Nanometer antibody targeting Nectin-4 and application thereof

Abstract

The invention discloses a nano antibody targeting to Nectin-4 and an application of the nano antibody. The nano antibody comprises a heavy chain variable region, the heavy chain variable region comprises CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 is SIAMA, the amino acid sequence of the CDR2 is SISGGGSTNYADSVKG, and the amino acid sequence of the CDR3 is DLDYGLGSGEENDY. The Nectin-4-targeted nano antibody disclosed by the invention has relatively strong binding force with a Nectin-4 antigen, and has the advantages of strong tumor targeting property, fast tumor enrichment and long retention.

Description

Technical Field

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

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

[0003] Nectin-4 antibody research is of great significance for improving cancer treatment effects, reducing treatment side effects, and promoting the development of personalized medicine and multimodal treatment strategies. Summary of the Invention

[0004] The present invention provides a nano-antibody targeting Nectin-4, which has a strong binding force with the Nectin-4 antigen and has the advantages of strong tumor targeting, rapid tumor enrichment and long-term retention.

[0005] Nanobodies, binding molecules, and antibody conjugates targeting nectin-4

[0006] On one hand, the present invention provides a nanobody targeting nectin-4, which comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is SIAMA (SEQ ID NO: 1), the amino acid sequence of CDR2 is SISGGGSTNYADSVKG (SEQ ID NO: 2), and the amino acid sequence of CDR3 is DLDYGLGSGENDY (SEQ ID NO: 3).

[0007] In some embodiments, the Nanobody comprises a heavy chain variable region comprising CDR1, CDR2 and CDR3 in the amino acid sequence shown in SEQ ID NO: 1;

[0008] The CDR1, CDR2 and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems.

[0009] The Nanobodies provided herein comprise any one or any combination of the above-mentioned CDR1, CDR2 and CDR3.

[0010] In some embodiments, in the aforementioned Nanobodies, the CDR1, CDR2 and CDR3 of the immunoglobulin single variable domain are defined according to the Kabat numbering system.

[0011] In some embodiments, the immunoglobulin single variable domain further comprises a framework region, preferably an alpaca-derived framework region or a human-derived framework region.

[0012] In some embodiments, the amino acid sequence of the heavy chain variable region in the aforementioned Nanobody is as shown in SEQ ID NO: 1, or has at least 80%, at least 85%, or at least 90% sequence identity.

[0013] In some embodiments, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 1, or has a sequence identity of at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% to SEQ ID NO: 1 and does not involve changes in the CDR sequences.

[0014] On the other hand, the present invention provides a binding molecule targeting nectin-4, wherein the binding molecule comprises the nanobody provided by the present invention, and the binding molecule is an Fc fusion antibody, a heavy chain antibody, a monoclonal VHH antibody, a bispecific antibody, a multispecific antibody or a VHH polymer.

[0015] In some embodiments of the invention, the binding molecule is a multivalent Nanobody comprising a plurality of said Nanobodies.

[0016] In some embodiments of the invention, the binding molecule is a heavy chain antibody.

[0017] In some embodiments, at least one heavy chain variable region in the aforementioned binding molecules is a VHH. In some embodiments, the binding molecules comprise one or more (e.g., 2, 3, 4, 5, 6) of the aforementioned heavy chain variable regions, which may be the same or different and may form VHH aggregates, such as dimers or multimers.

[0018] In some embodiments, the aforementioned binding molecules comprise an immunoglobulin Fc region.

[0019] In some embodiments, the immunoglobulin Fc region is derived from human or mouse, and / or the immunoglobulin Fc region is IgG Fc.

[0020] In some embodiments, the binding molecule further comprises a human immunoglobulin Fc region, for example, the Fc region is a human IgG1, IgG2 or IgG4 Fc region.

[0021] In some specific embodiments, the human immunoglobulin Fc region is the Fc region of wild-type IgG or a variant thereof.

[0022] In some embodiments of the present invention, the immunoglobulin Fc is IgG Fc, preferably derived from mouse or human.

[0023] In some embodiments, the Fc region is an Fc region that increases effector function, e.g., increases antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC) with increased effector function.

[0024] 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 / 296A 8D;280H / 290S / 298V;243L / 292P / 300L;243L / 292P / 300L / 396L;243L / 292P / 300L / 305I / 396L;236A / 239D / 332E;326A / 333A;326W / 333S;290E / 298G / 299A;290N / 298G / 299A;290E / 298G / 299A / 326E;or 290N / 298G / 299A / 326E;or any combination of any of the above positions. Said mutations are defined according to the EU numbering system.

[0025] 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.

[0026] In some embodiments, the Fc region contained in the aforementioned binding molecules can enable the binding protein to form a dimeric molecule and prolong the in vivo half-life of the binding protein.

[0027] In some embodiments, the heavy chain variable region and the Fc region of the aforementioned binding molecules are connected by a linker. The linker can be a non-functional amino acid sequence of 1-20 or more amino acids in length, free of secondary or higher structure. For example, the linker can be a flexible linker, such as G4S, GS, or GAP.

[0028] In some embodiments, the Fc region is linked behind the C-terminus of the Nanobody.

[0029] In some embodiments, the amino acid sequence of the immunoglobulin Fc is as shown in SEQ ID NO: 7, or has a sequence identity of at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% to SEQ ID NO: 7 and maintains the function of the immunoglobulin Fc region.

[0030] In some embodiments, the VHH antibody is directly linked to the Fc region, constituting a VHH-Fc antibody.

[0031] Another aspect of the present invention provides an antibody conjugate, which comprises the Nanobody provided by the present invention or the binding molecule provided by the present invention.

[0032] In some embodiments, the antibody conjugate comprises one or more selected from the group consisting of a His tag, a Cys, a cytokine, a lectin, and an enzyme.

[0033] In some embodiments, the antibody conjugate comprises the His tag sequence, which is a short sequence comprising consecutive histidine residues, such as 6 His, 8 His, and 10 His.

[0034] In some embodiments, the aforementioned antibody conjugate comprises a Cys at the C-terminus or the N-terminus.

[0035] In some embodiments, the antibody conjugate is a VHH-His-Cys antibody constructed by linking a Nanobody to a His-Cys tag.

[0036] In some specific embodiments, the aforementioned antibody conjugate comprises the amino acid sequence shown as HHHHHHC (SEQ ID NO: 9).

[0037] In some embodiments, the antibody conjugate has the amino acid sequence shown in SEQ ID NO: 8.

[0038] In some embodiments, the KD value of the aforementioned nanobodies, binding molecules or antibody conjugates herein for binding to nectin-4 may be ≤1×10 -7 M.

[0039] In some embodiments, the aforementioned nanobodies, binding molecules or antibody conjugates herein bind to tumor cells, such as the detection method described in Example 3.

[0040] In some embodiments, the aforementioned nanobodies, binding molecules or antibody conjugates herein have endocytic activity, such as the detection method in Example 4.

[0041] In some embodiments, the aforementioned Nanobodies, binding molecules or antibody conjugates herein are capable of inhibiting 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%.

[0042] In some embodiments, the Nanobodies herein comprise one or more amino acid substitutions, such as conservative amino acid substitutions, compared to SEQ ID NO: 1, e.g., comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions, which substitutions may occur in the CDR regions and / or in the FR regions.

