CN120271711A - Nectin-4 targeting antibodies and uses thereof - Google Patents
Nectin-4 targeting antibodies and uses thereof Download PDFInfo
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Abstract
The invention discloses an antibody targeting Nectin-4 and application thereof. An antibody targeting Nectin-4 comprises at least one immunoglobulin single variable domain comprising a CDR1, a CDR2 and a CDR3, the amino acid sequence of CDR1 being INVMG, the amino acid sequence of CDR2 being TITSGGSTNYADSVKG and the amino acid sequence of CDR3 being DRMDGSIWYDY. The antibody of the invention has stronger binding force with Nectin-4 antibody, and has the advantages of strong tumor targeting, quick tumor enrichment and long retention.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to an antibody targeting Nectin-4 and application thereof.
Background
Nectin-4 (also known as poliovirus receptor associated-4, PVRL-4) is an immunoglobulin-like molecule. Nectin-4 co-acts with other connexins such as Nectin-1, nectin-2 and Nectin-3 to participate in cell-cell adhesion. Nectin-4 is specifically enriched in human embryo and placenta tissue and expression is significantly reduced after adulthood compared to other Nectins. In recent years, nectin-4 has been found to be particularly overexpressed in a variety of malignant tumors such as breast, lung, colorectal, pancreatic, ovarian, and the like, and as a tumor-associated inducer. 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, poor prognosis, etc. (Subhajit et al., 2021).
Although Nectin-4 has shown potential as a therapeutic target in cancer therapy, there are still some problems to be overcome in the aspects of efficacy, safety, drug resistance and mechanism of action of existing Nectin-4 antibodies.
Disclosure of Invention
In order to solve the technical problems, the invention provides an antibody targeting Nectin-4 or an antigen binding fragment thereof, which has stronger binding force with the Nectin-4 antibody and has the advantages of strong tumor targeting, quick tumor enrichment and long retention.
Antibodies or antigen binding fragments thereof targeting Nectin-4
In one aspect, the invention provides an antibody or antigen binding fragment thereof that targets Nectin-4 comprising at least one immunoglobulin single variable domain, CDR1, CDR2 and CDR3, said CDR1 having an amino acid sequence of INVMG (SEQ ID NO: 1), said CDR2 having an amino acid sequence of TITSGGSTNYADSVKG (SEQ ID NO: 2) and said CDR3 having an amino acid sequence of DRMDGSIWYDY (SEQ ID NO: 3).
In some embodiments the antibody or antigen binding fragment thereof comprises at least one immunoglobulin single variable domain comprising CDR1, CDR2 and CDR3 of the amino acid sequence depicted as SEQ ID No. 1;
the CDR1, CDR2 and CDR3 are defined according to Kabat, IMGT, chothia, abM or Contact numbering system. The immunoglobulin single variable structure is one that specifically binds to the Nectin-4 antigen or a fragment thereof.
Provided herein are antibodies or antigen binding fragments thereof that target Nectin-4, comprising any one or any combination of CDR1, CDR2, and CDR3 described above.
In some embodiments, the aforementioned antibody, or antigen binding fragment thereof, the CDR1, CDR2, and CDR3 of the immunoglobulin single variable domain are defined according to the Kabat numbering system.
In some embodiments, the immunoglobulin single variable domain further comprises a framework region, preferably the framework region is an alpaca-derived framework region or a human-derived framework region.
In some embodiments, the amino acid sequence of the immunoglobulin single variable domain in the foregoing antibodies or antigen binding fragments thereof is as set forth in SEQ ID NO. 1, or has at least 80%, at least 85%, at least 90% sequence identity.
In some embodiments, the immunoglobulin single variable domain has an amino acid sequence as set forth 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 to SEQ ID NO. 1 and does not involve a change in CDR sequence.
In some embodiments, the antibody or antigen-binding fragment thereof is a nanobody, an Fc fusion antibody, a heavy chain antibody, a monoclonal VHH antibody, a bispecific antibody, a multispecific antibody, or a VHH multimer.
In some embodiments, the antibody is a nanobody.
In some embodiments, at least one immunoglobulin single variable domain in the foregoing antibodies or antigen binding fragments thereof is a VHH. In some embodiments, the antibody or antigen binding fragment thereof comprises one or more (e.g., 2, 3, 4, 5, 6) of the aforementioned immunoglobulin single variable domains, which may be the same or different, may form a VHH multimer, e.g., a dimer or multimer molecule.
In some embodiments, the antibody or antigen binding fragment thereof further comprises a His tag and/or Cys.
In some embodiments, the foregoing antibodies or antigen binding fragments thereof further comprise an immunoglobulin Fc region.
In some embodiments, the immunoglobulin Fc region is derived from a human or murine source.
In some embodiments, the immunoglobulin Fc region is an IgG Fc.
In some embodiments, the antibody or antigen binding fragment thereof comprises a human immunoglobulin Fc region. For example, the immunoglobulin Fc region is that of human IgG1, igG2, or IgG 4. In some embodiments, the human immunoglobulin Fc region is an Fc region of a wild type IgG or variant thereof.
In some embodiments, the Fc region is one that increases effector function, e.g., increases antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC) has increased effector function.
Exemplary IgG1 Fc regions include those having the following substitutions ：239D;239E;239K,241A;262A;264D;264L;264A;264S;265A;265S;265V;296A;296A;301A;332E;239D/332E;239D/330S/332E;239D/330L/332E;298A/333A/334A;247I/339D;247I/339Q;280H/290S;280H/290S/298D;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. The mutations are defined according to the EU numbering system.
Exemplary IgG1 Fc regions include those having 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;D280H/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 any of the above.
In some embodiments, the Fc region included in the foregoing antibodies or antigen binding fragments thereof may allow the binding protein to form a dimer molecule while extending the in vivo half-life of the binding protein.
In some embodiments, the VHH antibody is directly linked to the immunoglobulin Fc region, constituting a VHH-Fc antibody.
In some embodiments, the immunoglobulin single variable domain in the foregoing antibodies or antigen binding fragments thereof is linked to the Fc region by a linker. The linker may be a nonfunctional amino acid sequence 1-20 or more amino acids long, without secondary or more structure. For example, the linker is a flexible linker, such as G 4 S, GS, GAP, etc.
In some embodiments, the antibody or antigen-binding fragment thereof is a heavy chain antibody further comprising an immunoglobulin Fc region having an amino acid sequence as set forth in SEQ ID No. 7 or having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID No. 7 and maintaining the function of the heavy chain constant region.
In some embodiments, the foregoing antibodies, or antigen binding fragments thereof, further comprise the foregoing His tag sequences. The His tag sequence is a short sequence comprising continuous histidine residues, such as 6 His, 8 His, 10 His, etc.
In some embodiments, the foregoing antibodies, or antigen binding fragments thereof, comprise Cys at the C-terminus or N-terminus.
