Nectin-4-targeted nanobody and application thereof in preparation of product for treating bladder cancer

Abstract

The application discloses a Nectin-4 targeted nanobody and application thereof in preparation of a product for treating bladder cancer. 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 as SEQ ID NO:4; the amino acid sequence of the CDR2 is shown as SEQ ID NO:17; and the amino acid sequence of the CDR3 is shown as SEQ ID NO:8, 9 or 10. The Nectin-4 targeted nanobody provided by the application has strong binding force with Nectin-4 antigen, and has good endocytosis activity; the prepared related antibody conjugate has the advantages of strong tumor targeting, fast tumor enrichment and long retention time, is beneficial to research on diagnosis, prognosis and development of tumors, and provides an effective selection for treatment of tumors (for example, bladder cancer).

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a Nectin-4 targeted nanobody and its application in the preparation of products for treating bladder cancer. Background Technology

[0002] Nectin-4 is a type I transmembrane glycoprotein belonging to the Nectin family within the immunoglobulin superfamily. While Nectin-4 expression levels are low in normal tissues, it is significantly upregulated in malignant tumors such as bladder cancer. Particularly in bladder cancer, high Nectin-4 expression is closely associated with tumor invasiveness, metastatic potential, and poor prognosis. Therefore, Nectin-4 has become an important target for the treatment and diagnosis of bladder cancer.

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

[0004] In diagnostics, high expression levels of Nectin-4 can serve as a biomarker for bladder cancer, aiding in early diagnosis and prognostic assessment. Antibodies specifically recognizing Nectin-4 can sensitively detect its presence and expression levels in tumor tissue using methods such as immunohistochemistry. This contributes to improving the accuracy of bladder cancer diagnosis and the scientific basis of treatment decisions.

[0005] Therefore, developing Nectin-4 targeting antibodies with higher specificity and affinity is of great significance for improving the treatment efficacy 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 nanobody and its application in the preparation of products for treating bladder cancer.

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

[0008] A first aspect of the present invention provides a Nectin-4 targeted nanobody, the nanobody comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3; the amino acid sequence of CDR1 is INVMA (SEQ ID NO: 4); the amino acid sequence of CDR2 is SIX1X2GGSTNYADSVKG (SEQ ID NO: 17), where X1 is S or T and X2 is R, K or S; the amino acid sequence of 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 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 CDR1 is INVMA (SEQ ID NO: 4); the amino acid sequence of CDR2 is SITSGGSTNYADSVKG (SEQ ID NO: 5); and the amino acid sequence of CDR3 is DRLPDFMDY (SEQ ID NO: 8).

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

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

[0013] In some embodiments of the present invention, the frame region of the heavy chain variable region is an alpaca-derived frame region or a human-derived frame 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 any change to the CDR sequence.

[0015] A second aspect of the present invention provides a Nectin-4 binding molecule comprising a nanobody as described in the first aspect of the present invention.

[0016] In some embodiments of the present invention, the binding molecule is a multivalent nanobody comprising multiple 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 present 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 IgG1 Fc, and / or the IgG Fc is derived from mice or humans; 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] A third aspect of the present invention provides a chimeric antigen receptor comprising a nanobody as described in the first aspect of the present invention or a binding molecule as described in the second aspect of the present invention.

[0021] A fourth aspect of the invention provides a genetically modified cell comprising a chimeric antigen receptor as described in a third aspect of the invention.

[0022] In some embodiments of the present invention, the genetically modified cells are eukaryotic cells, preferably isolated human cells.

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

[0024] A fifth aspect of the present invention provides an isolated nucleic acid molecule that encodes a nanobody as described in the first aspect of the present invention, a binding molecule as described in the second aspect of the present invention, or a chimeric antigen receptor as described in the third aspect of the present invention.

[0025] A sixth aspect of the present invention provides a recombinant vector comprising a 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, granule, bacteriophage, or viral vector, and the viral vector is preferably a retroviral vector, lentiviral vector, adenovirus vector, or adeno-associated virus vector.

[0028] A seventh aspect of the present invention provides a transformant comprising a nucleic acid molecule as described in the fifth aspect of the present invention or a recombinant vector as described in the sixth aspect of the present invention; or expressing a nanobody as described in the first aspect of the present invention, a binding molecule as described in the second aspect of the present invention, or a chimeric antigen receptor as described in the third aspect of the present invention.

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

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

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

[0032] The antibody is obtained from the culture by culturing the transformant as described in the seventh aspect of the present invention.

[0033] A ninth aspect of the present invention provides an antibody conjugate comprising a nanobody as described in the first aspect of the present invention or a binding molecule as described in the second aspect of the present invention, and an effector molecule; said effector molecule is a radionuclide, 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 the following: toxins (e.g., small molecule toxins or enzyme-active toxins of bacterial, fungal, plant or animal origin), radioactive isotopes, toxic drugs, chemotherapeutic drugs, antibiotics, ribolysins and their derivatives.

[0035] In some embodiments of the present invention, the marker can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. The marker is selected from enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase) and 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)), acridine esters, magnetic beads, calorimetric markers (e.g., colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads), and biotin for binding avidin (e.g., streptavidin) modified with the above markers.

[0036] In some embodiments of the present invention, the C-terminus of the nanobody is attached with a His-Cys tag, and the amino acid sequence of the nanobody with the His-Cys tag attached to the C-terminus is, for example, 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 marker is IR808.

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

[0039] A tenth aspect of the present invention provides a pharmaceutical composition comprising a nanobody as described in the first aspect of the present invention, a binding molecule as described in the second aspect of the present invention, a chimeric antigen receptor as described in the third aspect of the present invention, a cell as described in the fourth aspect of the present invention, a transformant as described in the seventh aspect of the present invention, or an antibody conjugate as described in the ninth aspect of the present invention, and pharmaceutically acceptable excipients.

[0040] The eleventh aspect of the present invention provides a detection reagent for Nectin-4, the detection reagent comprising a nanobody as described in the first aspect of the present invention, a binding molecule as described in the second aspect of the present invention, or an 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 in in vivo imaging techniques, Western blot, enzyme-linked immunosorbent assay (ELISA), and / or flow cytometry.