[0043] In some embodiments, binding molecules or conjugates are provided that bind to or compete for binding to the same epitope as the heavy chain variable region in the Nanobodies herein described above.

[0044] In some embodiments, a binding molecule or antibody conjugate is provided that blocks the binding of the heavy chain variable region of the aforementioned Nanobodies herein to Nectin-4 (eg, human Nectin-4).

[0045] As previously stated, and herein, "at least 90% identity" encompasses at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity.

[0046] In some embodiments, the antibody conjugate comprises a Nanobody or binding molecule provided herein, and an effector molecule. The effector molecule is a molecule that exhibits a desired target activity, exemplified by the effector molecule being selected from radioisotopes, antitumor agents, immunomodulators, biological response modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.

[0047] In some embodiments, the effector molecule is a marker. By way of example, the marker can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. Such markers can be suitable for use in immunological assays (e.g., enzyme-linked immunosorbent assays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., IR808, Alexa 750)), acridinium ester compounds, magnetic beads, calorimetric labels such as 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-mentioned labels. The labels encompassed herein can be detected by methods known in the art. For example, radiolabels 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 a colored label.

[0048] In some embodiments, labels as described above may be attached to the Nanobodies or binding molecules herein via linkers of varying lengths to reduce potential steric hindrance.

[0049] In some embodiments, labels as described above may be linked to the Nanobodies or binding molecules herein via chelating agents, such as NOTA, DOTA and the like.

[0050] In some embodiments, the effector molecule is a cytotoxic drug.

[0051] In some embodiments, the antibody conjugate further comprises one or more of a radioisotope, an anti-tumor agent, an immunomodulatory cytotoxic drug, a chromophore, a fluorophore, a chemiluminescent compound, and a metal ion.

[0052] In some embodiments, the antibody conjugate is a conjugate of the Nanobody or the binding molecule coupled to IR808.

[0053] The C-terminus of the heavy chain variable region is connected to a His-Cys tag; and / or the antibody conjugate is an antibody probe, and the label of the antibody probe is IR808.

[0054] Nucleic acids and vectors

[0055] Another aspect of the present invention provides an isolated nucleic acid encoding a Nanobody as described above or a binding molecule as provided herein. The nucleic acid of the invention herein may be RNA, DNA or cDNA. According to some embodiments herein, the nucleic acid herein is an isolated nucleic acid.

[0056] The nucleic acid herein may also be in the form of a vector, may be present in a vector and / or may be part of a vector, such as a plasmid, cosmid, YAC or viral vector. The vector may in particular be an expression vector, i.e. a vector that can provide for the expression of a Nanobody or binding molecule in vitro and / or in vivo (i.e. in a suitable host cell, host organism and / or expression system).

[0057] Another aspect of the present invention provides a recombinant expression vector comprising the isolated nucleic acid provided by the present invention.

[0058] In some embodiments, the recombinant expression vector is a plasmid, phage or viral vector.

[0059] In some embodiments, the viral vector is a retroviral vector, an adenoviral vector, or an adeno-associated viral vector, for example a lentiviral vector.

[0060] The vectors herein are generally not naturally occurring. However, portions of the vectors may be naturally occurring. The recombinant expression vectors of the present invention may comprise any type of nucleotides, including but not limited to DNA and RNA: they may be single-stranded or double-stranded, synthetic or partially derived from natural sources, and they may contain natural, non-natural, or altered nucleotides. 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.

[0061] The expression vector generally comprises at least one nucleic acid as herein described, operably linked to one or more suitable expression control elements (e.g. promoters, enhancers, terminators, etc.). The selection of said elements and their sequences for expression in a particular host is within the skill of the art. Regulatory and other elements useful or necessary for the expression of the Nanobodies or binding molecules herein are, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.

[0062] The nucleic acids herein can be prepared or obtained by known means (eg, by automated DNA synthesis and / or recombinant DNA technology) based on the information of the amino acid sequences of the polypeptides herein, and / or can be isolated from suitable natural sources.

[0063] transformants

[0064] Another aspect of the present invention provides a transformant, which comprises the isolated nucleic acid provided by the present invention or the recombinant expression vector provided by the present invention.

[0065] Provided herein are recombinant host cells that express or are capable of expressing one or more Nanobodies or binding molecules herein and / or contain a nucleic acid or vector herein.

[0066] In some embodiments, the host cell of the transformant is a bacterial cell, a fungal cell, or a mammalian cell.

[0067] Examples of bacterial cells include Gram-negative bacterial strains such as Escherichia coli ( Escherichia coli ) strains, Proteus spp. ( Proteus ) strains and Pseudomonas spp. ( Pseudomonas ) strains) and Gram-positive bacterial strains (e.g. Bacillus spp. ( Bacillus ) strains, Streptomyces spp. ( Streptomyces ) strains, Staphylococcus spp. ( Staphylococcus ) strains and Lactococcus spp. ( Lactococcus) strains) cells.

[0068] Examples of fungal cells include Trichoderma ( Trichoderma ), Neurospora ( Neurospora ) and Aspergillus spp. ( Aspergillus ) or cells of species of the genus Saccharomyces ( Saccharomyces ) (e.g., Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) ), Schizosaccharomyces spp. ( Schizosaccharomyces ) (e.g., Schizosaccharomyces pombe ( Schizosaccharomyces pombe ) 、Pichia ( Pichia ) (e.g. Pichia pastoris ( Pichia pastoris ) and Pichia methanolica ( Pichia methanolica )) and Hansenula ( Hansenula ) of the species.

[0069] Examples of mammalian cells include HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0070] However, amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins may also be used herein.

[0071] The expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene editing (CRISPR-Cas system, ZFN system or TALEN system), transposon (Sleeping Beauty or PiggyBAC), gene gun, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, the cells are cultivated in culture medium and screened for suitable activity. The methods and conditions for culturing the transfected cells produced and for recovering the produced antibody molecules are known to those skilled in the art and can be varied or optimized according to the specific expression vector and host cell used based on this specification and methods known in the prior art. In addition, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. Markers can, for example, provide prototrophy, biocide resistance (e.g., antibiotics) or heavy metal (e.g., copper) resistance to auxotrophic hosts. Selectable marker genes can be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include splicing signals, as well as transcriptional promoters, enhancers, and termination signals.

[0072] Another aspect of the present invention provides a chimeric antigen receptor, which comprises the Nanobody provided by the present invention or the binding molecule provided by the present invention.

[0073] Another aspect of the present invention provides a genetically modified cell, wherein the cell comprises the chimeric antigen receptor provided by the present invention.

[0074] In some embodiments of the present invention, the genetically modified cell is a eukaryotic cell, preferably an isolated human cell.

[0075] In some embodiments of the present invention, the genetically modified cells are immune cells, such as T cells or NK cells.