In some embodiments, the antibody or antigen binding fragment thereof is constructed as a VHH-His-Cys antibody by linking the variable region of the antibody to a His-Cys tag.
In some embodiments, the foregoing antibodies, or antigen binding fragments thereof, comprise the sequence HHHHHHC (SEQ ID NO: 9).
In some embodiments, the antibody or antigen binding fragment thereof has the amino acid sequence shown in SEQ ID NO. 8.
In some embodiments, the antibodies or antigen binding fragments thereof described herein bind Nectin-4 with a KD value of 1X 10 -7 M or less.
In some embodiments, the antibodies or antigen binding fragments thereof described herein bind to a tumor cell. Such as the detection method in example 3.
In some embodiments, the antibodies or antigen binding fragments thereof described herein are endocytosed. Such as the detection method in example 4.
In some embodiments, the foregoing antibodies or antigen binding fragments thereof herein are capable of inhibiting tumor growth by at least about 10%, e.g., at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%.
In some embodiments, the antibodies or antigen-binding fragments thereof herein comprise one or more amino acid substitutions, e.g., conservative amino acid substitutions, e.g., comprising 1,2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions, as compared to SEQ ID NO:1, which substitutions may occur in the CDR regions and/or FR regions.
In some embodiments, the antibodies or antigen binding fragments thereof are provided that bind or compete for binding to the same epitope as the immunoglobulin single variable domain in the aforementioned antibodies or antigen binding fragments thereof targeting Nectin-4 herein.
In some embodiments, the antibodies or antigen-binding fragments thereof are provided that block binding of an immunoglobulin single variable domain in a Nectin-4-targeting antibody or antigen-binding fragment thereof described herein, previously, to Nectin-4 (e.g., human Nectin-4).
In some embodiments, antibodies or antigen binding fragments thereof are provided that target Nectin-4, whose binding to Nectin-4 (e.g., human Nectin-4) is blocked by an immunoglobulin single variable domain in a Nectin-4 binding protein of the foregoing text.
As previously mentioned, and as used herein, "at least 90% identical" includes 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% identical.
In another aspect the invention provides an antibody conjugate comprising an effector molecule and the Nectin-4 targeting antibody or antigen binding fragment thereof disclosed herein.
In some embodiments, the effector molecule is selected from one or more of cytokines, lectins, enzymes, radioisotopes, anti-tumor agents, immunomodulator cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, and metal ions.
In some embodiments, the antibody conjugate comprises the disclosed antibody or antigen binding fragment thereof that targets Nectin-4, further comprising one or more of a His tag, cys, a cytokine, a lectin, an enzyme, a radioisotope, an anti-tumor agent, an immunomodulatory cytotoxic drug, a chromophore, a fluorophore, a chemiluminescent compound, and a metal ion.
In some embodiments, antibody conjugates provided herein are antibody conjugates comprising an antibody or antigen-binding fragment thereof that targets Nectin-4 and an effector molecule. Effector molecules are molecules capable of exhibiting a desired target activity, illustratively selected from the group consisting of radioisotopes, antineoplastic agents, immunomodulators, biological response modifiers, lectins, cytotoxic drugs, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combination thereof.
In some embodiments, the effector molecule is a label. By way of example, the label may be any substance that is detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels can be suitable for immunological detection (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-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)), acridine esters, magnetic beads, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for incorporation of the above-described marker-modified avidin (e.g., streptavidin). The markers encompassed herein can be detected by methods known in the art. For example, the radiolabel may be detected using a photographic film or scintillation calculator, and the fluorescent label may be detected using a photodetector to detect the emitted light. Enzyme labels are typically 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 simply visualizing the coloured label.
In some embodiments, a label as described above may be attached to an antibody herein by linkers of different lengths to reduce potential steric hindrance.
In some embodiments, a label as described above can be attached to an antibody herein by a chelator, such as NOTA, DOTA, and the like.
In some embodiments, the effector molecule is a cytotoxic drug.
In some embodiments, the antibody conjugate has a His-Cys tag attached to the C-terminus of the antibody.
In some embodiments, the antibody conjugate has an amino acid sequence as set forth in SEQ ID NO. 8.
In some embodiments, the antibody conjugate is an antibody probe, preferably the label of the antibody probe is IR808.
In some embodiments, the antibody conjugate is a conjugate of the antibody or antigen binding fragment thereof coupled to IR 808.
Nucleic acids and vectors
In another aspect the invention provides an isolated nucleic acid encoding an antibody or antigen binding fragment thereof as described herein that targets Nectin-4. The nucleic acid herein may be RNA, DNA or cDNA. According to some embodiments herein, the nucleic acid herein is an isolated nucleic acid.
The nucleic acids herein may also be in the form of a vector, may be present in 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 which provides for expression of an antibody or antigen binding fragment thereof targeting Nectin-4 in vitro and/or in vivo (i.e. in a suitable host cell, host organism and/or expression system).
In another aspect, the invention provides a recombinant expression vector comprising the nucleic acid as provided herein. In some embodiments, the recombinant expression vector is a plasmid, phage, or viral vector.
In some embodiments, the viral vector is a retroviral vector, an adenoviral vector, or an adeno-associated viral vector, e.g., a lentiviral vector.
The vectors herein are not generally naturally occurring. However, portions of the vector may be naturally occurring. The recombinant expression vectors of the invention may comprise any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and which may contain natural, non-natural or altered nucleotides. Suitable vectors include those designed for expansion and amplification or for expression or both, examples of which include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmid 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 producer cells.
The expression vector typically comprises at least one nucleic acid herein operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). The choice of the element and its sequence for expression in a particular host is common knowledge to the skilled person. Regulatory elements useful or necessary for the expression of antibodies or antigen binding fragments thereof targeting Nectin-4 herein are, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.
The nucleic acids herein may be prepared or obtained by known means (e.g., by automated DNA synthesis and/or recombinant DNA techniques) based on information about the amino acid sequence of the polypeptides herein, and/or may be isolated from a suitable natural source.
Transformant body
In another aspect, the invention provides a transformant comprising the isolated nucleic acid or recombinant expression vector as provided herein.
Provided herein are recombinant host cells expressing or capable of expressing one or more antibodies or antigen-binding fragments thereof herein that target Nectin-4 and/or containing a nucleic acid or vector herein.
In some embodiments, the host cell of the transformant is a bacterial cell, a fungal cell, an insect cell, or a mammalian cell.
Bacterial cells include, for example, cells of gram-negative bacterial strains such as E.coli (ESCHERICHIA COLI) strains, proteus (Proteus) strains and Pseudomonas (Pseudomonas) strains, and gram-positive bacterial strains such as Bacillus (Bacillus) strains, streptomyces (Streptomyces) strains, staphylococcus (Staphylococcus) strains and Lactobacillus (Lactobacillus) strains.
