelial carcinoma. The drug binds to Nectin-4 on the surface of tumor cells, is internalized into cells, and then releases toxic small molecules, thereby achieving specific killing of tumor cells. However, existing Nectin-4 targeted therapies still have limited efficacy and large side effects in some cases.

[0004] In terms of diagnosis, high expression levels of Nectin-4 can be used as a biomarker for bladder cancer for early diagnosis and prognosis assessment of the disease. Antibodies that specifically recognize Nectin-4 can highly sensitively detect the presence and expression levels of Nectin-4 in tumor tissues through methods such as immunohistochemistry. This helps to improve the diagnostic accuracy of bladder cancer and the scientific nature of treatment decisions.

[0005] Therefore, the development of nectin-4 targeting antibodies with higher specificity and affinity is of great significance for improving the therapeutic effect and diagnostic accuracy of bladder cancer. Summary of the invention

[0006] In order to solve the technical problems existing in the prior art, the present invention provides a nectin-4 targeted nano antibody and its application in preparing a product for treating bladder cancer.

[0007] The present invention solves the above technical problems through the following technical solutions.

[0008] The first aspect of the present invention provides a nectin-4 targeted nanobody, which 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 INVMA (SEQ ID NO: 4); the amino acid sequence of the CDR2 is SIX1X2GGSTNYADSVKG (SEQ ID NO: 17), X1 is S or T, and X2 is R, K or S; the amino acid sequence of the CDR3 is DRLPDFMDY (SEQ ID NO: 8), VSPGPAGWLDY (SEQ ID NO: 9) or VSPSATGWYDY (SEQ ID NO: 10).

[0009] In some embodiments of the present invention, the amino acid sequence of the CDR2 is SITSGGSTNYADSVKG (SEQ ID NO: 5), SISRGGSTNYADSVKG (SEQ ID NO: 6) or SISKGGSTNYADSVKG (SEQ ID NO: 7).

[0010] In some embodiments of the present invention, the amino acid sequence of the CDR1 is INVMA (SEQ ID NO: 4); the amino acid sequence of the CDR2 is SITSGGSTNYADSVKG (SEQ ID NO: 5), and the amino acid sequence of the CDR3 is DRLPDFMDY (SEQ ID NO: 8);

[0011] The amino acid sequence of the CDR1 is INVMA (SEQ ID NO: 4); the amino acid sequence of the CDR2 is SISRGGSTNYADSVKG (SEQ ID NO: 6), and the amino acid sequence of the CDR3 is VSPGPAGWLDY (SEQ ID NO: 9); or,

[0012] The amino acid sequence of the CDR1 is INVMA (SEQ ID NO: 4); the amino acid sequence of the CDR2 is SISKGGSTNYADSVKG (SEQ ID NO: 7), and the amino acid sequence of the CDR3 is VSPSATGWYDY (SEQ ID NO: 10).

[0013] 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.

[0014] 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, 2 or 3, or has 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% sequence identity with SEQ ID NO: 1, 2 or 3 and does not involve changes in the CDR sequence.

[0015] 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.

[0016] In some embodiments of the invention, the binding molecule is a multivalent Nanobody comprising a plurality of said Nanobodies; or the binding molecule is a fusion protein, such as a multispecific antibody or a heavy chain antibody.

[0017] In some embodiments of the invention, the binding molecule is a heavy chain antibody.

[0018] In some embodiments of the present invention, the Fc of the heavy chain antibody is IgG Fc, preferably, the IgG Fc is IgG1 Fc, and / or, the IgG Fc is derived from mouse or human; more preferably, the IgG Fc is human IgG1 Fc.

[0019] In some embodiments of the present invention, the amino acid sequence of the Fc is as shown in SEQ ID NO: 11, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 11 and maintains the function of the Fc.

[0020] 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.

[0021] The fourth aspect of the present invention provides a genetically modified cell, wherein the cell comprises the chimeric antigen receptor as described in the third aspect of the present invention.

[0022] In some embodiments of the invention, the genetically modified cell is a eukaryotic cell, preferably an isolated human cell.

[0023] In some embodiments of the present invention, the genetically modified cells are immune cells, such as T cells or NK cells.

[0024] The fifth aspect of the present invention provides an isolated nucleic acid molecule, which encodes 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 Chimeric Antigen Receptor as described in the third aspect of the present invention.

[0025] The sixth aspect of the present invention provides a recombinant vector, which comprises the nucleic acid molecule as described in the fifth aspect of the present invention.

[0026] In some embodiments of the present invention, the recombinant vector is a recombinant expression vector or a recombinant replication vector.

[0027] 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.

[0028] 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, the binding molecule as described in the second aspect of the present invention, or the chimeric antigen receptor as described in the third aspect of the present invention.