[0076] Preparation method

[0077] On the other hand, the invention provides a method for preparing a nano antibody or binding molecule targeting Nectin-4, the method comprising: culturing the transformant provided by the present invention, and obtaining the nano antibody or binding molecule from the culture. Specifically, the target protein is expressed in the host cell as described above, and the target protein is separated from the host cell. Optionally, a purification step may be included, for example, purification using an A or G Sepharose FF column containing an adjusted buffer, washing away non-specifically bound components, and then eluting the bound antibodies using a pH gradient method, detecting with SDS-PAGE, and collecting. Optionally, conventional methods are used for filtration and concentration. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The obtained product needs to be immediately frozen, such as at -70°C, or freeze-dried.

[0078] The engineered nanobodies or binding molecules herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. Mammalian expression systems result in glycosylation of antibodies, especially at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are expanded and cultured in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes antibodies can be purified and collected using conventional techniques. Antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange.

[0079] Provided herein is a method for preparing an antibody conjugate, comprising linking the aforementioned nanobody or binding molecule and an effector molecule.

[0080] Composition

[0081] Another aspect of the present invention provides a pharmaceutical composition, comprising one or more of the nanoantibodies, binding molecules, nucleic acids, recombinant expression vectors, transformants, antibody conjugates and chimeric antigen receptors provided by the present invention; and pharmaceutically acceptable excipients.

[0082] In some embodiments, the pharmaceutical composition contains one or more of the Nanobodies, the binding molecules, the nucleic acids, the recombinant expression vectors, the transformants, the antibody conjugates and the chimeric antigen receptors as described above in an effective amount for treating, alleviating or preventing cancer.

[0083] In some embodiments, the pharmaceutically acceptable excipient is a pharmaceutically acceptable excipient, diluent, or carrier.

[0084] In some embodiments, a pharmaceutical composition is provided, which comprises the Nanobody, the binding molecule, the antibody conjugate or the chimeric antigen receptor as described above for diagnosing cancer and at least one pharmaceutically acceptable excipient, diluent or carrier.

[0085] In some embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of the Nanobody, the binding molecule, the antibody conjugate or the chimeric antigen receptor in a unit dose, or the amount of the Nanobody, the binding molecule, the antibody conjugate or the chimeric antigen receptor in a unit dose of the pharmaceutical composition is 0.1-2000 mg, and in some embodiments, 1-1000 mg.

[0086] In some embodiments, an article or product is provided comprising a Nanobody, a binding molecule, an antibody conjugate, or a chimeric antigen receptor. Optionally, the article comprises a container and a label. The container is, for example, a bottle, a syringe, or a test tube. The container holds a composition effective for treating a condition. The label on or associated with the container indicates that the composition is used to treat the selected condition. The composition contains the aforementioned Nanobody, a binding molecule, an antibody conjugate, or a chimeric antigen receptor.

[0087] In some embodiments, a pharmaceutical composition is provided, comprising the Nanobodies, binding molecules, antibody conjugates or chimeric antigen receptors described herein. The Nanobodies, binding molecules, antibody conjugates or chimeric antigen receptors may be in an effective amount for treating or alleviating a disease (e.g., cancer) or for diagnosing a disease (e.g., cancer), and the pharmaceutical composition may further comprise at least one pharmaceutically acceptable excipient, diluent or carrier.

[0088] use

[0089] Another aspect of the present invention provides the use of one or more of the nanobodies, binding molecules, nucleic acids, recombinant expression vectors, transformants, antibody conjugates, chimeric antigen receptors and pharmaceutical compositions provided by the present invention in the preparation of reagents or kits.

[0090] In some embodiments, the reagent or kit is used to detect Nectin-4 expression.

[0091] In some embodiments, the reagent or kit is used for cancer diagnosis.

[0092] In some embodiments, the reagents or kits are used in drug development.

[0093] In some embodiments of the present invention, the reagents or kits are used for in vivo imaging techniques, Western blot, enzyme-linked immunosorbent assay and / or flow cytometry.

[0094] Another aspect of the present invention provides the use of one or more of the nanobodies, binding molecules, nucleic acids, recombinant expression vectors, transformants, antibody conjugates, chimeric antigen receptors and pharmaceutical compositions provided herein in the preparation of a medicament for preventing and / or treating cancer; the cancer is a cancer associated with Nectin-4 expression.

[0095] Provided herein are methods for treating, alleviating, preventing, or diagnosing a disease or condition using the aforementioned Nanobodies, binding molecules, nucleic acids, recombinant expression vectors, transformants, antibody conjugates, chimeric antigen receptors, or pharmaceutical compositions.

[0096] In some embodiments, the aforementioned disease associated with nectin-4 expression is a proliferative disorder or any other disease or disorder characterized by uncontrolled cell growth (such as cancer, herein, cancer and tumor are used interchangeably), for example, a disease associated with nectin-4 expression or abnormal nectin-4 expression (such as cancer).

[0097] In some embodiments, the cancer is selected from one or more of breast cancer, lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, and ovarian cancer.

[0098] In some embodiments, the aforementioned cancer is breast cancer or bladder cancer.

[0099] Detection

[0100] Reagents or kits

[0101] On the other hand, the present invention provides a reagent or kit for detecting Nectin-4 and / or diagnosing cancers related to Nectin-4 expression, wherein the reagent or kit comprises one or more of the nanoantibodies, binding molecules, nucleic acids, recombinant expression vectors, transformants, antibody conjugates, chimeric antigen receptors and pharmaceutical compositions provided by the present invention.

[0102] In some embodiments, a kit is also provided, comprising the aforementioned Nanobodies, the binding molecules, the nucleic acids, the recombinant expression vectors, the transformants, the antibody conjugates, the chimeric antigen receptors, or the pharmaceutical compositions, and further comprising instructions for diagnostic use. The kit may also contain at least one additional reagent, such as a marker or an additional diagnostic agent. For in vivo use, the antibody or antibody conjugate may be formulated as a pharmaceutical composition.

[0103] Use of the Nanobodies, binding molecules, nucleic acids, recombinant expression vectors, transformants, antibody conjugates, chimeric antigen receptors, or pharmaceutical compositions provided herein for detection. Also provided herein are methods, systems, or devices for in vivo or in vitro detection of Nectin-4, comprising treating a sample with the aforementioned Nanobodies, binding molecules, nucleic acids, recombinant expression vectors, transformants, antibody conjugates, chimeric antigen receptors, or pharmaceutical compositions described herein.

[0104] Another aspect of the present invention provides a method for detecting nectin-4 expression for non-diagnostic purposes, the method comprising contacting a sample to be tested with one or more of the nanoantibodies, binding molecules, nucleic acids, recombinant expression vectors, transformants, antibody conjugates, chimeric antigen receptors, pharmaceutical compositions, and reagents or kits provided by the present invention to detect the expression level of nectin-4 in the sample to be tested.

[0105] In some embodiments of the present invention, the test sample is a cell or an animal.

[0106] 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.