Fungal cells include, for example, cells of species of Trichoderma (Trichoderma), neurospora (Neurospora) and Aspergillus (Aspergillus); or cells of species including Saccharomyces (e.g., saccharomyces cerevisiae (Saccharomyces cerevisiae)), schizosaccharomyces (e.g., schizosaccharomyces pombe (Schizosaccharomyces pombe)), pichia (Pichia) (e.g., pichia pastoris and Pichia methanolica (Pichia methanolica)), and Hansen (Hansenula).
Mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, heLa cells, COS cells, and the like.
However, amphibian cells, insect cells, plant cells, and any other cell used in the art for expression of heterologous proteins may also be used herein.
The expression vector may be transfected or introduced into a suitable host cell. Various techniques may be used for this purpose, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene editing (CRISPR-Cas system, ZFN system or TALEN system), transposons (sleep reliability or PiggyBAC), gene guns, lipid-based transfection or other conventional techniques. In the case of protoplast fusion, the cells are incubated in medium and screened for appropriate activity. Methods and conditions for culturing the resulting transfected cells and for recovering the resulting antibody molecules are known to those skilled in the art and may be varied or optimized depending on the particular expression vector and host cell used, based on the present specification and methods known in the art. Alternatively, 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. The marker may, for example, provide prototrophy, biocidal resistance (e.g., antibiotics) or heavy metal (e.g., copper) resistance to an auxotrophic host, and the like. The selectable marker gene may 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, transcriptional promoters, enhancers, and termination signals.
Preparation method
Provided herein is a method of making a Nectin-4-targeting antibody or antigen-binding fragment thereof, comprising obtaining the Nectin-4-targeting antibody or antigen-binding fragment thereof from a culture using the transformant provided herein. Specifically, the protein of interest is expressed in a host cell as described above, and isolated from the host cell. Alternatively, a purification step may be included, for example, purification with an A or G Sepharose FF column containing adjusted buffers, washing off non-specifically bound components, eluting the bound antibodies by pH gradient, detection by SDS-PAGE, and collection. Alternatively, the concentration may be carried out by filtration using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.
The antibodies or antigen-binding fragments engineered herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. Mammalian expression systems can lead to glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones were obtained by expressing antibodies that specifically bound to human antigens. Positive clones were expanded in serum-free medium of the bioreactor to produce antibodies. The antibody-secreting culture may be purified and collected using conventional techniques. The antibodies can be concentrated by filtration using conventional methods. The soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange.
Provided herein is a method for preparing an antibody conjugate comprising linking the aforementioned antibody or antigen-binding fragment thereof that targets Nectin-4 and an effector molecule.
In another aspect the invention provides a chimeric antigen receptor comprising an antibody or antigen binding fragment thereof according to one aspect of the invention that targets Nectin-4.
In another aspect the invention provides a genetically modified cell comprising a chimeric antigen receptor as provided herein.
In some embodiments of the invention, the genetically modified cell is a eukaryotic cell, preferably an isolated human cell.
In some embodiments of the invention, the genetically modified cell is an immune cell, such as a T cell or NK cell.
Composition and method for producing the same
In another aspect, the invention provides a pharmaceutical composition comprising one or more of the antibodies or antigen binding fragments thereof, the nucleic acids, the recombinant expression vectors, the transformants, the antibody conjugates, and the chimeric antigen receptor provided by the invention, and pharmaceutically acceptable excipients.
In some embodiments, the pharmaceutical composition comprises an amount of one or more of an antibody or antigen binding fragment thereof, the nucleic acid, the recombinant expression vector, the transformant, the antibody conjugate, and the chimeric antigen receptor as described above that is effective for the treatment, alleviation, or prevention of cancer.
In some embodiments, the pharmaceutically acceptable adjuvant is a pharmaceutically acceptable excipient, diluent or carrier.
In some embodiments, a pharmaceutical composition is provided comprising an antibody or antigen-binding fragment thereof that targets Nectin-4 as described above for diagnosing cancer and at least one pharmaceutically acceptable excipient, diluent or carrier.
In some embodiments, the pharmaceutical composition unit dose may contain from 0.01 to 99% by weight of the Nectin-4-targeting antibody or antigen-binding fragment thereof, or the pharmaceutical composition unit dose may contain from 0.1 to 2000mg, in some embodiments from 1 to 1000mg, of Nectin-4-targeting antibody or antigen-binding fragment thereof.
In some embodiments, an article or product is provided comprising the aforementioned Nectin-4-targeting antibody or antigen-binding fragment thereof. Optionally, the article comprises a container and a label. Containers such as bottles, syringes, and test tubes. The container contains a composition effective for treating a condition. A label on or associated with the container indicates that the composition is useful for treating the selected condition. The composition contains the aforementioned Nectin-4-targeting antibody or antigen binding fragment thereof.
In some embodiments, pharmaceutical compositions are provided comprising an antibody or antigen-binding fragment thereof herein that targets Nectin-4. The Nectin-4-targeting antibody or antigen-binding fragment thereof may be an amount effective to treat or ameliorate a disease (e.g., cancer) or to diagnose a disease (e.g., cancer), and the pharmaceutical composition may further comprise at least one pharmaceutically acceptable excipient, diluent or carrier.
Use of the same
In a further aspect, the invention provides the use of one or more of said antibody or antigen binding fragment thereof, said nucleic acid, said recombinant expression vector, said transformant, said antibody conjugate, said chimeric antigen receptor and said pharmaceutical composition provided by the invention, in the preparation of a reagent or kit.
In some embodiments, the reagent or kit is used to detect Nectin-4 expression.
In some embodiments, the reagent or kit is used for cancer diagnosis.
In some embodiments, the reagent or kit is used for drug development.
In some embodiments of the invention, the reagent or kit is used in vivo imaging techniques, western blot, enzyme linked immunosorbent assays and/or flow cytometry.
In another aspect, the invention provides the use of one or more of the antibody or antigen binding fragment thereof, the nucleic acid, the recombinant expression vector, the transformant, the antibody conjugate, the chimeric antigen receptor and the pharmaceutical composition provided by the invention in the preparation of a medicament for preventing and/or treating cancer associated with Nectin-4 expression.
Provided herein are methods of the foregoing antibodies or antigen-binding fragments thereof or antibody conjugates, polynucleotides, compositions (including pharmaceutical compositions) that target Nectin-4 for treating, ameliorating, preventing, diagnosing a disease or disorder.
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 (e.g., cancer, where cancer and tumor may be used interchangeably), such as Nectin-4 expression or a related disease associated with abnormal Nectin-4 expression (e.g., cancer).
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.
In some embodiments, the aforementioned cancer is breast cancer or bladder cancer.