[0029] In some embodiments of the present invention, the host cell used in constructing the transformant is selected from Escherichia coli cells, insect cells, yeast cells and mammalian cells.

[0030] In some embodiments of the present invention, the host cell is a 293 cell.

[0031] The eighth aspect of the present invention provides a method for preparing an antibody targeting nectin-4, the method comprising:

[0032] The transformant according to the seventh aspect of the present invention is cultured, and the antibody is obtained from the culture.

[0033] The ninth aspect of the present invention provides an antibody conjugate, which comprises the nanoantibody 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.

[0034] 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.

[0035] In some embodiments of the invention, the marker can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means. The marker 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, 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.

[0036] 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, as shown in SEQ ID NO: 14, 15 or 16.

[0037] In some embodiments of the present invention, the antibody conjugate is an antibody probe, and the label is IR808.

[0038] In some embodiments of the present invention, the antibody probe is obtained by connecting the nanobody with a His-Cys tag at the C-terminus to IR808.

[0039] The tenth aspect of the present invention provides a pharmaceutical composition, 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, 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 transformant as described in the seventh aspect of the present invention or the antibody conjugate as described in the ninth aspect of the present invention, and a pharmaceutically acceptable excipient.

[0040] 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.

[0041] 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.

[0042] The twelfth aspect of the present invention provides a kit for detecting Nectin-4 and / or diagnosing cancers associated with 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.

[0043] 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.

[0044] 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.

[0045] 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 prevention and / or treatment; the cancer is a cancer related to Nectin-4 expression.

[0046] 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.

[0047] 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.

[0048] In some embodiments of the present invention, the test sample is a cell or an animal.

[0049] 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.

[0050] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0051] The reagents and raw materials used in the present invention are commercially available.

[0052] The positive and progressive effects of the present invention are:

[0053] The nectin-4 targeted nanoantibodies provided by the present invention have strong binding force with the nectin-4 antigen and good endocytosis activity. The prepared related antibody conjugates have the advantages of strong tumor targeting, rapid tumor enrichment and long retention time, which is beneficial to the study of tumor diagnosis, prognosis and occurrence and development, and provides an effective option for the treatment of tumors (such as bladder cancer). BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The results of in vivo fluorescence imaging of anti-nectin-4 antibody conjugates 2-IR808, 4-IR808, and 260-IR808 are shown. DETAILED DESCRIPTION

[0055] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below:

[0056] "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.

[0057] 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.

[0058] 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 regions 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, antibodies produced by camelids that are then humanized, or antibodies obtained by 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):

[0059] - Only a single domain is required to bind the antigen with high affinity and selectivity, so that there is no need for the presence of two separate domains, nor is there a need to ensure that the two domains are present in the appropriate spatial conformation and configuration (for example, scFv generally requires the use of a specially designed linker);

[0060] - VHH domains can be expressed from a single gene and do not require post-translational folding or modification;

[0061] - VHH domains can be easily engineered into multivalent and multispecific formats;

[0062] - VHH domains are highly soluble and have no tendency to aggregate;

[0063] - 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;

[0064] - VHH domains are easy and relatively cheap to prepare, even on the scale required for production;

[0065] - The VHH domain is relatively small compared to conventional tetrapeptide chain structure 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 chain structure antibodies;

[0066] - VHH domains may display so-called cavity-binding properties (particularly due to their extended CDR3 loop compared to conventional VH domains), thereby being able to reach targets and epitopes that are inaccessible to conventional tetrapeptide structure antibodies.

[0067] 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.

[0068] Typically, the antibodies of the invention targeting nectin-4 will be expressed as preferably 10 -7 Up to 10 -10 mol / L (M), more preferably 10 -8 Up to 10 -10 mol / L, even more preferably 10 -9 Up 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 (i.e., nectin-4). Any value greater than 10 -4 KD values ​​of M are generally considered to indicate non-specific 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.

[0069] When "competition" 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 (e.g., antibody or immunologically functional fragment thereof) to be tested 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 Manual). Press); solid phase direct labeling RIA with 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 is on a solid surface or on a cell surface). Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cell 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: antigen binding proteins that bind to the same epitope as a reference antigen binding protein; and antigen binding proteins that bind to an epitope that is sufficiently close to the epitope bound by the reference antigen binding protein, and the two epitopes sterically hinder each other from binding. Additional details about methods for determining competitive binding are provided in the Examples of the present invention. Typically, when the competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of the specific binding of the reference antigen binding protein to the common antigen.In certain instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0070] 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 performed to obtain antibodies that compete with each other 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 WO03 / 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.

[0071] "Cross-reactivity" refers to, for example, the nectin-4 binding protein of the present invention with nectin-4 from 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 antigens 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.