[0107] In some embodiments, it can be used to detect the expression of Nectin-4 protein in tissue samples. In research and application scenarios for non-diagnostic and therapeutic purposes, the detection of Nectin-4 expression plays a key role. In basic research, scientists have deeply explored its role in cell adhesion, signal transduction and intercellular communication by detecting the expression pattern and function of Nectin-4 in different cell types, tissues or organs. In the field of drug development, the detection of Nectin-4 expression not only helps to determine the target of drug action, but is also used to evaluate the effect of drug candidates on Nectin-4 expression to measure its potential therapeutic effect. In disease model research, by changing the expression level of Nectin-4 in animal models, researchers can observe changes in the disease process and then understand the role of Nectin-4 in the occurrence and development of the disease. At the same time, the exploration of Nectin-4 as a potential biomarker, although not for diagnosis, helps researchers to more comprehensively grasp the biological characteristics of the disease. In terms of therapeutic target validation and cell therapy development, Nectin-4 expression detection provides important basis for preclinical research and clinical trials. For example, in CAR-T cell therapy, Nectin-4 expression detection is used to determine whether it is suitable as a target for tumor cells.

[0108] In some embodiments, the method comprises the following steps:

[0109] (1) contacting the sample with the reagent or kit;

[0110] (2) Complexes formed between the detection reagent and the sample;

[0111] (3) Detecting the complex.

[0112] In some embodiments, the method further comprises contacting a reference sample (e.g., a control sample) with the reagent. The extent of complex formation is determined by comparison with the reference sample. A change (e.g., a statistically significant change) in complex formation in the sample or subject compared to the control sample or subject indicates the presence of nectin-4 in the sample.

[0113] Another aspect of the present invention provides a system for detecting nectin-4 expression, the system comprising:

[0114] One or more of the nanoantibodies, binding molecules, nucleic acids, recombinant expression vectors, transformants, antibody conjugates, chimeric antigen receptors, pharmaceutical compositions, and reagents or kits provided by the present invention; for specifically binding to Nectin-4 protein;

[0115] A sample processing device, used for receiving and processing a sample to be tested, wherein the sample contains or may contain Nectin-4 protein;

[0116] a detection platform selected from one of an immunohistochemistry platform, an immunofluorescence platform, an enzyme-linked immunosorbent assay platform, a Western blot platform, and a flow cytometry platform;

[0117] A signal detection and quantification device for detecting the signal generated after the nanobody, binding molecule or antibody conjugate binds to the Nectin-4 protein in the sample, and performing quantitative analysis on the signal;

[0118] Data analysis and processing software for analyzing data output by the signal detection and quantification device to determine the expression level of Nectin-4 protein in the sample; and,

[0119] Controls and comparison kits, including positive and negative controls, are used to verify the accuracy and specificity of the assay system.

[0120] In some embodiments, the sample processing device includes one or more of the steps of fixation, permeabilization, sectioning, or cell lysis.

[0121] In some embodiments, the signal detection and quantification device comprises an enzyme substrate reaction detector, a fluorescence detector, a radioactivity counter, or an optical density scanner.

[0122] In some embodiments, the data analysis and processing software is capable of providing a visual representation of the expression level of the Nectin-4 protein.

[0123] Another aspect of the present invention provides a detection device for detecting Nectin-4 protein expression, the detection device comprising:

[0124] A nanobody, binding molecule or antibody conjugate immobilization module, used to immobilize the nanobody, binding molecule or antibody conjugate so as to specifically bind to the Nectin-4 protein;

[0125] A sample processing module is used to receive biological samples and perform pre-processing steps such as fixation, permeabilization, sectioning or cell lysis;

[0126] a detection module for binding the treated sample to the nanobody, binding molecule or antibody conjugate and detecting the binding event, wherein the detection module is selected from one of an immunohistochemistry detection module, an immunofluorescence detection module, an enzyme-linked immunosorbent assay detection module, a Western blot detection module and a flow cytometry detection module;

[0127] a signal detection and quantification unit, coupled to the detection module, for detecting the signal generated by the binding event and providing quantitative analysis of the signal;

[0128] a data analysis and processing unit, coupled to the signal detection and quantification unit, for receiving and analyzing signal data to determine the expression level of the Nectin-4 protein;

[0129] The control and comparison unit is used to provide positive and negative controls to verify the accuracy and specificity of the detection device.

[0130] In some embodiments, the Nanobody, binding molecule or antibody conjugate immobilization module comprises one or more microarrays of immobilized antibodies.

[0131] In some embodiments, the signal detection and quantification unit comprises at least one detector selected from the group consisting of an enzyme substrate reaction detector, a fluorescence detector, a radioactivity counter, and an optical density scanner.

[0132] In some embodiments, the data analysis and processing unit includes a user interface for displaying the visualization results of the Nectin-4 protein expression level.

[0133] Another aspect of the present invention provides a method for diagnosing, preventing, ameliorating or treating a disease, disorder or condition associated with nectin-4 expression, the method comprising administering to a subject in need thereof an effective amount of one or more of the nanobodies, binding molecules, nucleic acids, recombinant expression vectors, transformants, antibody conjugates, chimeric antigen receptors and pharmaceutical compositions provided herein.

[0134] In some embodiments, the disease, disorder, or condition associated with nectin-4 expression is selected from one or both of breast cancer and bladder cancer.

[0135] As used herein, the term "effective amount" refers to an amount of a drug or pharmaceutical agent that elicits the biological or pharmaceutical response of a tissue, system, animal, or human that is being sought, for example, by a researcher or clinician. Additionally, the term "therapeutically effective amount" refers to an amount that results in improved treatment, cure, prevention, or alleviation of a disease, condition, or side effect, or that reduces the rate of progression of a disease or condition, compared to a corresponding subject that has not received that amount. The term also includes within its scope amounts that are effective to enhance normal physiological function.

[0136] Drug combinations containing nanobodies, binding molecules, chimeric antigen receptors or antibody conjugates can be used for the prophylactic treatment of breast cancer and / or bladder cancer. For additional guidance on formulation, dosage, administration schedule and measurable treatment results, see Berkow et al. (2000) The Merck Manual of Medical Information and Merck & Co. Inc., Whitehouse Station, New Jersey; Ebadi (1998) CRC Desk Reference of Clinical Pharmacology.

[0137] On the other hand, the present invention provides one or more of the nanobodies, binding molecules, nucleic acids, recombinant expression vectors, transformants, antibody conjugates, chimeric antigen receptors and pharmaceutical compositions provided herein for use in diagnosing, preventing, improving or treating diseases, disorders or conditions associated with nectin-4 expression.

[0138] In some embodiments, the disease, disorder, or condition associated with nectin-4 expression is selected from one or both of breast cancer and bladder cancer.

[0139] In other embodiments, the in vivo detection method, system or device may include:

[0140] (1) administering the aforementioned binding molecule, the aforementioned nucleic acid, the aforementioned recombinant expression vector, the aforementioned transformant, the aforementioned antibody conjugate, the aforementioned chimeric antigen receptor, or the aforementioned pharmaceutical composition to a subject; and

[0141] (2) Detecting the formation of a complex between the aforementioned binding molecule, the aforementioned nucleic acid, the aforementioned recombinant expression vector, the aforementioned transformant, the aforementioned antibody conjugate, the aforementioned chimeric antigen receptor or the aforementioned pharmaceutical composition and the subject.

[0142] Term Definition

[0143] In order to make it easier to understand this document, some technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this document belongs.

[0144] "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.

[0145] 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.

[0146] In the present invention, "single domain antibody", "heavy chain variable region domain of heavy chain antibody", "VHH", "VHH domain" and "nanoantibody" are used interchangeably and all refer to nanoantibodies that specifically recognize and bind to nectin-4.