[0042] The twelfth aspect of the present invention provides a kit for detecting Nectin-4 and / or diagnosing Nectin-4 expression-related cancers, the kit comprising a nanobody as described in the first aspect of the present invention, a binding molecule as described in the second aspect of the present invention, an antibody conjugate as described in the ninth aspect of the present invention, or a 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 includes other reagents, such as fluorescently labeled secondary antibodies, for detecting Nectin-4 using in vivo imaging technology, Western blot, enzyme-linked immunosorbent assay and / or flow cytometry.

[0045] The thirteenth aspect of this invention provides the use of nanobodies as described in the first aspect of this invention, binding molecules as described in the second aspect of this invention, chimeric antigen receptors as described in the third aspect of this invention, cells as described in the fourth aspect of this invention, nucleic acid molecules as described in the fifth aspect of this invention, recombinant vectors as described in the sixth aspect of this invention, transformants as described in the seventh aspect of this invention, antibody conjugates as described in the ninth aspect of this invention, pharmaceutical compositions as described in the tenth aspect of this invention, and detection reagents as described in the eleventh aspect of this invention in the preparation of products for diagnosing cancer, or medicaments for prevention and / or treatment; wherein the cancer is a Nectin-4 expression-related cancer.

[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 a nanobody as described in the first aspect of the present invention, a binding molecule as described in the second aspect of the present invention, an antibody conjugate as described in the ninth aspect of the present invention, a detection reagent as described in the eleventh aspect of the present invention, or a kit as described in the twelfth aspect of the present invention, and detecting 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 employs in vivo imaging technology, Western blot, enzyme-linked immunosorbent assay (ELISA), and / or flow cytometry to detect Nectin-4.

[0050] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0051] The reagents and raw materials used in this invention are all commercially available.

[0052] The positive and progressive effects of this invention are as follows:

[0053] The Nectin-4-targeting nanobody provided by this invention has a strong binding affinity to the Nectin-4 antigen and exhibits good endocytic activity. The prepared antibody conjugates have the advantages of strong tumor targeting, rapid tumor enrichment, and long retention time, which is beneficial for studying the diagnosis, prognosis, and development of tumors and provides an effective option for the treatment of tumors (such as bladder cancer). Attached Figure Description

[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 Implementation

[0055] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the operational steps used herein, such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, are all conventional steps widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below:

[0056] “Nectin-4” or “PVRL-4” are used interchangeably and include variants, isotypes, species homologs of human Nectin-4, and analogs that share at least one common epitope with Nectin-4.

[0057] In this invention, the letters in the amino acid sequence represent 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 this invention, the terms "single-domain antibody," "heavy chain variable region domain of a heavy chain antibody," "VHH," "VHH domain," and "nanobody" are used interchangeably, all referring to nanobodies that specifically recognize and bind to Nectin-4. A nanobody is the variable region of a heavy chain antibody. Typically, a nanobody contains three CDRs and four FRs. A nanobody is the smallest functional antigen-binding fragment. The "VHH domain" includes, but is not limited to, naturally occurring antibodies produced by camelids, or antibodies produced by camelids that have been humanized, or antibodies obtained through phage display technology. The total number of amino acid residues in the VHH domain will typically be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences are also suitable for the purposes described in this invention. The VHH domain (alone or as part of a larger polypeptide) offers many significant advantages over the use of conventional VH and VL domains, scFv, or conventional antibody fragments (e.g., Fab- or F(ab')2- fragments):

[0059] - Only a single domain is needed to bind to the antigen with high affinity and high selectivity, so that there is no need for two separate domains, nor is it necessary to ensure that the two domains exist in the appropriate spatial conformation and configuration (for example, scFv generally requires the use of specially designed adapters).

[0060] The -VHH domain can be expressed by a single gene and does not require post-translational folding or modification;

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

[0062] - The VHH domain is highly soluble and has no tendency to aggregate;

[0063] The -VHH domain is highly stable to heat, pH, proteases and other denaturants or conditions, and therefore can be prepared, stored or transported without the use of refrigeration equipment, thus saving costs, time and the environment;

[0064] -VHH domains are easy to prepare and relatively inexpensive, even at the scale required for production.

[0065] The -VHH domain is relatively small compared to conventional tetrapeptide chain antibodies (approximately 15 kDa, or 1 / 10 the size of conventional IgG), thus exhibiting higher tissue penetration and allowing for higher dose administration compared to conventional tetrapeptide chain antibodies.

[0066] The -VHH domain can exhibit so-called cavity-binding properties (especially due to its elongated CDR3 loop compared to the conventional VH domain), thereby reaching targets and epitopes that are inaccessible to conventional tetrapeptide chain 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 antibody targeting Nectin-4 of the present invention will be preferably 10 as measured in Biacore, KinExA, or Fortibio assays. -7 Up to 10 -10 moles per liter (M), more preferably 10 -8 Up to 10 -10 moles per liter, or even more preferably 10 -9 Up to 10 -10 Or a lower dissociation constant (KD), and / or 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 it wants to bind to (i.e., Nectin-4). Any value greater than 10... -4 The KD value of M is generally considered to indicate nonspecific binding. The specific binding of antigen-binding proteins to antigens or epitopes can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assays) as described in this invention.