[0072] "Antigen" refers to a molecule used to immunize an immunologically active vertebrate to produce an antibody that recognizes the antigen, or to screen an expression library (e.g., a phage, yeast, or ribosome display library, in particular). In the present invention, antigens are defined more broadly to include target molecules specifically recognized by antibodies, as well as a portion or mimetic of a molecule used in an immunization process for producing antibodies or in a library screening for selecting antibodies. For example, for 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 variants and other variants of human nectin-4 are all referred to as antigens.

[0073] In the present invention, the amino acid sequences of complementarity determining regions (CDRs) listed are shown according to the definition of Kabat numbering convention. However, it is well known to those skilled in the art that the CDR of an antibody 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 loop, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), 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 appreciated 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 the complementarity determining regions as defined by any of the above-mentioned known schemes described in the present invention.

[0074] Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment system rules or combinations). Although the scope of the present invention is based on the sequences shown in the definition according to the Kabat numbering rules, the amino acid sequences corresponding to the definition rules of other CDRs should also fall within the scope of the present invention.

[0075] 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.

[0076] 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 usually composed of 1 to 15 amino acids selected from G and S, for example (G4S)3.

[0077] 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.

[0078] In the present invention, "fusion protein" refers to a protein formed by connecting two or more different protein or polypeptide sequences together by genetic engineering means. 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. Both have relatively independent 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, increases 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 cause the binding protein to form a dimeric molecule, while prolonging the in vivo half-life of the binding protein.

[0079] 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. The mutations are defined according to the EU numbering system.

[0080] 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; or K290N / S298G / T299A / K326E, or any combination of the foregoing positions.

[0081] In the present invention, "heavy chain antibodies" are antibodies derived from camelids or cartilaginous fish. Compared with the above-mentioned full-length antibodies, heavy chain antibodies lack light chains and heavy chain constant region 1 (CH1), and only contain two heavy chains composed of variable regions (VHH) and other constant regions, and the variable regions are connected to the constant regions through a hinge region-like structure. Each heavy chain of the camelid heavy chain antibody contains one variable region (VHH) and two constant regions (CH2 and CH3), and each heavy chain of the cartilaginous fish heavy chain antibody contains one variable region and five constant regions (CH1~CH5). The antigen-binding fragments of heavy chain antibodies include VHH and single-chain heavy chain antibodies. By fusing with the constant region of human IgG Fc, the heavy chain antibody can have CH2 and CH3 of human IgG Fc.

[0082] 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.

[0083] In some embodiments, the aforementioned anti-nectin-4 Nanobody or heavy chain antibody comprises a Cys at the C-terminus or N-terminus.

[0084] 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.

[0085] In some embodiments, the aforementioned anti-Nectin-4 nanobody or heavy chain antibody binds to tumor cells. For example, the detection method in Example 3.

[0086] In some embodiments, the aforementioned anti-nectin-4 nanobody or heavy chain antibody has endocytosis activity. For example, the detection method in Example 4.

[0087] 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%, about 80%.

[0088] In the present invention, heavy chain antibodies and antibodies are intended to distinguish different combinations of antibodies.

[0089] Without substantially affecting the activity of the antibody, a person skilled in the art may change one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the sequence 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 (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and addition of one or more (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. In the art, conservative substitution with amino acids with similar or similar properties usually does not change the function of the protein. For example, amino acids with similar properties are substituted in the FR and / or CDR regions of the variable region. Amino acid residues that can be conservatively substituted 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 will generally not change the function of the protein. They are all considered to be included in the scope of protection of the present invention.

[0090] Variant forms of the various antibodies described in the present invention include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of 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.

[0091] In some embodiments, the sequence of the variant of the present invention can have 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% identity with its source sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST, especially BLASTP or TBLASTN, using default parameters is used. The present invention also includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

[0092] The antibodies of the present invention can be prepared by conventional methods in the art, such as hybridoma technology well known in the art. Nanobodies and heavy chain antibodies of the present invention can be prepared by conventional methods in the art, such as phage display technology well known in the art. Alternatively, various antibodies of the present invention can be expressed in other cell lines. Sequences encoding various antibodies of the present invention can be used to transform suitable mammalian host cells. Transformation can be carried out by any known method, for example, including packaging polynucleotides in viruses (or viral vectors) and transducing host cells with viruses (or vectors). 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, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides 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, a variety of 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.

[0093] 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.