[0147] "Antibody" is used in the broadest sense to cover various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions), so long as they exhibit the desired antigen-binding activity.

[0148] In the present invention, "anti-Nectin-4 antibody" refers to an antibody targeting Nectin-4, which can be a nanobody, an antibody binding molecule or an antibody conjugate, all of which have a targeted binding effect.

[0149] In the present invention, the amino acid sequences of the complementarity determining regions (CDRs) listed are shown according to the definition of the Kabat numbering convention. However, it is well known to those skilled in the art that antibody CDRs can be defined by a variety of methods in the art, such as Chothia (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)) based on the three-dimensional structure of the antibody and the topology of the CDR loops, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability, AbM (University of Bath), Contact (University College London), the international ImMunoGeneTicsdatabase (IMGT, World Wide Web imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions as defined by any of the above-mentioned known schemes described in the present invention.

[0150] An "immunoglobulin variable domain" is essentially composed of four "framework regions," referred to in the art and hereinafter as "framework region 1" or "FR1," "framework region 2" or "FR2," "framework region 3" or "FR3," and "framework region 4" or "FR4," and three "complementarity determining regions" or "CDRs," respectively, "complementarity determining region 1" or "CDR1," "complementarity determining region 2" or "CDR2," and "complementarity determining region 3" or "CDR3." Thus, the general structure or sequence of an immunoglobulin variable domain can be represented as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. An immunoglobulin variable domain confers specificity for an antigen by having an antigen-binding site.

[0151] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which can be a heavy chain or light chain domain, including a VH, VHH or VL domain) that can form a functional antigen binding site without interacting with other variable domains (e.g., without the VH / VL interaction required between the VH and VL domains of conventional four-chain monoclonal antibodies). Examples of "immunoglobulin single variable domains" include nanobodies (including VHH, humanized VHH and / or camelized VH, such as camelized human VH), IgNAR, domains, (single domain) antibodies (e.g., dAbs™) that are VH domains or derived from VH domains, and (single domain) antibodies (e.g., dAbs™) that are VL domains or derived from VL domains. Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (e.g., VH or VHH domains) are generally preferred. A specific example of an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") as defined below.

[0152] "VHH domain", also known as heavy chain single domain antibody, VHH, VHH antibody fragment, VHH antibody, nanobody, is the variable domain of the antigen-binding immunoglobulin called "heavy chain antibody" (i.e., "antibody lacking light chain") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). "VHH domain" is used to distinguish the variable domain from the heavy chain variable domain (referred to herein as "VH domain") and the light chain variable domain (referred to herein as "VL domain") present in conventional tetrapeptide chain structure antibodies. VHH domains specifically bind epitopes without the need for additional antigen-binding domains (in contrast to the VH or VL domains in conventional tetrapeptide antibodies, in which the epitope is recognized by both the VL and VH domains). A VHH domain is a small, stable, and highly efficient antigen-recognition unit formed by a single immunoglobulin domain. The terms "heavy chain single-domain antibody," "VHH domain," "VHH," "VHH antibody fragment," "VHH antibody," and "domain" ("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) are used interchangeably. "VHH domain" includes, but is not limited to, natural antibodies produced by camelids, humanized antibodies produced by camelids, or those obtained through phage display technology.

[0153] As is well known in the art for VH and VHH domains, the total number of amino acid residues in each CDR may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual numbering of amino acid residues in the actual sequence. Other numbering systems or coding conventions include Chothia, IMGT, and AbM.

[0154] The total number of amino acid residues in a VHH domain will generally range from 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 herein.

[0155] VHH domains (alone or as part of a larger polypeptide) offer a number of significant advantages over the use of conventional VH and VL domains, scFv or conventional antibody fragments (e.g. Fab- or F(ab')2-fragments):

[0156] Only a single domain is required to bind the antigen with high affinity and selectivity, thus eliminating the need for two separate domains and ensuring that the two domains are in the appropriate spatial conformation and configuration (e.g., scFv generally requires the use of a specially designed linker);

[0157] - VHH domains can be expressed from a single gene and do not require post-translational folding or modification;

[0158] - VHH domains can be easily engineered into multivalent and multispecific formats;

[0159] - VHH domains are highly soluble and have no tendency to aggregate;

[0160] - 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;

[0161] - VHH domains are easy and relatively cheap to prepare, even on the scale required for production;

[0162] - The VHH domain is relatively small compared to conventional tetrapeptide-structured antibodies (approximately 15 kDa or 1 / 10 the size of conventional IgG), and therefore exhibits higher tissue penetration and can be administered at higher doses than conventional tetrapeptide-structured antibodies;

[0163] - VHH domains can display so-called cavity-binding properties (especially due to their extended CDR3 loop compared to conventional VH domains), thus being able to reach targets and epitopes that are inaccessible to conventional tetrapeptide-structured antibodies.

[0164] 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.

[0165] Typically, the anti-nectin-4 antibodies herein will be expressed as preferably 10 -7 to 10 -10 mol / L (M), more preferably 10 -8 to 10 -10 mol / L, even more preferably 10 -9 to 10 -10 or lower dissociation constant (KD), and / or with a dissociation constant of at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, more preferably at least 10 -10 The association constant (KA) of M binds to the antigen to be bound (ie, nectin-4). Any value greater than 10 -4 KD values ​​of M are generally considered to indicate nonspecific binding. Specific binding of an antigen-binding protein to an antigen or epitope can be determined in any suitable manner known in the art, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competition assays) as described herein.

[0166] When "competes" is used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, it means competition between antigen binding proteins, which is determined by the following assay: the antigen binding protein to be tested (e.g., antibody or immunologically functional fragment thereof) prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., nectin-4 antigen or fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, such as: solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid phase direct label assays, solid phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1992). Press); solid phase direct labeling RIA using an I-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology 176:546-552); and directly labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, the assay involves the use of purified antigen that binds to an unlabeled test antigen-binding protein and a labeled reference antigen-binding protein (the antigen being on a solid surface or on the surface of cells). Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cells in the presence of the test antigen-binding protein. Typically, the test antigen-binding protein is present in excess. Antigen binding proteins identified by competitive assays (competing antigen binding proteins) include those that bind to the same epitope as a reference antigen binding protein, and those that bind to an epitope sufficiently proximal to the epitope bound by the reference antigen binding protein that the two epitopes sterically interfere with each other's binding. Further details regarding methods for determining competitive binding are provided in the Examples herein. Typically, when a competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) specific binding of the reference antigen binding protein to the common antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more.In certain instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0167] Conventional techniques known to those skilled in the art can be used to competitively screen antibodies for binding to the same epitope. For example, competition and cross-competition studies can be conducted to obtain antibodies that compete with 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 WO 03 / 48731. Thus, conventional techniques known to those skilled in the art can be used to obtain antibodies that compete with the antibody molecules herein for binding to the same epitope on nectin-4.

[0168] "Cross-reactivity" refers to, for example, the nectin-4 binding protein herein with nectin-4 from a different species. For example, a single domain antibody or derivative protein herein that binds to human nectin-4 may also bind to nectin-4 from another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (e.g., SPR and ELISA), or binding or functional interaction with cells that physiologically express nectin-4. Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.