[0069] When “competition” is used in cases where antigen-binding proteins (e.g., neutralizing antigen-binding proteins or neutralizing antibodies) compete for the same epitope, it means that there is competition between antigen-binding proteins, which is determined by the following assay: the antigen-binding protein to be detected (e.g., an antibody or an immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand or a reference antibody) to a common antigen (e.g., Nectin-4 antigen or a fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another. These assays include: solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, for example, Stahli et al., 1983, Methods in Enzymology 9: 242-253); solid-phase direct biotin-avidin EIA (see, for example, Kirkland et al., 1986, J. Immunol. 137: 3614-3619), solid-phase direct labeling assay, and solid-phase direct labeling sandwich assay (see, for example, Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor). Press); solid-phase direct labeling of RIAs with I-125 label (see, for example, Morel et al., 1988, Molec. Immunol. 25: 7-15); solid-phase direct biotin-avidin EIA (see, for example, Cheung et al., 1990, Virology 176: 546-552); and directly labeled RIAs (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). Typically, the assay involves using a purified antigen (on a solid surface or cell surface) that binds to both an unlabeled detection antigen-binding protein and a labeled reference antigen-binding protein. Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cell in the presence of the target antigen-binding protein. Typically, the target antigen-binding protein is present in excess. Antigen-binding proteins identified by competitive assays (competitive 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 adjacent to the epitope of the reference antigen-binding protein, the two epitopes spatially hindering each other from binding. Further details regarding methods for determining competitive binding are provided in embodiments of the invention. Typically, when an excess of a competing antigen-binding protein is present, 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 some cases, the binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0070] Antibodies can be competitively screened for binding to the same epitope using conventional techniques known to those skilled in the art. For example, competitive and cross-competitive studies can be performed to obtain antibodies that compete or cross-competitively bind to the antigen. A high-throughput method for obtaining antibodies that bind to the same epitope based on their cross-competition is described in International Patent Publication WO03 / 48731. Therefore, antibodies that compete with the antibody molecules of this invention for binding to the same epitope on Nectin-4 can be obtained using conventional techniques known to those skilled in the art.

[0071] “Cross-reactivity” refers to, for example, the Nectin-4 binding protein of this invention and Nectin-4 from different species. For example, a single-domain antibody or derived protein of this invention that binds to human Nectin-4 may also bind to Nectin-4 from another species. Cross-reactivity is measured by detecting the specific reactivity with purified antigens in binding assays (e.g., SPR and ELISA), or by binding or functional interaction with cells physiologically expressing Nectin-4. Methods for determining cross-reactivity include standard binding assays as described in this invention, such as surface plasmon resonance (SPR) analysis, or flow cytometry.

[0072] "Antigen" refers to a molecule of a vertebrate used for immunization to generate antibodies that recognize the antigen, or for screening expression libraries (e.g., particularly phage, yeast, or ribosome display libraries). In this invention, antigen is defined more broadly to include target molecules specifically recognized by antibodies, as well as portions or mimics of molecules used in immunization processes to generate antibodies or in library screening to select antibodies. For example, for the antibodies of this invention that bind to human Nectin-4, monomers and multimers of human Nectin-4 (e.g., dimers, trimers, etc.), as well as truncated variants and other variants of human Nectin-4, are all referred to as antigens.

[0073] In this invention, the amino acid sequences of the listed complementarity determining regions (CDRs) are all as defined by the Kabat numbering rules. However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, 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 antibody sequence variability (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, imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. Those skilled in the art will understand that, unless otherwise specified, the terms “CDR” and “complementary determination region” for a given antibody or its region (e.g., variable region) should be understood to encompass the complementary determination region defined by any of the above-described known schemes as described in this invention.

[0074] Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations). Although the scope of protection claimed in this invention is based on the sequence defined according to the Kabat numbering rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

[0075] In this invention, "Nectin-4 binding molecules" are proteins that have the function of recognizing and binding Nectin-4, including but not limited to antibodies, antigen-binding fragments of antibodies, heavy chain antibodies, nanobodies, microbodies, multispecific antibodies, fusion proteins, affinity molecules, target binding regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines.

[0076] In this invention, a binding molecule containing two or more nanobodies is a multivalent nanobody; a binding molecule containing two or more nanobodies with different specificities is a multispecific nanobody. Multiple nanobodies are linked together via linkers. The linkers are typically composed of 1 to 15 amino acids selected from G and S, for example, (G4S)3.

[0077] In this invention, "multispecific antibody" refers to a class of antibodies that can simultaneously recognize and bind to at least two different antigenic epitopes, such as bispecific antibodies.

[0078] In this invention, a "fusion protein" refers to a protein formed by linking two or more different protein or polypeptide sequences together through genetic engineering. The structure of an Fc fusion protein (e.g., a heavy chain antibody) consists of two parts: the Fc region of an immunoglobulin and the nanobody described in this invention. Both have relatively independent structural domains and functions, and can influence their physicochemical properties and biological activities from different perspectives. In some embodiments, the Fc region is an Fc region that enhances effector function, for example, enhancing antibody-dependent cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC) with increased effector functions. In some embodiments, the Fc region included in the aforementioned Fc fusion protein can cause the binding protein to form a dimer molecule, while simultaneously prolonging the in vivo half-life of the binding protein.

[0079] An exemplary IgG1 Fc region includes substitutions having the following: 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; 2 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 the above positions. The mutations are defined according to the EU numbering system.

[0080] An exemplary IgG1 Fc region includes substitutions having the following: 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 280H / K290S / S298V; F243L / R292P / Y300L; F243L / R292P / Y300L / P396L; F243L / R292P / Y300L / V305I / P396L; G236A / S239D / I332E; K326A / E333A; K326W / E333S; K290E / S298G / T299A; K290N / S298G / T299A; K290E / S298G / T299A / K326E; or K290N / S298G / T299A / K326E, or any combination of the above positions.

[0081] In this invention, the "heavy chain antibody" is an antibody derived from camelids or cartilaginous fishes. Compared to the full-length antibodies described above, the heavy chain antibody lacks the light chain and the heavy chain constant region 1 (CH1), containing only two heavy chains composed of a variable region (VHH) and other constant regions. The variable region is connected to the constant region via a hinge-like structure. Each heavy chain of the camelid heavy chain antibody contains one variable region (VHH) and two constant regions (CH2 and CH3), while each heavy chain of the cartilaginous fish heavy chain antibody contains one variable region and five constant regions (CH1~CH5). The antigen-binding fragment of the heavy chain antibody includes the VHH and a single-chain heavy chain antibody. By fusing with the constant region of human IgG Fc, the heavy chain antibody can possess the CH2 and CH3 of human IgG Fc.