[0094] In the present invention, the term "recombinant vector" means a construct capable of delivering and expressing one or more target genes or sequences in a host cell in some embodiments. The term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct, when the construct comprises a nucleotide sequence encoding mRNA, protein, polypeptide or peptide, and the vector contacts the cell under conditions sufficient to allow mRNA, protein, polypeptide or peptide to be expressed in the cell, the construct permits the host cell to express mRNA, protein, polypeptide or peptide. The vector of the present invention is generally not naturally occurring. However, the part of the vector may be naturally occurring. The recombinant expression vector of the present invention may include any type of nucleotides, including but not limited to the following DNA and RNA: it may be single-stranded or double-stranded, synthesized or partially obtained from a natural source, and it may contain natural, non-natural or altered nucleotides. The recombinant expression vector may include naturally occurring or non-naturally occurring nucleotides, or these two types of connections. In exemplary aspects, the altered nucleotides or non-naturally occurring nucleotides are connected without hindering the transcription or replication of the vector.

[0095] The recombinant expression vector of the present invention can be any suitable recombinant expression vector, which can be used for transformation or transfection to deliver one or more genes or sequences of interest into any suitable host cell and preferably express the gene or sequence 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.

[0096] In the present invention, 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, an animal, a fungus, or an alga; or it can be a prokaryotic cell, such as a bacterium or a protozoan. 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 cell line), NS0 cells, and 293 cells.

[0097] 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. In general, a CAR comprises an extracellular domain, a transmembrane domain, and an intracellular domain. In exemplary aspects, the extracellular domain of a CAR comprises an antigen recognition region, which may be an scFV of an antigen-specific antibody.

[0098] In the present invention, the term "antibody conjugate" includes the aforementioned nano antibodies or binding molecules and effector molecules. Effector molecules are molecules that can exhibit the desired target activity, exemplified by the effector molecules selected from radioisotopes, antitumor agents, immunomodulators, biological response modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.

[0099] In some embodiments, the effector molecule is a marker. As an example, the marker can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. The marker 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 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 (e.g., streptavidin) modified with the above labels. The labels encompassed in 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.

[0100] In some embodiments, labels as described above can be attached to the antibodies of the invention via linkers of varying lengths to reduce potential steric hindrance.

[0101] 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.

[0102] In some embodiments, the anti-nectin-4 antibody further comprises the His tag sequence, which is a short sequence comprising consecutive histidine residues, such as 6 His, 8 His, and 10 His.

[0103] In some embodiments, the aforementioned anti-nectin-4 antibody comprises a Cys at the C-terminus or the N-terminus.

[0104] In some embodiments, the effector molecule is a cytotoxic drug.

[0105] In the present invention, the term "Antibody-Drug Conjugate" or "Antibody conjugate" generally refers to an antibody connected to a biologically active cytotoxic drug, nuclide or label through a stable linker. The "cytotoxic drug" may include toxins, such as small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin, toxic drugs, chemotherapeutic drugs, antibiotics or nucleolytic enzymes, or their derivatives.

[0106] In the present invention, the pharmaceutical composition may include a suitable pharmaceutically acceptable carrier such as a pharmaceutical excipient, such as a pharmaceutical carrier known in the art, a pharmaceutical excipient, including a buffer. "Pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic agents and absorption delay agents that are physiologically compatible. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. The pharmaceutical composition comprising the present invention can be prepared by mixing the antibody of the present invention with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)) having the desired purity, preferably in the form of a lyophilized preparation or an aqueous solution. In some embodiments, the pharmaceutical composition unit dosage may contain 0.01 to 99% by weight of the aforementioned nano antibody or the aforementioned binding molecule, or the amount of the aforementioned nano antibody or the aforementioned binding molecule in the pharmaceutical composition unit dose is 0.1-2000 mg, and in some embodiments is 1-1000 mg.

[0107] In some embodiments, an article or product is provided, comprising the aforementioned anti-Nectin-4 antibody or antibody conjugate. Optionally, the article comprises a container and a label. The container is, for example, a bottle, a syringe, and a test tube. The container holds a composition effective for treating a condition. The label on or connected to the container indicates that the composition is used to treat a selected condition. The composition contains the aforementioned anti-Nectin-4 antibody or antibody conjugate.

[0108] The pharmaceutical composition of the present invention can also include more than one active ingredient, which is required for the specific indication to be treated, preferably those active ingredients with complementary activities 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, and the matrix is ​​a formed article, such as a film or microcapsule form.

[0109] "Tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. "Cancer", "cancerous", "proliferative disorders" and "tumors" are not mutually exclusive when mentioned in the present invention. In some embodiments, the aforementioned cancer is breast cancer or bladder cancer.

[0110] In the present invention, the 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.

[0111] In the present invention, the term "effective amount" means the amount of a drug or pharmaceutical agent that elicits a biological or pharmaceutical response in a tissue, system, animal or human that is sought by, for example, a researcher or clinician. In addition, the term "effective amount" means an amount that causes improved treatment, cure, prevention or alleviation of a disease, condition or side effect, or an amount that reduces the rate of progression of a disease or condition, compared to a corresponding subject that has not received the amount. The term also includes within its scope an amount that effectively enhances normal physiological function.