Detection of
Reagents or kits
In a further aspect the present invention provides a reagent or kit for detecting Nectin-4 and/or diagnosing cancer associated with Nectin-4 expression, said reagent or kit comprising one or more of said antibody or antigen binding fragment thereof, said nucleic acid, said recombinant expression vector, said transformant, said antibody conjugate, said chimeric antigen receptor and said pharmaceutical composition as provided herein in accordance with the present invention, in contact with a test sample, and detecting the level of Nectin-4 expression in the test sample, said method being of non-diagnostic interest.
In some embodiments of the invention, the test sample is a cell or an animal.
In some embodiments of the invention, the method employs in vivo imaging techniques, western blot, ELISA and/or flow cytometry to detect Nectin-4.
In some embodiments, kits are also provided that comprise the aforementioned antibodies or antibody conjugates, polynucleotides, and may further comprise instructions for diagnostic use. The kit may also contain at least one additional reagent, such as a label or an additional diagnostic agent. For in vivo use, the antibody or antibody conjugate may be formulated as a pharmaceutical composition.
Provided herein are detection uses of antibodies or antigen binding fragments or antibody conjugates, polynucleotides, and compositions thereof that target Nectin-4. Also provided herein are methods, systems, or devices for in vivo or in vitro detection of Nectin-4 comprising treating a sample with an antibody or antigen-binding fragment thereof or antibody conjugate, polynucleotide, and composition as previously described herein that targets Nectin-4.
In a further aspect the invention provides a method of detecting Nectin-4 expression using one or more of the antibodies or antigen binding fragments thereof, the nucleic acids, the recombinant expression vectors, the transformants, the antibody conjugates, the chimeric antigen receptors, the pharmaceutical compositions and the reagents or kits provided herein, the method being of non-diagnostic interest.
In some embodiments, it can be used to detect Nectin-4 protein expression in tissue samples, and Nectin-4 expression detection plays a key role in research and application scenarios for non-diagnostic therapeutic purposes. In basic research, scientists have studied their role in cell adhesion, signaling and intercellular communication by examining the expression pattern and function of Nectin-4 in different cell types, tissues or organs. In the field of drug development, detection of Nectin-4 expression not only helps to determine the target point of drug action, but also serves to evaluate the effect of drug candidates on Nectin-4 expression to measure its potential efficacy. In disease model studies, researchers can observe changes in disease progression by altering the expression levels of Nectin-4 in animal models, and thus understand the role of Nectin-4 in the development and progression of disease. Meanwhile, nectin-4 is used as a potential biomarker for exploration, and is not used for diagnosis, but is helpful for researchers to more comprehensively grasp biological characteristics of diseases. In terms of therapeutic target validation and cell therapy development, detection of Nectin-4 expression provides an important basis for preclinical studies and clinical trials, for example in CAR-T cell therapies, to detect whether Nectin-4 is suitable as a target for tumor cells.
In some embodiments, the method comprises the steps of:
(1) Contacting the sample with an antibody or antigen binding fragment thereof that targets Nectin-4;
(2) Detecting a complex formed between an antibody or antigen binding fragment thereof that targets Nectin-4 and the sample;
(3) Detecting the complex.
In some embodiments, the method further comprises the step of contacting a reference sample (e.g., a control sample) with the reagent. The extent of complex formation was determined by comparison with a reference sample. A change in complex formation (e.g., a statistically significant change) in the sample or subject as compared to a control sample or subject indicates the presence of Nectin-4 in the sample.
In another aspect the invention provides a system for detecting expression of Nectin-4, said system comprising:
the antibody or one or more of antigen binding fragments thereof, the nucleic acid, the recombinant expression vector, the transformant, the antibody conjugate, the chimeric antigen receptor, the pharmaceutical composition and the reagent or kit provided by the invention are used for specifically binding to Nectin-4 protein;
sample processing means for receiving and processing a sample to be tested, said sample comprising or possibly comprising a Nectin-4 protein;
the detection platform is selected from one of an immunohistochemical platform, an immunofluorescence platform, an enzyme-linked immunosorbent assay platform, a western blotting platform and a flow cytometry platform;
The signal detection and quantification device is used for detecting a signal generated after an antibody or an antigen binding fragment thereof targeting the Nectin-4 is combined with Nectin-4 protein in a sample, and quantitatively analyzing the signal;
Data analysis and processing software for analyzing the data output by the signal detection and quantification device to determine the expression level of Nectin-4 protein in the sample, and,
Control and control components, including positive and negative controls, are used to verify the accuracy and specificity of the detection system.
In some embodiments, wherein the sample processing device comprises one or more of a fixation, permeation, sectioning, or cell lysis step.
In some embodiments, wherein the signal detection and quantification device comprises an enzyme substrate reaction detector, a fluorescence detector, a radiometer, or an optical density scanner.
In some embodiments, wherein the data analysis and processing software is capable of providing a visual representation of the expression level of Nectin-4 protein.
In another aspect, the invention provides a test device for detecting expression of Nectin-4 protein, the test device comprising:
An antibody or antigen binding fragment thereof targeting Nectin-4 immobilization module for immobilizing the antibody or antigen binding fragment thereof targeting Nectin-4 so as to specifically bind to Nectin-4 protein;
the sample processing module is used for receiving the biological sample and carrying out pretreatment steps such as fixation, infiltration, slicing or cell lysis;
the detection module is used for combining the treated sample with an antibody targeting Nectin-4 or an antigen binding fragment thereof and detecting a binding event, and is selected from one of an immunohistochemical detection module, an immunofluorescence detection module, an enzyme-linked immunosorbent assay detection module, a western blot detection module and a flow cytometry detection module;
a signal detection and quantification unit coupled to the detection module for detecting the signal generated by the binding event and providing a quantitative analysis of the signal;
A data analysis and processing unit coupled to the signal detection and quantification unit for receiving and analyzing the signal data to determine the expression level of the Nectin-4 protein;
And the control and control unit is used for providing positive control and negative control so as to verify the accuracy and the specificity of the detection device.
In some embodiments, the Nectin-4-targeting antibody or antigen binding fragment thereof immobilization module includes one or more microarrays of immobilized antibodies.
In some embodiments, wherein 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 radiometer, and an optical density scanner.
In some embodiments, the data analysis and processing unit includes a user interface for displaying a visual result of the Nectin-4 protein expression level.
In another aspect the invention provides a method of diagnosing, preventing, ameliorating or treating a disease, disorder or condition associated with the expression of Nectin-4, the method comprising administering to a subject in need thereof an effective amount of one or more of the Nectin-4 conjugate, the nucleic acid, the recombinant expression vector, the transformant, the pharmaceutical composition and the agent or kit as provided herein.
In some embodiments, the disease, disorder, or condition associated with expression of Nectin-4 is selected from one or both of breast cancer and bladder cancer.