[0082] In some implementations, the aforementioned nanobody is linked to the Fc region via a linker. The linker may be a non-functional amino acid sequence of 1-20 or more amino acids without secondary or higher structures.

[0083] In some implementations, the aforementioned anti-Nectin-4 nanobody or heavy chain antibody contains Cys at the C-terminus or N-terminus.

[0084] In some implementations, the KD value of the aforementioned anti-Nectin-4 nanobody or heavy chain antibody binding to Nectin-4 can be ≤1×10⁻⁶. -7 M.

[0085] In some implementations, the aforementioned anti-Nectin-4 nanobodies or heavy chain antibodies bind to tumor cells. For example, the detection method in Example 3.

[0086] In some implementations, the aforementioned anti-Nectin-4 nanobodies or heavy chain antibodies exhibit endocytic activity. For example, the detection method in Example 4.

[0087] In some implementations, the aforementioned anti-Nectin-4 nanobodies or heavy chain antibodies are able to inhibit tumor growth by at least about 10%, such as at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, or about 80%.

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

[0089] Without substantially affecting antibody activity, those skilled in the art can modify the sequence of the present invention by one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) to obtain variants of the antibody or its functional fragment sequence. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. In the art, conservative substitutions with amino acids of similar or comparable properties generally do not alter protein function. For example, substitutions of amino acids with similar properties 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 example, adding one or more amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein. These are all considered to be included within the scope of protection of this invention.

[0090] The variants of the various antibodies described in this invention include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the various antibodies of this invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the various antibodies of this invention.

[0091] In some embodiments, the sequence of the variants described in this invention may 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% homology with its source sequence. The sequence homology described in this invention can be measured using sequence analysis software, such as the computer program BLAST with default parameters, especially BLASTP or TBLASTN. This invention also includes molecules having antibody heavy chain variable regions with CDRs, provided that their CDRs have at least 90% (preferably at least 95%, most preferably at least 98%) homology with the CDRs identified herein.

[0092] The antibodies of the present invention can be prepared using methods conventional in the art, such as hybridoma techniques well known in the art. The nanobodies and heavy chain antibodies of the present invention can be prepared using methods conventional in the art, such as phage display techniques well known in the art. Alternatively, the various antibodies of the present invention can be expressed in other cell lines. Suitable mammalian host cells can be transformed with sequences encoding the various antibodies of the present invention. Transformation can be performed using any known method, including, for example, packaging polynucleotides in a virus (or viral vector) and transducing host cells 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, 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 suitable as hosts for expression are well known in the art, including but 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, young hamster kidney (BHK) cells, monkey kidney cells (COS) cells, and human hepatocellular carcinoma cells (e.g., HepG2). Particularly preferred cell lines are selected by identifying which cell lines exhibit high expression levels and produce antibodies with essential GPC3 binding properties.

[0093] In this invention, "nucleic acid molecule" refers to a nucleotide chain of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate capable of being incorporated into the chain by DNA or RNA polymerase.

[0094] In this invention, the term "recombinant vector" refers to a construct capable of being delivered to a host cell and, in some embodiments, expressing one or more target genes or sequences. The term "recombinant expression vector" refers to a genetically modified oligonucleotide or polynucleotide construct that, when the construct contains a nucleotide sequence encoding mRNA, protein, polypeptide, or peptide, and the vector is contacted with a cell under conditions sufficient to allow the mRNA, protein, polypeptide, or peptide to be expressed in the cell, permits the expression of the mRNA, protein, polypeptide, or peptide by the host cell. The vectors of this invention are generally not naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of this invention can contain any type of nucleotide, including but not limited to DNA and RNA that can be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural, or modified nucleotides. Recombinant expression vectors can contain naturally occurring or non-naturally occurring nucleotide linkages, or both. In an exemplary aspect, modified nucleotides or non-naturally occurring nucleotide linkages do not impede transcription or replication of the vector.

[0095] The recombinant expression vector of the present invention can be any suitable recombinant expression vector capable of being used to transform or transfect one or more genes or sequences of interest into any suitable host cell and preferably to express the genes or sequences in the host cell. Suitable vectors include those designed for amplification and expansion or for expression or both of the above, and examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors associated with cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0096] In this invention, the term "host cell" refers to any type of cell that may contain the nucleic acids or vectors described herein. In exemplary aspects, the host cell is a eukaryotic cell, such as a plant, animal, fungus, or algae; or it may be a prokaryotic cell, such as bacteria or protozoa. Easily transformable bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; members of the Bacillaceae family, 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), NSO cells, and 293 cells.

[0097] In this invention, the 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 an exemplary aspect, the extracellular domain of the CAR contains an antigen recognition region, which may be a scFV of an antigen-specific antibody.

[0098] In this invention, the term "antibody conjugate" includes the aforementioned nanobodies or conjugated molecules and effector molecules. An effector molecule is a molecule capable of exhibiting the desired target activity; for example, the effector molecule is selected from radioisotopes, antitumor agents, immunomodulators, bioresponse 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 fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. The marker is any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, 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)), acridine esters, magnetic beads, calorimetric markers (e.g., colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads), and biotin for binding avidin (e.g., streptavidin) modified with the above markers. The markers covered in this invention can be detected by methods known in the art. For example, radioactive markers can be detected using photographic film or a scintillation calculator, and fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme markers are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the enzyme's action on the substrate, and calorimetric markers are detected by simple, visually appealing colored markers.

[0100] In some implementations, the markers described above can be connected to the antibodies of the present invention via connectors of different lengths to reduce potential steric hindrance.

[0101] In some implementations, the markers described above can be linked to the antibodies of the present invention using chelating agents such as NOA, DOTA, etc.

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

[0103] In some implementations, the aforementioned anti-Nectin-4 antibody contains Cys at the C-terminus or N-terminus.

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

[0105] In this invention, the term "antibody-drug conjugate" or "antibody conjugate" generally refers to an antibody linked to a biologically active cytotoxic drug, radionuclide, or tag via a stable linker unit. The "cytotoxic drug" may include toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, toxic drugs, chemotherapeutic drugs, antibiotics, or ribolysins, or derivatives thereof.