[0112] The “subject” and “patient” described in the present invention refer to mammals, especially primates, and especially humans.

[0113] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0114] Example 1. Screening and preparation of anti-nectin-4 antibodies

[0115] In this example, human Nectin-4 (Acro, NE4-H52H3) with a His tag was used as an immune antigen to immunize alpacas. Peripheral blood was collected, PBMCs were separated from the peripheral blood, RNA was extracted from the PBMCs, and reverse transcription was performed to obtain total cDNA. A yeast library was constructed for antibody screening.

[0116] After two rounds of sorting, multiple unique VHH sequences binding to human nectin-4 antigen were obtained through monoclonal identification, sequencing and sequence analysis. The sequences of NB656-M1-2, NB656-M1-4 and NB656-M1-260 are shown below.

[0117] > NB656-M1-2 variable region

[0118] QVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMAWYRQAPGKQRELVASITSGGSTNYADSVKGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCNADRLPDFMDYWGKGTLVTVSS (SEQ ID NO: 1)

[0119] > NB656-M1-4 variable region

[0120] EVQLVESGGGLVQPGGSLSISCAASGSIFSINVMAWYRQAPGRQRELVASISRGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNTVSPGPAGWLDYWGQGTQVTVSS (SEQ ID NO: 2)

[0121] > NB656-M1-260 variable region

[0122] EVQLVESGGGLVQPGGSLRLSCAASGSSSSINVMAWYRQAPGKQRELVASISKGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNTVSPSATGWYDYWGQGTQVTVSS (SEQ ID NO: 3)

[0123] The CDR sequences of the above-mentioned nanobodies are shown in Table 1 below (Kabat numbering rules):

[0124] Table 1 CDR sequences

[0125]

[0126] The general formula of CDR of anti-nectin-4 antibody is: CDR1 is INVMA (SEQ ID NO: 4); CDR2 is SIX1X2GGSTNYADSVKG (SEQ ID NO: 17), X1 is S or T, X2 is R, K or S; CDR3 is DRLPDFMDY (SEQ ID NO: 8), VSPGPAGWLDY (SEQ ID NO: 9) or VSPSATGWYDY (SEQ ID NO: 10).

[0127] The above sequences were connected to the human IgG1 Fc (including the hinge region) fragment to construct a VHH-Fc antibody. The plasmid was constructed and transiently transfected into 293 cells. The cells were cultured to express the antibody 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). Dialyzed into 1× PBS buffer (pH 7.4). The sequence of human IgG1 Fc (including the hinge region) is as follows:

[0128] >Fc

[0129] EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 11)

[0130] >BS025-PC HC

[0131] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSSLLQMNSLRDEDTAVYYCARAYYYGMDVWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 12)

[0132] > BS025-PC LC

[0133] DIQMTQSPSSVSASVGDRVTITCRASQGISGWLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO: 13)

[0134] Example 2. Identification of the affinity between anti-nectin-4 antibodies and the antigen nectin-4

[0135] The binding ability of anti-nectin-4 antibody to nectin-4 antigen protein was detected by ELISA and BLI methods.

[0136] 1.ELISA

[0137] Experimental method: Take 100μl / well of buffer (50mM NaHCO3, pH 9.6) containing Human Nectin-4 his (Acro, NE4-H52H3), Cyno Nectin-4 his (Acro, NE4-C52H4), and Mouse Nectin-4 his (Acro, NE4-M52H3) with a final concentration of 2μg / mL, and coat the antigen at 4℃ overnight. After coating, wash three times with PBST solution. Block with 5% milk at 37℃ for 1h. Wash once with PBST, dilute the protein from 100nM in a 1:5 gradient (5% milk dilution), add 100μL of the gradient diluted antibody protein to the ELISA plate wells, and incubate at 37℃ for 1h. The isotype control antibody (Isotype) used was purchased from Syd labs, with the catalog number PA007165.m2a. Wash with PBST 5 times, add the secondary antibody (Anti-Human IgG Fc, HRP, 1:10K) diluted with 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 at 50μL / well to terminate the reaction, and OD 450 Reading and calculation of EC 50 .

[0138] Experimental results: The results are shown in Table 2, which show that the anti-Nectin-4 antibodies have a strong binding affinity with the Nectin-4 antigen, and are stronger than the control antibody BS025-PC.

[0139] Table 2 ELISA detection of anti-nectin-4 antibody binding antigen

[0140]

[0141] 2. BLI Method

[0142] Experimental method: First, immerse the biosensor in PBST buffer for 15 minutes for equilibrium, then immerse it in a known concentration of antibody (2μg / ml), then immerse the sensor with the solidified antigen in the buffer for baseline equilibrium, then immerse the biosensor with a known concentration of antigen (200nM, 100nM, 50nM or 25nM) in the sample solution containing the antibody to be tested, and finally immerse the sensor bound to the antibody to be tested in the buffer for dissociation. The Octet instrument monitors the biosensor biofilm layer thickness in real time during the experiment, and the kinetic constants of the sample to be tested can be obtained. The specific steps are as follows:

[0143] (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.