[0169] "Antigen" refers to a molecule used to immunize an immunocompetent vertebrate to produce antibodies that recognize the antigen, or to screen an expression library (e.g., particularly a phage, yeast, or ribosome display library). As used herein, antigen is defined in a broader sense to include target molecules specifically recognized by antibodies, as well as portions or mimetics of molecules used in immunization procedures for antibody production or library screening for antibody selection. For example, with respect to antibodies herein that bind to human nectin-4, monomers and multimers (e.g., dimers, trimers, etc.) of human nectin-4, as well as truncated and other variants of human nectin-4, are all referred to as antigens.

[0170] "Epitope" refers to a site on an antigen that binds to an immunoglobulin or antibody. An epitope can be formed by adjacent amino acids, or non-adjacent amino acids juxtaposed by tertiary folding of a protein. Epitopes formed by adjacent amino acids are generally retained after exposure to a denaturing solvent, while epitopes formed by tertiary folding are generally lost after treatment with a denaturing solvent. An epitope generally comprises at least 3-15 amino acids in a unique spatial conformation. Methods for determining which epitope is bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays. Methods for determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.

[0171] A "conservative substitution" refers to a substitution with another amino acid residue having properties similar to the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, and aspartic acid and glutamic acid have similar properties in that they have acidic side chains. In addition, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, and alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have non-polar side chains. In addition, tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be apparent to those skilled in the art that even when substituting an amino acid residue in a group that exhibits similar properties as described above, it will not exhibit specific changes in properties.

[0172] "Homology," "identity," or "sequence identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in the two compared sequences is occupied by the same nucleotide or amino acid monomer, for example, if every position in two DNA molecules is occupied by the same nucleotide, then the molecules are homologous at that position. The percent homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100%. For example, if 6 out of 10 positions in the two sequences match or are homologous when the sequences are optimally aligned, then the two sequences are 60% homologous. Generally, a comparison is made when the two sequences are aligned to achieve the maximum percent homology.

[0173] "Nucleic acid molecule" refers to DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.

[0174] "Vector" or "recombinant expression vector" refers to a construct capable of delivering and, in some embodiments, expressing one or more genes or sequences of interest in a host cell. 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 conjugated to a cationic condensing agent, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as production cells.

[0175] "Host cell" includes individual cells or cell cultures that can be or have been recipients of vectors for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and due to natural, accidental or intentional mutations, the progeny may not necessarily be identical (in morphology or genomic DNA complement) to the original parent cell. Host cells include cells transfected and / or transformed in vivo with the polynucleotides herein. "Cell," "cell line," and "cell culture" are used interchangeably, and all such names include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be precisely identical in terms of DNA content. Mutant progeny having the same function or biological activity as that screened for in the originally transformed cell are included. Host cells can include microorganisms (e.g., bacteria), plants, or animal cells. Bacteria readily transformed include Enterobacteriaceae ( enterobacteriaceae ) members, such as Escherichia coli ( Escherichia coli ) or Salmonella ( Salmonella ) strains; Bacillaceae ( Bacillaceae ) For example, Bacillus subtilis ( Bacillus subtilis ); Pneumococcus ( Pneumococcus ); Streptococcus ( Streptococcus ) and Haemophilus influenzae ( Haemophilus influenzae Suitable microorganisms include Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) and Pichia pastoris ( Pichia pastoris Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line), NS0 cells, and 293 cells.

[0176] A "pharmaceutical composition" refers to a mixture containing one or more antibodies described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, and other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitating absorption of the active ingredient and thereby exerting its biological activity.

[0177] "Tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0178] "Cancer," "cancerous," "proliferative disorder," and "tumor" as referred to herein are not mutually exclusive.

[0179] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. "And / or" should be taken as specifically disclosing that each of the two specified features or components has or does not have the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein includes "A and B," "A or B," "A" (alone), and "B" (alone). Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc. should be understood to have an inclusive sense, rather than an exclusive or exhaustive sense; that is, the sense of "including but not limited to."

[0180] In the present invention, application scenarios of "non-diagnostic purposes" include but are not limited to: for example, in vitro detection of the presence or absence of antigens (proteins containing the extracellular region of nectin-4) in the laboratory; or as a positive antibody to screen other antibodies targeting nectin-4; or competing with other antibodies targeting nectin-4 to detect whether there is competition between antibodies, that is, whether the antigen epitopes are the same or similar, etc.

[0181] As used herein, the term "effective amount" refers to an amount of a drug or pharmaceutical agent that elicits the biological or pharmaceutical response of a tissue, system, animal, or human that is being sought, for example, by a researcher or clinician. Furthermore, the term "effective amount" refers to an amount that results in improved treatment, cure, prevention, or alleviation of a disease, condition, or side effect, or reduces the rate of progression of a disease or condition, compared to a corresponding subject that has not received that amount. The term also includes within its scope amounts that are effective to enhance normal physiological function.

[0182] "Subject" and "patient" herein refer to mammals, especially primates, and especially humans.

[0183] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0184] The reagents and raw materials used in the present invention are commercially available.

[0185] The positive progress of the present invention is that the antibody of the present invention can specifically bind to human nectin, cyno nectin-4 and mouse nectin-4 with high affinity, and can also bind to nectin-4 on the surface of human breast cancer cells MCF-7 with high affinity. The antibody also has the advantages of strong tumor targeting, rapid tumor enrichment and long-term retention. BRIEF DESCRIPTION OF THE DRAWINGS

[0186] Figure 1 In vivo fluorescence imaging of the anti-nectin-4 antibody conjugate 198-IR808. DETAILED DESCRIPTION

[0187] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0188] The following embodiments are used to further describe the present invention, but these embodiments are not intended to limit the scope of the present invention.

[0189] Experimental methods in the Examples or Test Examples of the present invention, where specific conditions are not specified, were generally performed under conventional conditions or according to the conditions recommended by the raw material or product manufacturers. See Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; and Contemporary Methods in Molecular Biology, Ausubel et al., Greene Publishing Associates, Wiley Interscience, NY. Reagents whose sources are not specified were commercially available.

[0190] Example 1. Screening and Preparation of Anti-Nectin-4 Antibodies

[0191] In this example, alpacas were immunized using His-tagged human nectin-4 (Acro, NE4-H52H3). Peripheral blood was collected, and PBMCs were isolated. RNA from the PBMCs was extracted and reverse transcribed to obtain total cDNA. A yeast library was constructed for antibody screening.

[0192] After two rounds of sorting, multiple unique VHH sequences binding to the human nectin-4 antigen were obtained through monoclonal identification, sequencing, and sequence analysis. The sequence of NB656-M1-198 is shown below.

[0193] >NB656-M1-198 variable region

[0194] EVQLVESGGGLVQPGGSLRLSCAASGSIFSSIAMAWYRQAPGKQRELVASISGGGSTNYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCNADLDYGLGSGENDYWGQGTQVTVSS (SEQ ID NO: 1)

[0195] The CDR sequences are shown in Table 1 below.