[0106] In this invention, the pharmaceutical composition may contain a suitable pharmaceutically acceptable carrier, such as pharmaceutical excipients, including buffers, as known in the art. "Pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextran and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. The pharmaceutical composition comprising the invention can be prepared by mixing the antibody of the invention, having 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 an aqueous solution. In some specific embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of the aforementioned nanobody or the aforementioned conjugated molecule per unit dose, or the amount of the aforementioned nanobody or the aforementioned conjugated molecule per unit dose of the pharmaceutical composition may be 0.1-2000 mg, and in some specific embodiments, 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, for example, is a bottle, syringe, or test tube. The container contains a composition effective in treating a condition. A label on or attached to the container indicates that the composition is intended to treat the selected condition. The composition contains the aforementioned anti-Nectin-4 antibody or antibody conjugate.

[0108] The pharmaceutical compositions of the present invention may also comprise more than one active ingredient required for a specific indication to be treated, preferably those active ingredients having 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 agents, small molecule drugs, or immunomodulators. The active ingredients are suitably combined in amounts effective for the intended use. Sustained-release formulations can be prepared, suitable examples of which include a semi-permeable matrix of a solid hydrophobic polymer containing the antibody of the present invention, said matrix being a shaped article, such as a film or microcapsule.

[0109] "Tumor" refers to all neoplasmic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," "proliferative disorder," and "tumor" are not mutually exclusive when used in this invention. In some embodiments, the aforementioned cancer is breast cancer or bladder cancer.

[0110] In this invention, the "non-diagnostic purpose" application scenarios include, but are not limited to: for example, detecting the presence of antigens (proteins containing the extracellular region of Nectin-4) in vitro in the laboratory; or using it 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 the antibodies, i.e., whether the antigen epitopes are the same or similar, etc.

[0111] In this invention, the term "effective amount" refers to the amount of a drug or agent that elicits a biological or pharmaceutical response in a tissue, system, animal, or human, as sought by, for example, an investigator or clinician. Furthermore, the term "effective amount" refers to the amount that causes improved treatment, cure, prevention, or reduction of disease, symptom, or side effects, or reduces the rate of progression of a disease or condition, compared to a corresponding subject who did not receive that amount. Within its scope, the term also includes amounts that effectively enhance normal physiological function.

[0112] In this invention, "subject" and "patient" refer to mammals, especially primates, and particularly humans.

[0113] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

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

[0115] In this embodiment, human Nectin-4 (Acro, NE4-H52H3) with a His tag was used as an immunogen for alpaca immunization. Peripheral blood was collected, PBMCs were isolated 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, single-clone identification, sequencing, and sequence analysis yielded multiple unique VHH sequences binding to the human Nectin-4 antigen. The sequences of NB656-M1-2, NB656-M1-4, and NB656-M1-260 are shown below.

[0117] NB656-M1-2 Variable Area

[0118] QVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMAWYRQAPGKQRELVASITSGGSTNYADSVKGRFAISRDNAKNTVYLQMNSLKPEDTAVYYCNADRLPDFMDYWGKGTLVTVSS (SEQ ID NO: 1)

[0119] NB656-M1-4 Variable Area

[0120] EVQLVESGGGLVQPGGSLSISCAASGSIFSINVMAWYRQAPGRQRELVASISRGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNTVSPGPAGWLDYWGQGTQVTVSS (SEQ ID NO: 2)

[0121] NB656-M1-260 Variable Area

[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 Sequence

[0125]

[0126] The general formula of the CDR for anti-Nectin-4 antibody is as follows: CDR1 is INVMA (SEQ ID NO: 4); CDR2 is SIX1X2GGSTNYADSVKG (SEQ ID NO: 17), where X1 is S or T and 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 ligated to the human IgG1 Fc (including the hinge region) fragment to construct the VHH-Fc antibody. The plasmid was constructed, transiently transfected into 293 cells, and the cells were cultured to express the antibody. The antibody was purified using a Protein A column, washed with 1×PBS buffer, and eluted with 0.1 M glycine buffer (pH 2.5). The antibody was then dialyzed into 1×PBS buffer (pH 7.4). The human IgG1 Fc (including the hinge region) sequence 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. Affinity determination of anti-Nectin-4 antibody with 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 Methods: 100 μL / well of buffer (50 mM NaHCO3, pH 9.6) containing 2 μg / mL Human Nectin-4 his (Acro, NE4-H52H3), Cyno Nectin-4 his (Acro, NE4-C52H4), and Mouse Nectin-4 his (Acro, NE4-M52H3) at 4°C was used to coat the antigen. After coating, the plate was washed three times with PBST solution. Blocking was performed with 5% milk at 37°C for 1 h. After washing once with PBST, the protein was serially diluted 1:5 from 100 nM (using 5% milk). 100 μL of the serially diluted antibody protein was added to each well of the ELISA plate and incubated at 37°C for 1 h. The isotype control antibody (Isotype) used was purchased from Syd Labs, catalog number PA007165.m2a. Wash five times with PBST, then add the corresponding amount of secondary antibody (Anti-Human IgG Fc, HRP, 1:10K) diluted with blocking buffer. Wash the ELISA plate incubated with the secondary antibody five times with PBST, add 100 μL of TMB single-component chromogenic solution to each well, and incubate at 37°C for 7 min. Stop the reaction by adding 50 μL / well of 1M HCl. OD 450 Readings, calculating EC 50 .

[0138] Experimental results: The results are shown in Table 2. The results show that the anti-Nectin-4 antibodies all have a strong binding force to the Nectin-4 antigen, and the binding force is stronger than that of 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, the biosensor was immersed in PBST buffer for equilibration for 15 minutes. Then, it was immersed in a sample solution containing a known concentration of antibody (2 μg / ml). Next, the sensor with the immobilized antigen was immersed in buffer for baseline equilibration. Then, the biosensor with the immobilized antigen at a known concentration (200 nM, 100 nM, 50 nM, or 25 nM) was immersed in a sample solution containing the antibody to be tested. Finally, the sensor bound to the antibody was immersed in buffer for dissociation. The kinetic constants of the sample could be obtained by real-time monitoring of the biofilm thickness of the biosensor during the experiment using an Octet instrument. Specific steps are as follows:

[0143] (1) Turn on the power and operating software of the Octet K2 instrument (Sartorius) to initialize the instrument. This process takes about 1 minute.