[0144] (2) Add 200 μL PBST to the well plate to pre-wet the sensor (the sensor is ProA sensor-Sartorius), pre-wet for 15 minutes;

[0145] (3) Programming: solidify the antibody for 60 seconds, bind the antigen for 180 seconds, and dissociate for 240 seconds;

[0146] (4) Prepare samples according to the set procedure and add the samples to the detection plate;

[0147] (5) Place the sensor and sample plate into the instrument together and perform tests in sequence;

[0148] (6) After the test is completed, recycle the sensor, clean the sample plate, and turn off the instrument power.

[0149] (7) Use the analysis software DataAnalysis12 to perform fitting, export images and fitted affinity constant values.

[0150] 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.

[0151] Table 3 Detection of binding ability of anti-nectin-4 antibodies

[0152]

[0153] Example 3. In vitro cell binding assay of anti-nectin-4 antibodies

[0154] Experimental purpose: To detect the binding ability of anti-nectin-4 antibody using human breast cancer cell line MCF-7 (Punosai, CL-0149).

[0155] 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 the PCR plate, 50μL per well; dilute the antibody to be tested with 1×PBS buffer to a starting concentration of 400nM, take 50μL / well and add it to the plate to make the starting concentration of the antibody 200nM, 1:5-fold gradient, make 7 gradient dilutions, and incubate in a 4℃ refrigerator for 1 hour; after the incubation is completed, wash the cells 3 times with 1×PBS buffer, add 100μL of fluorescent secondary antibody (647Anti-Human IgG Fc) to each well, and incubate in a 4℃ refrigerator for 1 hour; after the incubation is completed, wash the cells once with 1×PBS buffer, transfer the cells to a 96-well cell plate, and analyze them on a flow cytometer.

[0156] Experimental results: The results are shown in Table 4, which show that the anti-nectin-4 antibodies NB656-M1-2, NB656-M1-4 and NB656-M1-260 all have strong binding affinity with the nectin-4 antigen on human breast cancer MCF-7 cells, and are stronger than the control antibody BS025-PC.

[0157] Table 4 EC binding of anti-nectin-4 antibody to cell surface antigen nectin-4 50

[0158]

[0159] Example 4. Endocytic activity of anti-nectin-4 antibodies

[0160] Experimental purpose: To detect the endocytic activity of anti-nectin-4 antibody using human breast cancer cell line MCF-7.

[0161] 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 5cells / μL, aliquot the cells into PCR plates, 50μL per well; dilute the antibody to be tested to 4μg / mL with 1×PBS buffer, take 50μL / well and add it to the plate, so that the final concentration of the antibody is 2μg / mL, and set 5 wells for each antibody; incubate the antibody and cells in a 4°C refrigerator for 1 hour; after the incubation is completed, wash the cells once with 1×PBS buffer, add 200μL 1×PBS buffer to resuspend the cells, transfer the antibodies and cells to a 96-well cell plate according to the time gradient of 0h, 0.5h, 1h, 2h and 4h, and incubate at 37°C; after the incubation is completed, wash the cells once with 1×PBS buffer, add 100μL fluorescent secondary antibody (647 Anti-Human IgGFc) to each well; incubate in a 4°C refrigerator for 1 hour; after the incubation is completed, wash the cells once with 1×PBS buffer, add 200μL 1×PBS buffer to resuspend the cells, transfer the cells to a 96-well cell plate, and analyze them on a flow cytometer.

[0162] Experimental results: As shown in Table 5, the anti-nectin-4 antibody has endocytic activity.

[0163] Table 5 Endocytic activity of anti-nectin-4 antibody in MCF-7 cells

[0164]

[0165] Example 5. Anti-Nectin-4 Antibody

[0166] The variable region of the antibody was connected to the His-Cys tag to construct a VHH-His-Cys antibody. The plasmid was constructed and transformed into the Escherichia coli BL21 (DE3) strain. The monoclonal colony was selected and activated in LB medium at 37°C overnight. The culture was transferred to 300 mL of LB medium at a ratio of 1:100 and cultured. When the OD value reached 0.6, IPTG was added to a final concentration of 1 mmol / L and induced at 30°C for 6 h. The culture system obtained above was centrifuged at 6,000 rpm for 5 min to collect the bacteria. The collected bacteria were resuspended in 1× PBS buffer, polymyxin was added to a final concentration of 0.5 mM, and the bacteria were broken at 37°C for 2 h, and then centrifuged at 8,000 rpm for 30 min. The filtered supernatant was combined with Ni Sepharose (GE) for 2 h. The impurities were 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 buffer was replaced with 0.1M NaHCO3 (pH 8.3). The protein purity was determined to be >90% by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The sequences of NB656-M1-2-His-Cys, NB656-M1-4-His-Cys, and NB656-M1-260-His-Cys are as follows:

[0167] > NB656-M1-2-His-Cys

[0168] QVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMAWYRQAPGKQRELVASITSGGSTNYADSVKGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCNADRLPDFMDYWGKGTLVTVSSHHHHHHC (SEQ ID NO: 14)

[0169] > NB656-M1-4-His-Cys

[0170] EVQLVESGGGLVQPGGSLSISCAASGSIFSINVMAWYRQAPGRQRELVASISRGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNTVSPGPAGWLDYWGQGTQVTVSSHHHHHHC (SEQ ID NO: 15)

[0171] > NB656-M1-260-His-Cys

[0172] EVQLVESGGGLVQPGGSLRLSCAASGSSSSINVMAWYRQAPGKQRELVASISKGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNTVSPSATGWYDYWGQGTQVTVSSHHHHHHC (SEQ ID NO: 16)

[0173] Example 6. Anti-Nectin-4 Antibody Conjugate

[0174] The anti-nectin-4 antibody conjugate is obtained by coupling the anti-nectin-4 antibody with anthocyanin dye derivative IR808 (also known as MHI-808). The specific preparation steps are as follows: 1 mg of anti-nectin-4 antibodies NB656-M1-2-His-Cys, NB656-M1-4-His-Cys, and NB656-M1-260-His-Cys were sampled and diluted to 1 mg / ml with buffer (20 mM Tris-HCl, pH 8.0), and 2.8 μL TCEP (100 mM) was added to prevent protein aggregation. According to the molar ratio of MHI-808: antibody = 2:1, about 32 μL of MHI-808 (5 mg / ml) was added to every 1 mg of protein, and the reaction was carried out at 4°C for 30 minutes. The sample was dialyzed in 20 mM Tris-HCl, pH 8.0 buffer (dialysis bag 5KD), and the solution was changed once every 3-4 hours, for a total of 5 changes.

[0175] The anti-nectin-4 antibody conjugates 2-IR808, 4-IR808, and 260-IR808 were obtained according to the above method.

[0176] Example 7. Targeting of anti-nectin-4 antibody conjugates

[0177] Objective: To investigate the tumor targeting and sustained tumor accumulation of anti-nectin-4 antibodies using bladder cancer cells T24 (Punosai, CL-0277).

[0178] Experimental method: First, resuspended T24 cells (5×10 6 cells) were injected subcutaneously into BALB / cNj-Foxn1 nu / Gpt mice (Jicui Yaokang, strain number: D000521, female) right hind limbs to construct T24 xenograft models. When the tumor volume reached about 200-400mm 3At the same time, 10 T24 xenograft mice were intravenously injected with the corresponding anti-nectin-4 antibody conjugates (concentration of 1 mg / mL, 100 μL each), and the mice were imaged in vivo using an in vivo imaging system (IVIS) at different time points (1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h and 96h).

[0179] Experimental results: The results showed that the anti-nectin-4 antibody conjugates 2-IR808, 4-IR808, and 260-IR808 have the advantages of strong tumor targeting, rapid tumor enrichment, and long retention time ( Figure 1 ).

Claims

1. A nanobody targeting nectin-4, characterized in that: 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: 4; the amino acid sequence of the CDR2 is shown in SEQ ID NO: 17; the amino acid sequence of the CDR3 is shown in SEQ ID NO: 8, 9 or 10; Preferably, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 5, 6 or 7; More preferably, the amino acid sequence of the CDR1 is shown in SEQ ID NO: 4; the amino acid sequence of the CDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: 8; The amino acid sequence of the CDR1 is shown in SEQ ID NO: 4; the amino acid sequence of the CDR2 is shown in SEQ ID NO: 6, and the amino acid sequence of the CDR3 is shown in SEQ ID NO: 9; or, The amino acid sequence of the CDR1 is shown in SEQ ID NO: 4; the amino acid sequence of the CDR2 is shown in SEQ ID NO: 7, and the amino acid sequence of the CDR3 is shown in SEQ ID NO:

10.

2. The Nanobody according to claim 1, characterized in that The framework region of the heavy chain variable region is an alpaca-derived framework region or a human-derived framework region; Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, 2 or 3, or has 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% sequence identity with SEQ ID NO: 1, 2 or 3 and does not involve changes in the CDR sequence.