[0196] Table 1 CDR sequence listing: (Kabat numbering rules)

[0197]

[0198] The above sequences were linked to human IgG1 Fc fragments (including the hinge region) to construct VHH-Fc antibodies. Plasmids were constructed and transiently transfected into 293 cells. The cells were cultured for antibody expression and purified using a protein A column. The column was washed with 1× PBS buffer and eluted with 0.1 M glycine buffer (pH 2.5). The column was then dialyzed into 1× PBS buffer (pH 7.4). The sequence of human IgG1 Fc (including the hinge region) is as follows:

[0199] >IgG1 Fc

[0200] EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 7)

[0201] >BS025-PC HC

[0202] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSSLLQMNSLRDEDTAVYYCARAYYYGMDVWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5)

[0203] > BS025-PC LC

[0204] DIQMTQSPSSVSASVGDRVTITCRASQGISGWLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ IDNO: 6)

[0205] Example 2. Affinity determination of anti-nectin-4 antibodies and nectin-4 antigen

[0206] The binding ability of anti-nectin-4 antibody to nectin-4 antigen protein was detected by ELISA and BLI.

[0207] 1.ELISA

[0208] Experimental Methods: Human nectin-4 his (Acro, NE4-H52H3), cyno nectin-4 his (Acro, NE4-C52H4), and mouse nectin-4 his (Acro, NE4-M52H3) were coated at a concentration of 2 μg / mL in 50 mM NaHCO₃, pH 9.6 (100 μl / well) overnight at 4°C. Plates were washed three times with PBST. Blocking was performed with 5% milk at 37°C for 1 hour. After washing once with PBST, the proteins were serially diluted 1:5 from 100 nM in 5% milk. 100 μL of these diluted antibodies were added to the microtiter plate wells and incubated at 37°C for 1 hour. Isotype control antibodies were purchased from Syd Labs, catalog number PA007165.m2a. Wash with PBST 5 times, add the secondary antibody (Anti-Human IgG Fc, HRP, 1:10K) diluted in blocking solution. Wash the ELISA plate incubated with secondary antibody 5 times with PBST, add 100μl TMB single component colorimetric solution to each well and incubate at 37℃ for 7min, add 1M HCL to stop the reaction at 50μl / well, and the OD 450 Reading, calculation of EC 50 .

[0209] Experimental results: The results are shown in Table 2, which show that the NB656-M1-1984 antibody has a strong binding affinity to the nectin-4 antigen, and is stronger than the control antibody BS025-PC.

[0210] Table 2. ELISA detection of anti-nectin-4 antibody binding to antigen

[0211]

[0212] 2. BLI

[0213] Experimental method: First, immerse the biosensor in PBST buffer for 15 minutes to equilibrate, then immerse it in a solution containing a known concentration of antibody (2μg / ml). Then, immerse the sensor with the immobilized antigen in the buffer for baseline equilibrium. Then, immerse the biosensor with the immobilized antigen at a known concentration (200nM, 100nM, 50nM, or 25nM) in a sample solution containing the test antibody. Finally, immerse the sensor bound to the test antibody in the buffer for dissociation. The Octet instrument monitors the biofilm thickness of the biosensor in real time during the experiment to obtain the kinetic constant of the test sample. The specific steps are as follows:

[0214] (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.

[0215] (2) Add 200 μL PBST to the well plate to pre-wet the sensor (the sensor is ProA sensor - Sartorius) for 15 minutes;

[0216] (3) Programming: antibody solidification for 60 s, antigen binding for 180 s, and dissociation for 240 s;

[0217] (4) Prepare the sample according to the set procedure and add the sample to the detection plate;

[0218] (5) Place the sensor and sample plate into the instrument together and perform the test in sequence;

[0219] (6) After the test is completed, recycle the sensor, clean the sample plate, and turn off the instrument power.

[0220] (7) Use the analysis software DataAnalysis12 to perform fitting, export the images and the fitted affinity constant values.

[0221] 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.

[0222] Table 3 Detection of binding ability of anti-nectin-4 antibodies

[0223]

[0224] Example 3. In vitro cell binding assay of anti-nectin-4 antibodies

[0225] Experimental purpose: To detect the binding ability of anti-nectin-4 antibodies using human breast cancer cells MCF-7 (Punosai, CL-0149).

[0226] Experimental method: MCF-7 cells were cultured in Purnosel special medium, washed twice with 1×PBS, and resuspended in 1×PBS to a cell concentration of 3×10 5 cells / μL, aliquot the cells into a PCR plate, 50 μL per well; dilute the test antibody with 1× PBS to a starting concentration of 200 nM, 1:5-fold dilution, make 7 serial dilutions, take 50 μL / well and add it to the plate and incubate in a 4°C refrigerator for 1 hour; after the incubation is completed, wash the cells 3 times with 1× PBS, add 100 μL of fluorescent secondary antibody (647 Anti-HumanIgG Fc) to each well, and incubate in a 4°C refrigerator for 1 hour; after the incubation is completed, wash the cells once with 1× PBS, transfer the cells to a 96-well cell plate, and analyze on a flow cytometer.

[0227] Experimental results: The results are shown in Table 4, which show that the anti-nectin-4 antibodies have strong binding affinity to the nectin-4 antigen, and are stronger than the control antibody BS025-PC.

[0228] Table 4 EC binding of anti-nectin-4 antibodies to cell surface antigen nectin-4 50

[0229]

[0230] Example 4. Endocytic activity of anti-nectin-4 antibodies

[0231] Experimental purpose: To detect the endocytic activity of anti-nectin-4 antibody using human breast cancer cells MCF-7.

[0232] Experimental method: After washing the cells twice with 1× PBS buffer, resuspend the cells in 1× PBS buffer to a cell concentration of 3×10 5Cells / μL were dispensed into PCR plates, 50 μL per well, the test antibody was diluted to 4 μg / mL with 1× PBS buffer, and 50 μL / well was added to the plate to make the final concentration of the antibody 2 μg / mL. Five wells were set for each antibody; the antibody and cells were incubated in a 4°C refrigerator for 1 hour; after the incubation was completed, the cells were washed once with 1× PBS buffer, and 200 μL of 1× PBS buffer was added to resuspend the cells. The antibodies and cells were transferred to a 96-well cell plate according to the time gradient of 0h, 0.5h, 1h, 2h and 4h, and incubated at 37°C; after the incubation was completed, the cells were washed once with 1× PBS buffer, and 100 μL of fluorescent secondary antibody (647 Anti-Human IgG Fc) was added to each well; incubated in a 4°C refrigerator for 1 hour; after the incubation was completed, the cells were washed once with 1× PBS buffer, and 200 μL of 1× PBS buffer was added to resuspend the cells, and the cells were transferred to a 96-well cell plate and analyzed on a flow cytometer.

[0233] Experimental results: As shown in Table 5, the anti-nectin-4 antibody has endocytic activity.