As used herein, the term "effective amount" means the amount of a drug or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought, for example, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means an amount that results in improved treatment, cure, prevention, or alleviation of a disease, disorder, or side effect, or a reduction in the rate of progression of a disease or condition, as compared to a corresponding subject that does not receive such an amount. The term also includes within its scope an amount effective to enhance normal physiological function.
Pharmaceutical combinations containing antibodies or antigen binding fragments thereof that target Nectin-4 can be used for the prophylactic treatment of breast cancer and/or bladder cancer, see Berkow et al (2000) The Merck Manual of Medical Information (Merck handbook of medical information) and Merck&Co.Inc., Whitehouse Station, New Jersey; Ebadi (1998) CRC Desk Reference of Clinical Pharmacology（ clinical pharmacology, et al for additional guidance regarding formulations, dosages, administration protocols, and measurable therapeutic results.
In a further aspect the invention provides one or more of the antibodies or antigen binding fragments thereof, the nucleic acids, the recombinant expression vectors, the transformants, the antibody conjugates, the chimeric antigen receptors, the pharmaceutical compositions and the reagents or kits as provided herein for use in the diagnosis, prevention, amelioration or treatment of a disease, disorder or condition associated with expression of Nectin-4.
In some embodiments, the disease, disorder, or condition associated with expression of Nectin-4 is selected from one or both of breast cancer and bladder cancer.
In other embodiments, in vivo detection methods, systems, or devices may include:
(1) Administering to a subject the aforementioned Nectin-4-targeting antibody or antigen-binding fragment or antibody conjugate, polynucleotide, and composition
(2) Detecting the formation of a complex between the binding of the aforementioned antibody or antibody conjugate, polynucleotide, and the composition and the subject.
Definition of terms
For easier understanding of the present document, certain technical and scientific terms are specifically defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this document belongs.
"Nectin-4" or "PVRL-4" is used interchangeably and includes variants, isoforms, species homologs of human Nectin-4, and analogs having at least one epitope in common with Nectin-4.
In the present invention, the letters in the amino acid sequence represent single letter abbreviations for amino acids known in the art, such as 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.
In the present invention, "single domain antibody", "heavy chain antibody heavy chain variable region domain", "VHH domain" and "nanobody" are used interchangeably and refer to nanobodies that specifically recognize and bind to Nectin-4.
"Antibody" is used in its broadest sense and covers a variety of 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.
In the present invention, the amino acid sequences of the listed complementarity determining regions (complementarity determining region, CDRs) are all shown according to the definition of the Kabat numbering convention. However, it is well known to those skilled in the art that CDRs of an antibody can be defined in a variety of 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)）,) are based on Kabat (Kabat et al Sequences of Proteins of Immunological Interest, 4 th edition , U.S. Department of Health and Human Services, National Institutes of Health (1987)）,AbM（University of Bath）,Contact （University College London）, International ImMunoGeneTics database (IMGT, world Wide Web IMGT. Cines. Fr /), and North CDR definitions based on neighbor-propagating clusters (affinity propagation clustering) using a large number of crystal structures unless otherwise specified.
"Immunoglobulin variable domain" refers to a region consisting essentially of four "framework regions" known in the art and hereinafter referred to 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" of "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of an immunoglobulin variable domain can be expressed as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Immunoglobulin variable domains confer specificity for antigens by having antigen binding sites.
An "immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which may be a heavy or light chain domain, including a VH, VHH, or VL domain) that can form a functional antigen binding site without interaction with other variable domains (e.g., without VH/VL interactions as required between VH and VL domains of a conventional four chain monoclonal antibody). Examples of "immunoglobulin single variable domains" include nanobodies (including VHHs, humanized VHHs, and/or camelized VH, e.g., camelized human VH), igNAR, domains, (single domain) antibodies that are VH domains or are derived from VH domains (e.g., dAbsTM), and (single domain) antibodies that are VL domains or are derived from VL domains (e.g., dAbsTM). 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.
"VHH domain", also known as a heavy chain single domain antibody, VHH antibody fragment, VHH antibody, nanobody, is the variable domain （Hamers-Casterman C,Atarhouch T,Muyldermans S,Robinson G,Hamers C,Songa EB,Bendahman N,Hamers R.："Naturally occurring antibodies devoid of light chains";Nature363,446-448（1993））. of an antigen-binding immunoglobulin known as a "heavy chain antibody" (i.e., an "antibody lacking a light chain") is used to distinguish the variable domain from the heavy chain variable domain (which is referred to herein as a "VH domain") and the light chain variable domain (which is referred to herein as a "VL domain") present in conventional tetrapeptide chain structural antibodies. The VHH domain specifically binds to the epitope without the need for additional antigen binding domains (this is in contrast to VH or VL domains in conventional tetrapeptide chain structural antibodies, in which case the epitope is recognized by the VL domain along with the VH domain). VHH domains are small stable and efficient antigen recognition units formed from a single immunoglobulin domain. "heavy chain single domain antibody", "VHH domain", "VHH antibody fragment", "VHH antibody" and "domain" ("Nanobody" is a trademark of Ablynx n.v. company, ghent, belgium) are used interchangeably. "VHH domains" include, but are not limited to, natural antibodies produced by camelids, either humanized or screened by phage display techniques.
As is well known in the art for VH domains and VHH domains, the total number of amino acid residues in each CDR may be different and may not correspond to the total number of amino acid residues indicated by Kabat numbering (i.e., one or more positions according to Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by Kabat numbering). This means that in general, 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 rules include Chothia, IMGT, abM.
The total number of amino acid residues in the VHH domain will typically range from 110 to 120, often between 112 and 115. It should be noted, however, that smaller and longer sequences may also be suitable for the purposes described herein.
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):
Only a single domain is required to bind antigen with high avidity and high selectivity, so that neither the presence of two separate domains nor the assurance that the two domains are present in the appropriate spatial conformation and configuration is required (e.g. scFv typically require the use of specifically designed linkers);
The VHH domain can be expressed from a single gene and does not require post-translational folding or modification;
VHH domains can be easily engineered into multivalent and multispecific formats;
-VHH domains are highly soluble and have no aggregation tendency;
VHH domains are highly stable to heat, pH, proteases and other denaturants or conditions, and thus can be prepared, stored or transported without the use of refrigeration equipment, thereby achieving cost, time and environmental savings;
VHH domains are easy to prepare and relatively inexpensive, even on the scale required for production;
The VHH domain is relatively small compared to conventional tetrapeptide chain structural antibodies (about 15kDa or 1/10 of the size of conventional IgG), thus exhibiting higher tissue permeability and can be administered in higher doses compared to conventional tetrapeptide chain structural antibodies;
VHH domains can exhibit so-called cavity binding properties (especially due to their extended CDR3 loop compared to conventional VH domains) so that targets and epitopes not reachable by conventional tetrapeptide chain structural antibodies can be reached.