[0144] (2) Add 200 μL of PBST to the well plate to pre-wet the sensor (the sensor is ProA sensor-Sartorius) for 15 min and set aside.

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

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

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

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

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

[0150] Experimental results: The results are shown in Table 3, which show that the anti-Nectin-4 antibodies all have a strong binding affinity to the Nectin-4 antigen.

[0151] Table 3. Detection of anti-Nectin-4 antibody binding ability

[0152]

[0153] Example 3. In vitro cell binding assay of anti-Nectin-4 antibody

[0154] Experimental objective: To detect the binding ability of anti-Nectin-4 antibody using human breast cancer cells MCF-7 (Pronosa, CL-0149).

[0155] Experimental method: Wash cells twice with 1×PBS buffer, and resuspend cells in 1×PBS buffer to a cell concentration of 3×10⁻⁶. 5 Cells / μL, dispense 50μL of cells into each well of a PCR plate; dilute the antibody to be tested with 1×PBS buffer to a starting concentration of 400nM, add 50μL / well to the plate to make the starting antibody concentration 200nM, perform 7 serial dilutions at a 1:5 ratio, and incubate at 4°C for 1 hour; after incubation, wash the cells 3 times with 1×PBS buffer, add 100μL of fluorescent secondary antibody (647Anti-Human IgG Fc) to each well, and incubate at 4°C for 1 hour; after incubation, wash the cells once with 1×PBS buffer, transfer the cells to a 96-well cell plate, and analyze by flow cytometry.

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

[0157] Table 4. Binding of anti-Nectin-4 antibody to cell surface antigen Nectin-4 (EC) 50

[0158]

[0159] Example 4. Endocytotic activity of anti-Nectin-4 antibody

[0160] Experimental objective: To detect the endocytic activity of anti-Nectin-4 antibody using human breast cancer cells MCF-7.

[0161] Experimental method: Wash cells twice with 1×PBS buffer, and resuspend cells in 1×PBS buffer to a cell concentration of 3×10⁻⁶. 5Cells / μL, dispensed into PCR plates, 50μL per well; diluted the antibody to be tested to 4μg / mL with 1×PBS buffer, added 50μL / well to the plate to make the final antibody concentration 2μg / mL, with 5 wells for each antibody; antibody and cells were incubated at 4°C for 1 hour; after incubation, the cells were washed once with 1×PBS buffer, and resuspended in 200μL of 1×PBS buffer. The antibody and cells were transferred to 96-well cell plates at time gradients of 0h, 0.5h, 1h, 2h, and 4h, and incubated at 37°C; after incubation, the cells were washed once with 1×PBS buffer, and 100μL of fluorescent secondary antibody (647 Anti-Human IgGFc) was added to each well; incubated at 4°C for 1 hour; after incubation, the cells were washed once with 1×PBS buffer, and resuspended in 200μL of 1×PBS buffer. The cells were then transferred to 96-well cell plates and analyzed by flow cytometry.

[0162] Experimental results: The results are shown in Table 5. The anti-Nectin-4 antibody has endocytic activity.

[0163] Table 5. Endocytotic 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 linked to a His-Cys tag to construct the VHH-His-Cys antibody. The plasmid was constructed and transformed into *E. coli* BL21(DE3) strain. Single colonies were selected and activated overnight at 37°C in LB medium. The colonies were then transferred at a 1:100 ratio to 300 mL of LB medium and cultured. 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 h. The culture system was centrifuged at 6,000 rpm for 5 min to collect the cells. The collected cells were resuspended in 1×PBS buffer, and polymyxin was added to a final concentration of 0.5 mM. The cells were lysed at 37°C for 2 h, followed by centrifugation at 8,000 rpm for 30 min. The filtered supernatant was conjugated with Ni Sepharose (GE) for 2 h. 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 using ultrafiltration tubes, and the buffer was replaced with 0.1M NaHCO3 (pH 8.3). Protein purity was then confirmed to be >90% by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The sequences 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 conjugating an anti-Nectin-4 antibody with anthocyanin dye derivative IR808 (also known as MHI-808). The specific preparation steps are as follows: Take 1 mg of anti-Nectin-4 antibodies NB656-M1-2-His-Cys, NB656-M1-4-His-Cys, and NB656-M1-260-His-Cys, dilute to 1 mg / ml with buffer (20 mM Tris-HCl, pH 8.0), add 2.8 μL TCEP (100 mM) to prevent protein aggregation, add about 32 μL of MHI-808 (5 mg / ml) for every 1 mg of protein according to the molar ratio of MHI-808:antibody = 2:1, react at 4℃ for 30 min, and dialyze in 20 mM Tris-HCl, pH 8.0 buffer (dialysis bag 5KD), change the buffer every 3-4 hours, for a total of 5 times.

[0175] Anti-Nectin-4 antibody conjugates 2-IR808, 4-IR808, and 260-IR808 were obtained using the method described above.

[0176] Example 7. Targeting of the anti-Nectin-4 antibody conjugate

[0177] Experimental objective: To detect the tumor targeting and persistent tumor accumulation of anti-Nectin-4 antibody using bladder cancer cells T24 (Pronosai, CL-0277).