3. A nectin-4 binding molecule, characterized in that: The binding molecule comprises the Nanobody as claimed in claim 1 or 2; Preferably, the binding molecule is a multivalent Nanobody comprising a plurality of the Nanobodies; or the binding molecule is a fusion protein, such as a multispecific antibody or a heavy chain antibody; Further preferably, the binding molecule is a heavy chain antibody; More preferably, the Fc of the heavy chain antibody is IgG Fc, preferably, the IgG Fc is IgG1 Fc, and / or, the IgG Fc is derived from mouse or human; more preferably, the IgG Fc is human IgG1 Fc; Further more preferably, the amino acid sequence of the Fc of the heavy chain antibody is as shown in SEQ ID NO: 11, or has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO: 11 and maintains the function of the Fc.

4. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises the Nanobody as claimed in claim 1 or 2 or the binding molecule as claimed in claim 3.

5. A genetically modified cell, characterized in that The cell comprises the chimeric antigen receptor of claim 4; Preferably, the genetically modified cells are eukaryotic cells, preferably isolated human cells; More preferably, the genetically modified cells are immune cells, such as T cells or NK cells.

6. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule encodes the Nanobody as claimed in claim 1 or 2, the binding molecule as claimed in claim 3, or the Chimeric Antigen Receptor as claimed in claim 4.

7. A recombinant vector, characterized in that: The recombinant vector comprises the nucleic acid molecule according to claim 6; Preferably, the recombinant vector is a recombinant expression vector or a recombinant replication vector; More preferably, 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.

8. A transformant, characterized in that The transformant comprises the nucleic acid molecule of claim 6 or the recombinant vector of claim 7; or, expresses the Nanobody of claim 1 or 2, the binding molecule of claim 3 or the chimeric antigen receptor of claim 4; Preferably, the host cell used in constructing the transformant is selected from Escherichia coli cells, insect cells, yeast cells and mammalian cells; More preferably, the host cell is 293 cell.

9. A method for preparing an antibody targeting nectin-4, characterized in that: The method comprises: The transformant according to claim 8 is cultured, and the antibody is obtained from the culture.

10. An antibody conjugate, characterized in that The antibody conjugate comprises the Nanobody according to claim 1 or 2 or the binding molecule according to claim 3, and an effector molecule; the effector molecule is a nuclide, a cytotoxic drug or a marker; Preferably, 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; The marker can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemical means; the marker is preferably 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, Alexa 750)), acridinium ester compounds, magnetic beads, calorimetric labels (e.g., colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex) beads, and biotin for binding to avidins (e.g., streptavidin) modified with the above labels; Further preferably, 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, as shown in SEQ ID NO: 14, 15 or 16; More preferably, the antibody conjugate is an antibody probe, and the label is IR808; Further more preferably, the antibody probe is obtained by connecting the nanobody with a His-Cys tag at the C-terminus to IR808.

11. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the Nanobody as claimed in claim 1 or 2, the binding molecule as claimed in claim 3 or the antibody conjugate as claimed in claim 10, and a pharmaceutically acceptable excipient.

12. A Nectin-4 detection reagent, characterized in that: The detection reagent comprises the Nanobody according to claim 1 or 2, the binding molecule according to claim 3, or the antibody conjugate according to claim 10; Preferably, the Nectin-4 detection reagent is used for in vivo imaging technology, Western blot, enzyme-linked immunosorbent assay and / or flow cytometry.

13. 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 claim 1 or 2, the binding molecule according to claim 3, the antibody conjugate according to claim 10, or the detection reagent according to claim 12; Preferably, the cancer is selected from one or more of breast cancer, lung cancer, colorectal cancer, pancreatic cancer, bladder cancer and ovarian cancer; and / or, The kit may also include 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.

14. Use of the Nanobody according to claim 1 or 2, the binding molecule according to claim 3, the chimeric antigen receptor according to claim 4, the cell according to claim 5, the nucleic acid molecule according to claim 6, the recombinant vector according to claim 7, the transformant according to claim 8, the antibody conjugate according to claim 10, the pharmaceutical composition according to claim 11 or the detection reagent according to claim 12 in the preparation of a product for diagnosing cancer, or a drug for prevention and / or treatment; the cancer is a cancer associated with Nectin-4 expression; Preferably, the cancer is selected from one or more of breast cancer, lung cancer, colorectal cancer, pancreatic cancer, bladder cancer and ovarian cancer.

15. A method for detecting nectin-4 for non-diagnostic purposes, characterized in that: The method comprises contacting a sample to be tested with the Nanobody according to claim 1 or 2, the binding molecule according to claim 3, the antibody conjugate according to claim 10, the detection reagent according to claim 12 or the kit according to claim 13 to detect the expression level of Nectin-4 in the sample to be tested; Preferably, the test sample is a cell or an animal; and / or The method uses in vivo imaging technology, Western blot, enzyme-linked immunosorbent assay and / or flow cytometry to detect nectin-4.

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