Methods for obtaining VHH binding to a specific antigen or epitope 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.2645-2652,17June,2009 and WO94/04678.
Typically, an antibody or antigen binding fragment thereof herein that targets Nectin-4 will bind to the antigen to be bound (i.e., nectin-4) with an association constant (KA) 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 M, preferably from 10 -7 to 10 -10 moles/liter (M), more preferably from 10 -8 to 10 -10 moles/liter, even more preferably from 10 -9 to 10 -10 or lower, as measured in a Biacore or KinExA or Fortibio assay. Any KD value greater than 10 -4 M is generally considered to indicate non-specific binding. Specific binding of an antigen binding protein to an antigen or epitope can be determined in any suitable manner known, including, for example, surface Plasmon Resonance (SPR) assays, scatchard assays, and/or competitive binding assays (e.g., radioimmunoassays (RIA), enzyme Immunoassays (EIA), and sandwich competitive assays) described herein.
When "competing" for the case of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, it is meant that competition is between antigen binding proteins, as determined by an assay in which the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) to be detected 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 an antigen binding protein competes with another, such as solid phase direct or indirect Radioimmunoassay (RIA), solid phase direct or indirect Enzyme Immunoassay (EIA), sandwich competition assays (see, e.g., stahli et al, 1983,Methodsin 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 labeling assays, Solid phase direct labeling sandwich assays (see, e.g., harlow and Lane,1988,Antibodies,A Laboratory Manual (antibodies, laboratory Manual), cold Spring Harbor Press), direct labeling of RIA with the solid phase of an I-125 label (see, e.g., morel et al, 1988, molecular. Immunol. 25:7-15), solid phase direct biotin-avidin EIA (see, e.g., cheung, et al, 1990, virology 176:546-552), and direct labeled RIA (Moldenhauer et al, 1990, scand. J. Immunol. 32:77-82). typically the assay involves the use of a purified antigen (on a solid surface or cell surface) that is capable of binding to a detection antigen binding protein that is unlabeled and a reference antigen binding protein that is labeled. The amount of label bound to the solid surface or cell is measured in the presence of the antigen binding protein to be tested to measure competitive inhibition. Typically, the antigen binding protein to be tested is present in excess. Antigen binding proteins identified by a competition assay (competing antigen binding proteins) include antigen binding proteins that bind to the same epitope as the reference antigen binding protein, and antigen binding proteins that bind to an epitope adjacent to the epitope bound by the reference antigen binding protein that are spatially blocked from each other. Additional details regarding methods for assaying competitive binding are provided in the examples herein. Typically, when the competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more of the specific binding of the reference antigen binding protein to the cognate antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
Antibodies can be screened for binding to the same epitope competitively using conventional techniques known to those skilled in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete for binding to an antigen. High throughput methods for obtaining antibodies binding to the same epitope based on their cross-competition are described in International patent publication WO 03/48731. Thus, antibodies that compete with the antibody molecules herein for binding to the same epitope on Nectin-4 can be obtained using conventional techniques known to those skilled in the art.
"Cross-reactive" refers to, for example, nectin-4 binding proteins herein and Nectin-4 from a different species. For example, a single domain antibody or derived protein herein that binds human Nectin-4 may also bind Nectin-4 of another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen, or binding or functional interaction with cells physiologically expressing Nectin-4, in binding assays (e.g., SPR and ELISA). Methods of determining cross-reactivity include standard binding assays as described herein, such as Surface Plasmon Resonance (SPR) analysis, or flow cytometry.
"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., a phage, yeast, or ribosome display library, among others). Antigens are defined more broadly herein, including target molecules specifically recognized by antibodies, as well as including portions or mimics of molecules used in an immunization process for producing antibodies or in a library screen for selecting antibodies. For example, for the 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 referred to as antigens.
An "epitope" refers to a site on an antigen that binds to an immunoglobulin or antibody. Epitopes can be formed by contiguous amino acids, or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by adjacent amino acids are typically maintained after exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost after treatment with denaturing solvents. Epitopes typically comprise at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation detection assays, among others. Methods for determining the spatial conformation of an epitope include techniques in the art and those described herein, such as X-ray crystallography, two-dimensional nuclear magnetic resonance, and the like.
"Conservative substitution" refers to the substitution of another amino acid residue that has similar properties 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 nonpolar side chains. In addition, tyrosine, phenylalanine, tryptophan and histidine have similar properties in that they have aromatic side chains. Thus, it will be apparent to those skilled in the art that even when amino acid residues in groups exhibiting similar properties as described above are substituted, it will not exhibit a particular change in properties.
"Homology", "identity" or "sequence identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptides. When a position in both comparison sequences is occupied by the same nucleotide or amino acid monomer, for example if each position of 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 matched or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, in the optimal alignment of sequences, if there are 6 matches or homologies at 10 positions in the two sequences, then the two sequences are 60% homologous. In general, a comparison is made when two sequences are aligned to give the greatest percent homology.
"Nucleic acid molecule" refers to DNA molecules and RNA molecules. The nucleic acid molecule may 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.
"Vector" or "recombinant expression vector" means 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 cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.
"Host cell" includes individual cells or cell cultures which may or may not be the recipient of the vector used to incorporate the polynucleotide insert. Host cells include progeny of a single host cell, and due to natural, accidental, or deliberate mutation, 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 designations include progeny thereof. It should also be understood that all offspring may not be exactly identical in terms of DNA content due to deliberate or unintentional mutations. Including mutant progeny having the same function or biological activity as screened in the original transformed cell. Host cells may include microbial (e.g., bacterial), plant, or animal cells. Bacteria that are susceptible to transformation include members of the enterobacteriaceae (enterobacteriaceae), such as strains of E.coli (ESCHERICHIA COLI) or Salmonella (Salmonella), the Baciliatae (Baciliatae) such as Bacillus subtilis (Bacillus subtilis), pneumococci (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, 293 cells.
"Pharmaceutical composition" means a mixture comprising one or more antibodies described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.
"Tumor" refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
"Cancer," "cancerous," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.
"Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not. "and/or" should be taken to specifically disclose each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "a and/or B" herein includes "a and B", "a or B", "a" (alone) and "B" (alone). Throughout the specification and claims, the words "comprise," "have," "include," and the like are to be construed as having an inclusive rather than an exclusive or exhaustive meaning, i.e., a meaning of "including but not limited to," unless the context clearly dictates otherwise.
In the present invention, the application scenario of "non-diagnostic purpose" includes, but is not limited to, in vitro detection of the presence or absence of antigen (protein comprising the extracellular domain of Nectin-4) in a laboratory, or screening of other antibodies targeting Nectin-4 as positive antibodies, or competition binding with other antibodies targeting Nectin-4, detection of whether competition exists between antibodies, i.e., whether the epitope of antigen is the same or similar, etc.