[0178] Experimental method: First, resuspended T24 cells (5×10⁻⁶) 6 (1 cell) subcutaneously injected into BALB / cNj-Foxn1 nu A T24 xenograft model was established in the right hind limb of female / Gpt mice (Jicui Yaokang, strain number: D000521). When the tumor volume reached approximately 200-400 mm², a xenograft model was established. 3Ten T24 xenograft mice were intravenously injected with the corresponding anti-Nectin-4 antibody conjugate (concentration of 1 mg / mL, 100 μL per mouse). In vivo fluorescence imaging of the mice was performed 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 had 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 is a heavy chain variable region, which includes CDR1, CDR2, and CDR3; wherein... The amino acid sequence of CDR1 is shown in SEQ ID NO: 4; the amino acid sequence of CDR2 is shown in SEQ ID NO: 5; and the amino acid sequence of CDR3 is shown in SEQ ID NO:

8. The amino acid sequence of CDR1 is shown in SEQ ID NO: 4; the amino acid sequence of CDR2 is shown in SEQ ID NO: 6; and the amino acid sequence of CDR3 is shown in SEQ ID NO: 9; or, The amino acid sequence of CDR1 is shown in SEQ ID NO: 4; the amino acid sequence of CDR2 is shown in SEQ ID NO: 7; and the amino acid sequence of CDR3 is shown in SEQ ID NO:

10.

2. The nanobody as described in claim 1, characterized in that, The frame region of the heavy chain variable region is an alpaca-derived frame region or a human-derived frame region.

3. The nanobody as described in claim 2, characterized in that, The amino acid sequence of the heavy chain variable region has at least 75% sequence identity with SEQ ID NO: 1, 2 or 3 and does not involve any change in the CDR sequence.

4. The nanobody as described in claim 2, characterized in that, The amino acid sequence of the heavy chain variable region has at least 80% sequence identity with SEQ ID NO: 1, 2 or 3 and does not involve any change in the CDR sequence.

5. The nanobody as described in claim 2, characterized in that, The amino acid sequence of the heavy chain variable region has at least 85% sequence identity with SEQ ID NO: 1, 2 or 3 and does not involve any change in the CDR sequence.

6. The nanobody as described in claim 2, characterized in that, The amino acid sequence of the heavy chain variable region has at least 90% sequence identity with SEQ ID NO: 1, 2 or 3 and does not involve any change in the CDR sequence.

7. The nanobody as described in claim 2, characterized in that, The amino acid sequence of the heavy chain variable region has at least 95% sequence identity with SEQ ID NO: 1, 2 or 3 and does not involve any change in the CDR sequence.

8. The nanobody as described in claim 2, characterized in that, The amino acid sequence of the heavy chain variable region has at least 96% sequence identity with SEQ ID NO: 1, 2 or 3 and does not involve any change in the CDR sequence.

9. The nanobody as described in claim 2, characterized in that, The amino acid sequence of the heavy chain variable region has at least 97% sequence identity with SEQ ID NO: 1, 2 or 3 and does not involve any change in the CDR sequence.

10. The nanobody according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region has at least 98% sequence identity with SEQ ID NO: 1, 2 or 3 and does not involve any change in the CDR sequence.

11. The nanobody as described in claim 2, characterized in that, The amino acid sequence of the heavy chain variable region has at least 99% sequence identity with SEQ ID NO: 1, 2 or 3 and does not involve any change in the CDR sequence.

12. The nanobody as described in claim 2, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1, 2 or 3.

13. A Nectin-4 binding molecule, characterized in that, The binding molecule comprises a nanobody as described in any one of claims 1-12, wherein the binding molecule is a heavy chain antibody.

14. The binding molecule as described in claim 13, characterized in that, The Fc of the heavy chain antibody is IgG1 Fc, and / or the Fc is derived from mouse or human.

15. The binding molecule as described in claim 14, characterized in that, The Fc is human IgG1 Fc.

16. The binding molecule as described in claim 13, characterized in that, The amino acid sequence of the Fc of the heavy chain antibody has at least 85% sequence identity with SEQ ID NO: 11 and maintains the function of the Fc.

17. The binding molecule as claimed in claim 13, characterized in that, The amino acid sequence of the Fc of the heavy chain antibody has at least 90% sequence identity with SEQ ID NO: 11 and maintains the function of the Fc.

18. The binding molecule as claimed in claim 13, characterized in that, The amino acid sequence of the Fc of the heavy chain antibody has at least 95% sequence identity with SEQ ID NO: 11 and maintains the function of the Fc.

19. The binding molecule as claimed in claim 13, characterized in that, The amino acid sequence of the Fc of the heavy chain antibody has at least 96% sequence identity with SEQ ID NO: 11 and maintains the function of the Fc.

20. The binding molecule as claimed in claim 13, characterized in that, The amino acid sequence of the Fc of the heavy chain antibody has at least 97% sequence identity with SEQ ID NO: 11 and maintains the function of the Fc.

21. The binding molecule as claimed in claim 13, characterized in that, The amino acid sequence of the Fc of the heavy chain antibody has at least 98% sequence identity with SEQ ID NO: 11 and maintains the function of the Fc.

22. The binding molecule as described in claim 13, characterized in that, The amino acid sequence of the Fc of the heavy chain antibody has at least 99% sequence identity with SEQ ID NO: 11 and maintains the function of the Fc.

23. The binding molecule as described in claim 13, characterized in that, The amino acid sequence of the Fc of the heavy chain antibody is shown in SEQ ID NO:

11.

24. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises a nanobody as described in any one of claims 1-12.

25. A genetically modified cell, characterized in that, The genetically modified cells contain the chimeric antigen receptor as described in claim 24.

26. The gene-modified cell as described in claim 25, characterized in that, The genetically modified cells are eukaryotic cells.

27. The gene-modified cell as described in claim 26, characterized in that, The eukaryotic cells are isolated human cells.

28. The gene-modified cell as described in claim 26, characterized in that, The genetically modified cells are immune cells.

29. The gene-modified cell as described in claim 28, characterized in that, The immune cells are T cells or NK cells.

30. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes a nanobody as described in any one of claims 1-12, a binding molecule as described in any one of claims 13-23, or a chimeric antigen receptor as described in claim 24.

31. A recombinant vector, characterized in that, The recombinant vector comprises the nucleic acid molecule as described in claim 30.

32. The recombinant vector as described in claim 31, characterized in that, The recombinant vector is a recombinant expression vector or a recombinant replication vector.