[0234] Table 5 Endocytic activity of anti-nectin-4 antibodies in MCF-7 cells

[0235]

[0236] Example 5. Anti-Nectin-4 Antibody

[0237] The variable region of the antibody was linked to a His-Cys tag to construct a VHH-His-Cys antibody. The plasmid was constructed and transformed into Escherichia coli BL21(DE3) strain. Single colonies were selected and activated overnight in LB medium at 37°C. The cells were transferred to 300 mL of LB medium at a ratio of 1:100. When the OD value reached 0.6, IPTG was added to a final concentration of 1 mmol / L and expression was induced at 30°C for 6 hours. The resulting culture was centrifuged at 6,000 rpm for 5 minutes to collect the cells. The collected cells were resuspended in PBS, polymyxin was added to a final concentration of 0.5 mM, and the cells were disrupted at 37°C for 2 hours. The supernatant was then centrifuged at 8,000 rpm for 30 minutes. The supernatant was filtered and bound to Ni Sepharose (GE) for 2 hours. The supernatant was washed with Tris buffer containing 40 mM imidazole, and the target protein was eluted with 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 sequence of NB656-M1-198-His-Cys is as follows:

[0238] >NB656-M1-198-His-Cys

[0239] EVQLVESGGGLVQPGGSLRLSCAASGSIFSSIAMAWYRQAPGKQRELVASISGGGSTNYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCNADLDYGLGSGENDYWGQGTQVTVSSHHHHHHC (SEQ ID NO: 8)

[0240] Example 6. Anti-Nectin-4 Antibody Conjugate

[0241] The anti-nectin-4 antibody conjugate is obtained by coupling the anti-nectin-4 antibody to the anthocyanin dye derivative IR808 (also known as MHI-808). The specific preparation steps are as follows: 1 mg of the anti-nectin-4 antibody NB656-M1-198-His-Cys was diluted to 1 mg / ml in 20 mM Tris-HCl, pH 8.0 buffer. 2.8 μL of 100 mM TCEP was added to prevent protein aggregation. At a molar ratio of MHI-808:protein of 2:1, approximately 32 μL of MHI-808 (5 mg / ml) was added per 1 mg of protein. The reaction was carried out at 4°C for 30 minutes. The sample was then dialyzed against 20 mM Tris-HCl, pH 8.0 buffer (5 kD dialysis bag). The buffer was changed every 3-4 hours for a total of five changes.

[0242] The anti-nectin-4 antibody conjugate 198-IR808 was obtained according to the above method.

[0243] Example 7. Targeting of anti-nectin-4 antibody conjugates

[0244] Objective: To investigate the tumor targeting and sustained tumor accumulation of anti-nectin-4 antibodies using bladder cancer cells T24 (Punosai, CL-0277).

[0245] Experimental method: First, T24 cells were cultured using the Purnos special medium. T24 cells (5×10 6 The T24 xenograft model was established by subcutaneously injecting 100 cells into the right hind limb of mice (BALB / cNj-Foxn1nu / Gpt, NO.D000521, female, Jicui Yaokang). When the tumor volume reached approximately 200-400 mm 3At the same time, 10 T24 xenograft mice were intravenously injected with the corresponding anti-nectin-4 antibody conjugate (concentration of 1 mg / ml, 100 μl each), and the mice were imaged in vivo with an in vivo imaging system (IVIS) at different time points (1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h and 96h).

[0246] Experimental results: The results showed that the anti-nectin-4 antibody conjugate 198-IR808 has the advantages of strong tumor targeting, rapid tumor accumulation and long-term retention (see Figure 1 ).

Claims

1. A nanobody targeting Nectin-4, the nanobody comprising a heavy chain variable region, the heavy chain variable region comprising CDR1, CDR2 and CDR3, characterized in that, The amino acid sequence of the CDR1 is SIAMA, the amino acid sequence of the CDR2 is SISGGGSTNYADSVKG, and the amino acid sequence of the CDR3 is DLDYGLGSGENDY.

2. The nanobody according to claim 1, characterized in that, The heavy chain variable region further comprises a framework region, preferably the framework region is a camelid-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, 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 and does not involve changes in the CDR sequence.

3. A binding molecule targeting Nectin-4, characterized in that, The binding molecule comprises the nanobody as claimed in claim 1 or 2, and the binding molecule is an Fc fusion antibody, a heavy chain antibody, a monoclonal VHH antibody, a bispecific antibody, a multispecific antibody or a VHH aggregate; Preferably, the binding molecule further comprises an immunoglobulin Fc region; the immunoglobulin Fc region is preferably derived from human or murine, and / or, the immunoglobulin Fc region is preferably IgG Fc, such as IgG1 Fc; More preferably, the amino acid sequence of the immunoglobulin Fc is as shown in SEQ ID NO: 7, 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: 7 and maintains the function of the immunoglobulin Fc region; and / or, The VHH antibody is directly linked to the Fc region to form a VHH-Fc antibody.

4. An isolated nucleic acid, characterized in that, The nucleic acid encodes the nanobody as claimed in claim 1 or 2 or the binding molecule as claimed in claim 3.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as claimed in claim 4; Preferably, the recombinant expression vector is selected from plasmid, phage and viral expression vectors.

6. A transformant, characterized in that, The transformant comprises the recombinant expression vector as claimed in claim 5; Preferably, the host cell of the transformant is selected from Escherichia coli cells, insect cells and mammalian cells, such as 293 cells.

7. A method for preparing a nanobody or binding molecule targeting Nectin-4, characterized in that, The method comprises culturing the transformant as claimed in claim 6 and obtaining the nanobody or the binding molecule from the culture.

8. An antibody conjugate, characterized in that, The antibody conjugate comprises the nanobody as claimed in claim 1 or 2, or, the binding molecule as claimed in claim 3; Preferably, the antibody conjugate further comprises one or more selected from cytokines, lectins, enzymes, radioisotopes, anti-tumor agents, immunomodulators, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds and metal ions; and / or, a His-Cys tag is linked to the C-terminus of the nanobody or the binding molecule in the antibody conjugate, preferably the antibody conjugate has the amino acid sequence as shown in SEQ ID NO: 8; and / or, the antibody conjugate is an antibody probe, and the label of the antibody probe is preferably IR808.

9. 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.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a nanobody as claimed in claim 1 or 2, a binding molecule as claimed in claim 3, an antibody conjugate as claimed in claim 8, or a chimeric antigen receptor as claimed in claim 9; and a pharmaceutically acceptable excipient.

11. A reagent or kit for detecting Nectin-4 and / or diagnosing cancer related to Nectin-4 expression, characterized in that, The reagent or kit comprises one or more of a nanobody as claimed in claim 1 or 2, a binding molecule as claimed in claim 3, an antibody conjugate as claimed in claim 8, and a pharmaceutical composition as claimed in claim 10.

12. Use of one or more of a nanobody as claimed in claim 1 or 2, a binding molecule as claimed in claim 3, an antibody conjugate as claimed in claim 8, a chimeric antigen receptor as claimed in claim 9, and a pharmaceutical composition as claimed in claim 10 in the preparation of a product for diagnosing cancer or a drug for preventing and / or treating cancer; the cancer is a cancer related to Nectin-4 expression; Preferably, the cancer is selected from one or more of breast cancer, lung cancer, colorectal cancer, pancreatic cancer, bladder cancer, and ovarian cancer.

13. A method for detecting Nectin-4 expression for non-diagnostic purposes, characterized in that, The method comprises contacting a test sample with one or more of a nanobody as claimed in claim 1 or 2, a binding molecule as claimed in claim 3, an antibody conjugate as claimed in claim 8, and a pharmaceutical composition as claimed in claim 10, and detecting the expression level of Nectin-4 in the test sample.

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