In the present invention, the term "effective amount" means an amount of a drug or pharmaceutical agent that elicits the biological or pharmaceutical response in a tissue, system, animal or human that is being sought, for example, by a researcher or clinician. Furthermore, the term "effective amount" means an amount that results in improved treatment, cure, prevention, or alleviation of a disease, disorder, or side effect, or a reduction in the rate of progression of a disease or condition, as compared to a corresponding subject that does not receive such an amount. The term also includes within its scope an amount effective to enhance normal physiological function.
"Subject" and "patient" herein mean a mammal, particularly a primate, particularly a human.
For easier understanding of the present document, certain technical and scientific terms are specifically defined below. The above-mentioned preferred conditions may be arbitrarily combined to obtain each preferred embodiment of the present invention, except on the basis of common knowledge in the art.
The reagents and materials used in the present invention are commercially available.
The antibody disclosed by the invention has the positive progress effects that the antibody can be specifically combined with Human Nectin, cyno Nectin-4 and Mouse Nectin-4 with high affinity, and can be combined with Nectin-4 on the surface of Human breast cancer cells MCF-7 with high affinity, and the antibody also has the advantages of strong tumor targeting, quick tumor enrichment and long retention.
Drawings
FIG. 1 is an in vivo fluorescence imaging of anti-Nectin-4 antibody conjugate 56-IR 808.
Detailed Description
The invention will be further described with reference to the following examples, which are not intended to limit the scope of the invention.
The experimental methods of the present invention, which do not address specific conditions in the examples or test cases, are generally performed under conventional conditions or under conditions recommended by the manufacturer of the raw materials or goods. See Sambrook et al, molecular cloning, a laboratory Manual, cold spring harbor laboratory, current methods of molecular biology, ausubel et al, greene publishing Association, WILEY INTERSCIENCE, NY. The reagents of specific origin are not noted and are commercially available conventional reagents.
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
Example 1 screening and preparation of anti-Nectin-4 antibodies
In this example alpaca immunization was performed using His-tagged human Nectin-4 (Acro, NE4-H52H 3)) as an immunizing antigen. Peripheral blood is taken, PBMC in the peripheral blood is separated, RNA in the PBMC is extracted, and total cDNA is obtained through reverse transcription. Screening of antibodies was performed by constructing a yeast pool.
After two rounds of sorting are finished, a plurality of VHH unique sequences combined with human Nectin-4 antigen are obtained through monoclonal identification, sequencing and sequence analysis. The NB656-M1-56 sequences are shown below.
NB656-M1-56 variable region
EVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVATITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTADRMDGSIWYDYWGQGTQVTVSS（SEQ ID NO: 1）
The CDR sequences are shown in table 1 below,
Table 1 CDR sequence Listing (Kabat numbering convention)
The sequences were each ligated to a human IgG1 Fc (including the hinge region) fragment to construct a VHH-Fc antibody. Plasmid construction, transient 293 cells, cell culture to express antibodies, protein A column purification, 1 XPBS buffer washing, 0.1M glycine buffer (pH 2.5) elution. Dialyzed into 1 XPBS buffer (pH 7.4). Wherein the human IgG1 Fc (comprising the hinge region) sequence is as follows:
> IgG1 Fc
EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK（SEQ ID NO: 7）
>BS025-PC HC
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYNMNWVRQAPGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLSLQMNSLRDEDTAVYYCARAYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK（SEQ ID NO: 5）
> BS025-PC LC
DIQMTQSPSSVSASVGDRVTITCRASQGISGWLAWYQQKPGKAPKFLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPPTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC（SEQ ID NO: 6）
example 2 affinity identification of anti-Nectin-4 antibodies with antigen Nectin-4
The ability of anti-Nectin-4 antibodies to bind to Nectin-4 antigen protein was tested by ELISA and BLI.
1.ELISA
Experimental methods Human Nectin-4 his(Acro, NE4-H52H3), Cyno Nectin-4 his (Acro, NE4-C52H4),Mouse Nectin-4 his (Acro, NE4-M52H3),（50mM NaHCO3,pH 9.6）100μL/ wells were coated individually at a concentration of 2. Mu.g/mL, and antigen coated overnight at 4 ℃. The PBST solution was used for three washes. The blocking was carried out with milk at a concentration of 5% for 1h at 37 ℃. The proteins were diluted 1:5 in a gradient from 100nM (5% milk dilution) and 100. Mu.L of the gradient diluted antibody protein was added to the wells of the microplate and incubated at 37℃for 1h. The isotype control antibody (Isotype) used was purchased from Syd labs under the designation PA0077165.m2 a. Washed 5 times with PBST, corresponding to the secondary antibody (Anti-Human IgG Fc, HRP, 1:10K) after dilution with blocking solution. The secondary antibody incubated ELISA plate was washed 5 times with PBST, incubated for 7min at 37℃with 100. Mu.L of TMB single component color development solution per well, terminated with 50. Mu.L/well of 1M HCL, OD 450 read, and EC 50 calculated.
The results of the experiment are shown in Table 2, and show that the anti-NB 656-M1-56 antibodies have stronger binding force with Nectin-4 antibody and are stronger than the control antibody BS025-PC.
TABLE 2 ELISA detection of antigen binding by anti-Nectin-4 antibodies
2. BLI
The experimental method comprises the steps of firstly immersing a biosensor into a PBST buffer solution for balancing for 15 minutes, immersing the biosensor containing an antibody with a known concentration (2 mug/ml), immersing the sensor solidified with the antigen into the buffer solution for balancing a baseline, immersing the biosensor solidified with the antigen with a known concentration (200 nM, 100nM, 50nM or 25 nM) into a sample solution containing the antibody to be detected, and finally immersing the sensor combined with the antibody to be detected into the buffer solution for dissociation. The dynamic constant of the sample to be tested can be obtained by real-time monitoring of the thickness of the biological film layer of the biosensor in the experimental process through an Octet instrument. The method comprises the following specific steps:
(1) Opening the power supply and operation software of an Octet K2 instrument (Sidoris) to initialize the instrument, wherein the process takes about 1min;
(2) 200 mu L of PBST is added into the pore plate to perform sensor prewetting (the sensor is ProA sensor-Sidories), and the sensor is prewetted for 15min for standby;
(3) Programming, solidifying the antibody for 60s, combining the antigen for 180s, and dissociating for 240s;
(4) Preparing a sample according to a set program, and adding the sample into a detection pore plate;
(5) The sensor and the sample plate are put into an instrument together, and detection is carried out sequentially;
(6) And after the detection is finished, the sensor is recovered, the sample plate is cleaned, and the power supply of the instrument is turned off.
(7) Fitting was performed using analysis software DATAANALYSIS, resulting in a picture and fitting affinity constant values.