33. The recombinant vector as described in claim 32, characterized in that, The recombinant vector is a plasmid, bacteriophage, or viral vector.

34. The recombinant vector as described in claim 33, characterized in that, The viral vector is a retroviral vector, an adenovirus vector, or an adeno-associated virus vector.

35. The recombinant vector as described in claim 34, characterized in that, The retroviral vector is a lentiviral vector.

36. The recombinant vector as described in claim 32, characterized in that, The recombinant carrier is a clay particle.

37. A transformant, characterized in that, The transformant comprises the nucleic acid molecule as described in claim 30 or the recombinant vector as described in any one of claims 31-36; or expresses the nanobody as described in any one of claims 1-12, the binding molecule as described in any one of claims 13-23, or the chimeric antigen receptor as described in claim 24.

38. The transformant as described in claim 37, characterized in that, The host cells used in the construction of the transformant are selected from Escherichia coli cells, insect cells, yeast cells, and mammalian cells.

39. The transformant as described in claim 38, characterized in that, The host cell was 293 cells.

40. A method for preparing an antibody targeting Nectin-4, characterized in that, The method includes: The antibody is obtained from the culture by culturing the transformant as described in any one of claims 37-39.

41. An antibody conjugate, characterized in that, The antibody conjugate includes a nanobody as described in any one of claims 1-12 or a binding molecule as described in any one of claims 13-23, and an effector molecule; the effector molecule is a marker.

42. The antibody conjugate according to claim 41, characterized in that, The markers can be detected by electrical, optical, or chemical means.

43. The antibody conjugate according to claim 41, characterized in that, The marker can be detected by fluorescence.

44. The antibody conjugate as described in claim 41, characterized in that, The markers can be detected by photochemical methods.

45. The antibody conjugate as described in claim 41, characterized in that, The markers can be detected by spectroscopic, biochemical, or immunological methods.

46. ​​The antibody conjugate as described in claim 41, characterized in that, The markers are selected from enzymes, radionuclides, fluorescent dyes, acridine esters, magnetic beads, calorimetric markers, and biotin used to bind avidin modified with the above markers.

47. The antibody conjugate as described in claim 46, characterized in that, The enzyme is selected from horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase; and / or, The radionuclides are selected from 3 H, 125 I, 35 S, 14 C 32 P; and / or, The fluorescent dye is selected from fluorescein isothiocyanate, fluorescein, tetramethylrhodamine isothiocyanate, phycoerythrin, Texas Red, rhodamine, quantum dots, and cyanine dye derivatives; and / or, The calorimetric marker is selected from colloidal gold, colored glass, plastic beads, latex beads; and / or, The avidin is streptavidin; The cyanine dye derivative is either IR808 or Alexa 750.

48. The antibody conjugate as described in claim 47, characterized in that, The plastic beads are polystyrene beads or polypropylene beads.

49. The antibody conjugate according to any one of claims 41-48, characterized in that, The C-terminus of the nanobody is attached with a His-Cys tag.

50. The antibody conjugate as described in claim 49, characterized in that, The amino acid sequence of a nanobody with a His-Cys tag attached to its C-terminus is shown in SEQ ID NO: 14, 15 or 16.

51. The antibody conjugate as described in claim 50, characterized in that, The antibody conjugate is an antibody probe, and the label is IR808.

52. The antibody conjugate as described in claim 51, characterized in that, The antibody probe was obtained by linking the nanobody with a His-Cys tag to IR808 at its C-terminus.

53. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a nanobody as described in any one of claims 1-12, a conjugating molecule as described in any one of claims 13-23, or an antibody conjugate as described in any one of claims 41-52, and a pharmaceutically acceptable excipient.

54. A reagent for detecting Nectin-4, characterized in that, The detection reagent comprises a nanobody as described in any one of claims 1-12, a binding molecule as described in any one of claims 13-23, or an antibody conjugate as described in any one of claims 41-52.

55. The detection reagent as described in claim 54, characterized in that, The detection reagents are used in in vivo imaging techniques, Western blot, enzyme-linked immunosorbent assay (ELISA), and / or flow cytometry.

56. A kit for detecting Nectin-4 and / or diagnosing Nectin-4 expression-related cancers, characterized in that, The kit comprises a nanobody as described in any one of claims 1-12, a binding molecule as described in any one of claims 13-23, an antibody conjugate as described in any one of claims 41-52, or a detection reagent as described in claim 54 or 55, wherein the cancer is selected from one or more of breast cancer, lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, and ovarian cancer.

57. The kit as described in claim 56, characterized in that, The kit also includes other reagents for detecting Nectin-4 using in vivo imaging technology, Western blot, enzyme-linked immunosorbent assay (ELISA), and / or flow cytometry.

58. The kit as described in claim 57, characterized in that, The other reagents are fluorescently labeled secondary antibodies.

59. The use of the nanobody as described in any one of claims 1-12, the binding molecule as described in any one of claims 13-23, the nucleic acid molecule as described in claim 30, the recombinant vector as described in any one of claims 31-36, the transformant as described in any one of claims 37-39, the antibody conjugate as described in any one of claims 41-52, the pharmaceutical composition as described in claim 53, or the detection reagent as described in claim 54 or 55 in the preparation of a product for diagnosing cancer; wherein the cancer is a Nectin-4 expression-related cancer; and wherein, The cancer is selected from one or more of breast cancer, lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, and ovarian cancer.

60. A method for detecting Nectin-4 for non-diagnostic purposes, characterized in that, The method includes contacting a sample to be tested with a nanobody as described in any one of claims 1-12, a binding molecule as described in any one of claims 13-23, an antibody conjugate as described in any one of claims 41-52, a detection reagent as described in claim 50, or a kit as described in any one of claims 56-58, to detect the expression level of Nectin-4 in the sample to be tested.

61. The method as described in claim 60, characterized in that, The sample to be tested is a cell or an animal; and / or, The method employs in vivo imaging technology, Western blot, enzyme-linked immunosorbent assay (ELISA), and / or flow cytometry to detect Nectin-4.

Images