The results of the experiment are shown in Table 3, and show that the anti-Nectin-4 antibodies have strong binding force with the Nectin-4 antibodies.
TABLE 3 detection of anti-Nectin-4 antibody binding Capacity
Example 3 in vitro cell binding assay of anti-Nectin-4 antibodies
Experimental purposes the ability of human breast cancer cells MCF-7 (Punuocele, CL-0149) to bind anti-Nectin-4 antibodies was used.
The experimental method comprises culturing MCF-7 cells with a culture medium special for pramoxine, washing the cells with 1 XPBS for 2 times, resuspending the cells with 1 XPBS to obtain a cell concentration of 3×10 5 cells/μl, dispensing the cells into PCR plates with 50 μl per well, diluting the antibody to be tested with 1 XPBS to a concentration of 200 nM, starting with 1:5-fold dilution, performing 7 gradient dilutions, adding 50 μl per well into the plates, incubating for 1 hr at 4 ℃ in a refrigerator, washing the cells with 1 XPBS for 3 times after incubation, adding 100 μl of fluorescent secondary antibody (647 Anti-Human IgG Fc) per well, incubating for 1 hr at 4 ℃ in a refrigerator, washing the cells with 1 XPBS for 1 time after incubation, transferring the cells to 96-well cell plates, and performing an analysis by a flow cytometer.
The results of the experiment are shown in Table 4, and show that the anti-Nectin-4 antibodies have stronger binding force with the Nectin-4 antibodies on the MCF-7 cells of the human breast cancer cells and are stronger than the control antibody BS025-PC.
TABLE 4 binding of anti-Nectin-4 antibodies to the cell surface antigen Nectin-4 EC 50
EXAMPLE 4 endocytosis of anti-Nectin-4 antibodies
The aim of the experiment was to detect endocytosis of anti-Nectin-4 antibodies using human breast cancer cells MCF-7.
The experimental method comprises the steps of washing cells with 1 XPBS buffer for 2 times, resuspending the cells with 1 XPBS buffer to obtain the cell concentration of 3×10 5 cells/mu L, split charging the cells into PCR plates, diluting the antibody to be detected with 1 XPBS buffer to 4 mu g/mL, taking 50 mu L/well and adding the antibody to the plates to obtain the final concentration of 2 mu g/mL, setting 5 holes for each antibody, incubating the antibody and the cells in a refrigerator at 4 ℃ for 1 hour, washing the cells with 1 XPBS buffer for 1 time after the incubation is completed, adding 200 mu L of 1 XPBS buffer to resuspend the cells, transferring the antibody and the cells into 96-well cell plates according to time gradients of 0h, 0.5h, 1h, 2h and 4h, incubating at 37 ℃, washing the cells with 1 XPBS buffer for 1 time after the incubation is completed, adding 100 mu L of fluorescent secondary antibody (64 647 Anti-Human Fc) to the plate at 4 ℃ for 1 hour, washing the cells with 1 XPBS buffer for 1 time after the incubation is completed, transferring the cells to the cell suspension to the 96-well, and carrying out cell flow assay on the cell suspension by the cell flow analyzer.
The results of the experiment are shown in Table 5, and the anti-Nectin-4 antibody was endocytosed.
TABLE 5 endocytic Activity of anti-Nectin-4 antibodies in MCF-7 cells
Example 5 anti-Nectin-4 antibodies
The variable region of the antibody was linked to a His-Cys tag to construct a VHH-His-Cys antibody. Plasmid was constructed and transformed into E.coli BL21 (DE 3) strain, and monoclonal colonies were picked and activated in LB medium at 37℃overnight. Transfer to 300mL LB medium at a ratio of 1:100, culture, and when OD value reaches 0.6, adding IPTG to a final concentration of 1mmol/L, inducing expression at 30deg.C 6 h. The culture system obtained above was centrifuged at 6,000rpm for 5min to collect the cells, the collected cells were resuspended in PBS, polymyxin was added to a final concentration of 0.5mM at 37℃to break 2 h, and then centrifuged at 8,000rpm for 30min. The filtered supernatant was bound to Ni Sepharose (GE) 2 h. The target protein was eluted with a Tris buffer containing 40mM imidazole and with a buffer containing 250mM imidazole. The protein was concentrated by ultrafiltration using ultrafiltration tubes and buffer was replaced with 0.1M NaHCO 3 (pH 8.3), and protein purity >90% was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) testing. Wherein, the NB656-M1-56-His-Cys sequence is as follows:
>NB656-M1-56-His-Cys
EVQLVESGGGLVQPGGSLRLSCAASGSIFSINVMGWYRQAPGKQRELVATITSGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTADRMDGSIWYDYWGQGTQVTVSSHHHHHHC（SEQ ID NO: 8）
EXAMPLE 6 anti-Nectin-4 antibody conjugates
The anti-Nectin-4 antibody conjugate is obtained by coupling an anti-Nectin-4 antibody with an anthocyanin dye derivative IR808 (also known as MHI-808). The preparation method comprises the specific steps of sampling 1mg of an anti-Nectin-4 antibody NB656-M1-56-His-Cys, diluting to 1mg/ml with buffer 20mM Tris-HCl, diluting to pH8.0, adding 2.8 mu L of TCEP (100 mM), preventing protein aggregation, adding about 32 mu L of MHI-808 (5 mg/ml) per 1mg of protein according to the molar ratio of MHI-808 to anti-protein=2:1, reacting for 30min at 4 ℃, dialyzing (dialysis bag 5 KD) in 20mM Tris-HCl, and performing liquid exchange for 3-4h for 1 time, wherein the total liquid exchange is 5 times.
Anti-Nectin-4 antibody conjugate 56-IR808 was obtained as described above.
Example 7 targeting of anti-Nectin-4 antibody conjugates
Experimental objective tumor targeting and tumor persistence of anti-Nectin-4 antibodies were detected using bladder cancer cells T24 (Punuocele, CL-0277).
Experimental method first, T24 cells were cultured using a medium dedicated to pranoss, and T24 cells (5×10 6 cells) resuspended in PBS were subcutaneously injected into the right hind limb of mice (BALB/cNj-Foxn 1 nu/Gpt, no. d000521, female, collectable drug) to construct a T24 xenograft model. When the tumor volume reached about 200-400mm 3, 10T 24 xenograft mice were injected intravenously with the corresponding anti-Nectin-4 antibody conjugate (at a concentration of 1mg/ml, 100 μl each), respectively, and mice were subjected to in vivo fluorescence imaging at different time points (1 h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, and 96 h) using an In Vivo Imaging System (IVIS).
The experimental results show that the anti-Nectin-4 antibody conjugate 56-IR808 has the advantages of strong tumor targeting, fast tumor enrichment and long retention as shown in the figure 1.
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